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מכון טכנולוגי לישראל –הטכניון
Technion – Israel Institute of Technology
ספריות הטכניוןThe Technion Libraries
ייקובס'ג ואן'וג ארווין ש"ע מוסמכים ללימודי הספר בית
Irwin and Joan Jacobs Graduate School
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© שמורות הזכויות כל
חיבור זה או , להפיץ באינטרנט, לאחסן במאגר מידע, לתרגם, להדפיס, )במדיה כלשהי(אין להעתיק
ת או רוביק, הוראה, בקטעים קצרים מן החיבור למטרות לימוד "שימוש הוגן"למעט , כל חלק ממנו .שימוש מסחרי בחומר הכלול בחיבור זה אסור בהחלט. מחקר
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Elucidation of aminoglycosides modes of activity
in eukaryotes: towards improved therapeutic
derivatives
Moran Shalev Ben-Ami
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Elucidation of aminoglycosides modes of activity
in eukaryotes: towards improved therapeutic
derivatives
Research Thesis
In Partial Fulfillment of the Requirements
For the Degree of
Doctor of Philosophy
Moran Shalev Ben-Ami
Submitted to the senate of the
Technion - Israel Institute of Technology
Tammuz 5772 Haifa June 2013
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This research was conducted under the supervision of
Prof. Timor Baasov and Prof. Noam Adir
At the Schulich Faculty of Chemistry, Technion, Israel
The Generous Financial Help Of the Technion and Mr. Seymour Schulich Is
Gratefully Acknowledged
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List of Publications
Publication in scientific journals
Shalev M., Kondo J., Kopelyanskiy D., Jaffe C.L., Adir N., and Baasov T. (2013).
Identification of the molecular attributes required for Aminoglycoside activity
against Leishmania. Proceedings of the National Academy of Sciences USA.
Accepted. IF 9.681; Ranked 3 out of 56 in Multidisciplinary Sciences.
Hariton A., Shalev M., Adir N., Belausov E. and Altstein M. (2013). Structural and
functional differences between cloned and expressed Heliothis peltigera and
Spodoptera littoralis PK/PBAN receptors. Biochimica et Biophysica Acta – general
subjects. Accepted. doi: 10.1016/j.bbagen.2013.06.041. IF 5.000; Ranked 89 out of
290 in Biochemistry & Molecular Biology. Performed the structural studies
including homology model building and structural analysis.
Xue X., Mutyam V., Tang L.P., Biswas S., Du M., Jackson L.A., Dai Y., Belakhov
V., Shalev M., Chen F., Schacht J., Bridges R., Baasov T., Hong J., Bedwell D.M.,
and Rowe S.M. (2013). Synthetic Aminoglycosides Efficiently Suppress CFTR
Nonsense Mutations and Are Further Enhanced by the CFTR Potentiator
Ivacaftor. American Journal of Respiratory Cell and Molecular Biology. Under
review. IF 5.125; Ranked 76 out of 290 in Biochemistry & Molecular Biology.
Ranked 7 out of 50 in Respiratory System. Performed AG monitoring in mice
serum samples.
Shalev M., Kandasamy J., Skalka N., Belakhov V., Rosin-Arbesfeld R., and Baasov
T. (2012). Development of generic immunoassay for the detection of a series of
aminoglycosides with 6′-OH group for the treatment of genetic diseases in
biological samples. Journal of Pharmaceutical and Biomedical Analysis. 75:33–40.
IF 2.979; Ranked 20 out of 73 in Analytical Chemistry.
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Presentations in scientific conferences
Oral and Poster presentations: Shalev M., Kondo J., Kopelyanskiy D., Jaffe C.L.,
Adir N., and Baasov T. Identification of the molecular attributes required for
Aminoglycoside activity against Leishmania. Eurocarb 17, Tel-Aviv, 2013.
Oral and Poster presentations: Shalev M., Kondo J., Kopelyanskiy D., Jaffe C.L.,
Adir N., and Baasov T. Structural insights of aminoglycoside activity in protozoan
parasites. The Israeli Medicinal Chemistry Conference, Rehovot, June 2013.
Oral presentation: Shalev M., Kondo J., Kopelyanskiy D., Jaffe C.L., Adir N., and
Baasov T. Identification of the molecular attributes required for Aminoglycoside
activity against Leishmania. ICA (Israel Crystallography Association), Haifa, May
2013.
Poster presentation: Shalev M., Kondo J., Kopelyanskiy D., Jaffe C.L., Adir N., and
Baasov T. Structural insights of aminoglycoside activity in protozoan parasites.
(Awarded 1st prize) ICS (Israel Chemical Society) meeting, Tel Aviv, Feb 2013.
Poster presentation: Shalev M., Adir N., and Baasov T. Structural insights into
the mechanism of aminoglycoside dependent stop-codon read-through in human
ribosomes. ICS (Israel Chemical Society) meeting, Tel-Aviv, Feb 2011.
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Table of Contents
List of Figures and Tables.............................................................................................
List of Publications……………………………………………………………………
Abstract ............................................................................................................................... 1
List of Abbreviations .......................................................................................................... 3
1. Introduction ..................................................................................................................... 6
1.1 Aminoglycosides....................................................................................................... 6
1.2 Aminoglycosides Mechanism of Action in Bacteria ................................................ 7
1.3. Aminoglycosides and Genetic Diseases ................................................................ 10
1.4. The Read-Through Mechanism ............................................................................. 12
1.5 Aminoglycosides Activity against Parasitic Protozoa - Leishmania ...................... 16
1.6 Aminoglycosides Toxicity ...................................................................................... 17
1.7 Challenges in the Design of Novel Drugs Targeting the Eukaryotic Ribosome .... 18
1.7.1 Development of Read-Through Inducers ......................................................... 18
1.7.2 Development of Anti-Protozoal Derivatives ................................................... 21
1.8 Aminoglycosides and the Eukaryotic Ribosome - Current Knowledge and
Alternative Model Systems ..................................................................................... 22
2. Research Goals and Perspectives .................................................................................. 27
3. Materials and Methods ................................................................................................. 29
3.1 Materials ................................................................................................................. 29
3.2. General Techniques ............................................................................................... 29
3.2.1 X-ray Analysis ................................................................................................. 29
3.2.2 NMR, MS and TLC ......................................................................................... 29
3.3 RNA Purification and Crystallization ..................................................................... 30
3.3.1 RNA Purification ............................................................................................. 30
3.3.2 RNA Annealing ............................................................................................... 31
3.3.3 Crystallization .................................................................................................. 31
3.4. RNA-models: Crystal Handling, Data Collection, Structure Determination and
Refinement .................................................................................................................... 32
3.4.1 Standard Crystallization Procedures and Data Handling ................................. 32
3.4.2 Additional Crystallization Techniques (Seeding, Soaking) ............................. 32
3.5 Yeast 80S Ribosomes Crystallization and Crystal Structure Determination .......... 33
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3.6 Leishmania Cell Culture and Promastigote Viability Assay (IC50). ....................... 35
3.7 The Development of a Highly Sensitive Immunoassay for 6'-OH AG Detection .. 36
3.7.1 Preparation of Polyclonal Abs ......................................................................... 36
3.7.1.1 Preparation of 6'-(R)-methyl-2',3-diazido-1-N-[5-oxopentanoic acid]- ... 37
paromamine (compound B, Figure 3.1). ............................................................... 37
3.7.1.2 Preparation of Immunization Conjugate (compound E, Figure 3.1). ....... 37
3.7.1.3 Immunization and Antiserum Production. ................................................ 38
3.7.2 The Development of a Highly Sensitive Immunoassay ................................... 38
3.7.2.1 Preparation of NB82-OVA Coating Antigen. ........................................... 38
3.7.2.2 Preparation of NB82/84/124-GTA-OVA Coating Antigens .................... 39
3.7.2.3 NB82 Indirect Competitive ELISA .......................................................... 40
3.7.2.4 Cross Reactivity Experiments ................................................................... 40
3.7.2.5 NB82/84/124 Indirect Competitive ELISA .............................................. 41
3.7.2.6 Data Analysis ............................................................................................ 41
3.7.3 Determination of NB Compounds Levels in Mice Serum ............................... 42
4. Results and Discussion ................................................................................................. 43
4.1 Deciphering Aminoglycosides Mechanisms of Action in Eukaryotes ................... 43
4.1.1 rRNA Purification, Complex Generation and Crystallization ......................... 44
4.1.2 Crystal Structure Determination of Human A-site rRNA Complexes with
AG ............................................................................................................................. 46
4.1.2.1 The Double A-site Model ......................................................................... 46
4.1.2.2 The Single A-site Model ........................................................................... 62
4.1.3 Crystal Structure Determination of 80S Yeast Ribosomes in Complex with
AG ............................................................................................................................. 66
4.1.4 Leishmania A-site Crystal Structure Determination ........................................ 69
4.1.4.1 The Binding Mode of G418 to the Leishmanial A-site - ‘ON’ State........ 75
4.1.4.2. The Binding Mode of Apramycin to the Leishmanial A-site - ‘OFF’
State....................................................................................................................... 77
4.1.5. In-vitro Inhibition of L. donovani and L. major Promastigote Growth by
AG. ............................................................................................................................ 79
4.1.6. Aminoglycosides Mechanisms of Action in Leishmania ............................... 82
4.2. The Development and Employment of a Highly Sensitive Immunoassay for the
Detection of AG in Biological Samples................................................................. 84
4.2.1 Preparation of Immunization Conjugate .......................................................... 86
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4.2.2 Development of an Indirect Competitive ELISA ............................................ 88
4.2.3 Development of Heterologous-Assays ............................................................ 93
4.2.4 Determination of Selected NB Compounds Serum Levels in Mice ................ 94
5. Summary and Conclusions ........................................................................................... 98
Appendix ........................................................................................................................... 99
References ....................................................................................................................... 103
I ............................ ...........................................................................................תקציר מחקר
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List of Figures and Tables
Figures:
Figure 1.1: Chemical structures of natural AG derivatives……………………………..…… 7
Figure 1.2: Molecular glance at the bacterial decoding site…………………………...…..… 9
Figure 1.3: The molecular basis of aminoglycosides action in the bacterial ribosome…….... 9
Figure 1.4: The read-through mechanism…………………………………………......…… 11
Figure 1.5: Schematic view of translation and translation termination…………..………... 14
Figure 1.6: A molecular glance at the decoding site conformation upon RF binding to the
bacterial A-site………………………………………………………………...……. 15
Figure 1.7: Bacterial vs. Human mithochondrial A-site……………………………..….… 17
Figure 1.8: Structures of novel semi-synthetic AG that induce translational read-through of
premature stop codons………………………………………………………...……. 21
Figure 1.9: Structures of novel semi-synthetic AG exhibiting some anti-protozoal
properties……………………………………………………………………...……. 22
Figure 1.10: Two dimensional representation of the bacterial vs. eukaryotic cytoplasmic
decoding sites (A-sites)……………………………………………………….…….. 25
Figure 1.11: Superposition of Paromomycin bound to a minimal A-site model and to the
70S ribosome apparatus….……………………………………………………….… 26
Figure 1.12: The eukaryotic A-site models used for the structural exploration of AG
binding site……………………………………………………………………….…. 26
Figure 3.1: Synthetic scheme for the preparation of immunization\coating conjugates…… 36
Figure 4.1: The rRNA constructs used to explore AG interactions…………..…………… 43
Figure 4.2: RNA deprotection and purification………………………………………….… 44
Figure 4.3: Crystals emerged from an initial screen of eukaryotic rRNA duplex in complex
with NB54…………………………………………………………………………... 45
Figure 4.4: Crystals obtained from an optimization screen of eukaryotic rRNA duplex in
complex with NB54………………………………………………………………… 46
Figure 4.5: Structures of rRNA crystals that were crystallized in the presence of a potent
read-through inducer – NB54………………………………………………………. 48
Figure 4.6: NB74-human cytoplasmic A-site complex………………………………..…... 53
Figure 4.7: B-factor analysis of NB54-Cyt1 and NB74-Cyt2 …………………………….. 53
Figure 4.8: Crystal packing……………………………………………………………..….. 54
Figure 4.9: A. Crystals Apramycin-eukaryotic-rRNA duplex…………………………..…. 55
Figure 4.10: Crystal packing of the Apra-cyt complex………………………………...….. 56
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Figure 4.11: G418-human cytoplasmic A-site complex at pH 5.0…………………………. 58
Figure 4.12: Analysis of the pH 5.0 ‘ON’ state structure………………………………..… 61
Figure 4.13: Crystals emerged from single eukaryotic A-site rRNA duplex in complex
with AG……………………………………………………………………………. 63
Figure 4.14: Apramycin bound to the extended single A-site model…………………….... 63
Figure 4.15: Asymmetric unit and crystal packing of crystals emerged of complexes of
the read-through inducers………………………………………………………..…. 65
Figure 4.16: Crystallization of yeast 80S ribosomes…………………………………….… 67
Figure 4.17: Secondary structures of the bacterial, leishmanial and human cytoplasmic
A-sites………………………………………………………………………………. 69
Figure 4.18: Crystals emerged from crystallization experiments of complexes of the
leishmanial A-site model with G418 and Apramycin…………………………..…. 71
Figure 4.19: 2D and 3D representations of the double A-site complexes as obtained in the
X-ray crystal structures…………………………………………………………..…. 71
Figure 4.20: Electron density maps of the Apra-Leish and G418-Leish structures……….. 72
Figure 4.21: Surface representation of apramycin bound to the double A-site rRNA
construct corresponding to its putative binding site in leishmanial ribosomes…….. 72
Figure 4.22: Crystal structure visualization of G418 or Apramycin - leishmanial A-site
rRNA complexes……………………………………………………………………. 74
Figure 4.23: Description of the contacts between G418 and the leishmanial A-site…….…. 76
Figure 4.24: Description of the contacts between Apramycin and the leishmanial A-site..... 78
Figure 4.25: Surface representations of AG binding sites in leishmanial ribosomes…...…. 82
Figure 4.26: General scheme of AG activities in Leishmania………………………….… 83
Figure 4.27: Chemical structures of standard and semi-synthetic AG derivatives used in
the study…………………………………………………………………………..… 86
Figure 4.28: Antiserum activity…………………………………………………………….. 88
Figure 4.29: Antigen recognition (Chessboard) assay……………………………………… 89
Figure 4.30: Standard curve of NB82 (Homologous assay)………………………………... 89
Figure 4.31: Homologous assay cross reactivity…………………………………………… 92
Figure 4.32: Serum levels of NB84 and NB124 following a single dose IP injection in
male mice……………………………………………………………………...……. 96
Tables:
Table 4.1: Crystal data, data collection and data refinement statistics – NB54…………….. 47
Table 4.2: Crystal data, data collection and data refinement statistics……………………... 50
Table 4.3: Crystal data, data collection and data refinement statistics……………………... 52
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Table 4.4: Crystal data, data collection and data refinement statistics for the Apra-cyt
crystal……………………………………………………………………………… 56
Table 4.5: Crystal data, data collection and data refinement statistics ……………………. 59
Table 4.6: Crystal data, data collection and data refinement statistics…………………..… 64
Table 4.7: Crystal data, data collection and data refinement statistics…………………….. 68
Table 4.8: Crystal data, data collection and data refinement statistics…………………….. 73
Table 4.9: In-vitro inhibition of Leishmania promastigotes by natural AG……………… 80
Table 4.10: In-vitro inhibition of Leishmania promastigotes by synthetic AG and
toxicity measurements in HEK293 cells (LC50)………………………………….... 81
Table 4.11: Concentrations at 50% inhibition (I50) and limits of detection (I20) of various
AG using the homologous ELISA with NB82-OVA as a coating antigen....….…. 90
Table 4.12: Concentrations at 50% inhibition (I50) and limits of detection (I20) of
representative members using the heterologous ELISAs…………………………. 94
Table 4.13: Concentrations at 50% inhibition (I50) and limits of detection (I20) of
representative members in mice serum…………………………………………… 95
Table 4.14: Recovery of NB82, NB84 and NB124 from whole blood samples…………... 95
Table I: Crystal screen and initial crystallization conditions determination……………… 99
Table II: Crystal optimization matrix……………………………………………………. 101
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Abstract
Aminoglycosides (AG) are mostly known as highly potent, broad-spectrum
antibiotics that exert their antibacterial activities by selectively targeting the decoding A-
site of bacterial ribosomes, leading to aberrant protein synthesis. Recent studies
suggested that the action of AG is not limited to bacterial ribosomes only; such studies
demonstrated the ability of some AG to interfere with several aspects of eukaryotic
translation. As such, AG were marked as excellent candidates for the treatment of
nonsense mutations mediated human genetic disorders and as alternative treatments for
infections caused by human parasitic protozoa. Nevertheless, despite the great potential
of using AG for such additional therapeutic purposes, their use is quite restricted due to
relatively high toxicity values in human and the information regarding their mechanisms
of action/toxicity in eukaryotes is rather obscure.
Over the last few years several synthetic derivatives where suggested to overcome
some of the enhanced toxicity issues as well as exhibited some improved activity and
selectivity in eukaryotes; many of these derivatives were synthesized in our lab. These
derivatives bear a great potential to serve as therapeutic candidates. However, the newly
synthesized derivatives also demonstrated the necessity to further understand the
molecular mechanisms in which AG confer their biological activity in eukaryotic cells for
further rational drug design.
The present work aimed at exploring AG mechanism(s) of action in eukaryotes at
the molecular level, by solving the X-ray crystal structures of AG bound to their putative
binding sites in eukaryotes - the eukaryotic ribosome A-site where tRNA selection occurs
at the ribosome. These studies were based on the high conservation of the decoding site
among all living kingdoms and the already published literature regarding their ability to
interfere with eukaryotic translation. We present here the crystal structures of different
AG molecules bound to RNA molecules serving as models of the human ribosomal A-
site at resolutions ranging between 2.5-3.0 Å. In addition, crystal structures of two
different AG molecules bound to a model of the Leishmania ribosomal A-site are also
presented: Geneticin (G418), a potent AG for the treatment of leishmaniasis at a 2.65Å
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resolution, and Apramycin, shown to be a strong binder to the leishmanial ribosome
lacking an anti-leishmanial activity at 1.4Å resolution. The structural data, coupled with
in-vitro inhibition measurements on two strains of Leishmania, provides for the first time
insight as to the source of the difference in inhibitory activity of different AG. The
combined structural and biological data sets the ground for rational design of new and
more specific AG derivatives as potential therapeutic agents against leishmaniasis and
nonsense mediated genetic disorders.
In addition to the structural studies, a highly sensitive immunoassay for the
determination and quantification of synthetic AG in biological samples was developed.
The assay has been developed by the generation of highly specific polyclonal antibodies in
rabbits. The assay's efficiency and reproducibility have been demonstrated in treated mice
serum samples. Such assay could be of a great use in the further development of these
therapeutic compounds, allowing the monitoring of AG activities in-vivo. Such tools could
also be exploited for testing some additional mechanistic aspects of AG activities in
eukaryotes such as their permeability characteristics and could be of a great help towards
improving their therapeutic indexes.
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List of Abbreviations
2-DOS - 2-deoxystreptamine
2'-ACE - Acid-labile orthoester protecting group
aa - Amino acid
aa-tRNA - aminoacylated tRNA
Ab - Antibody
AG - Aminoglycoside
AHB - (s)-4-amino-2-hydroxybutanoyl
Apra-Leish - Apramycin bound to leishmanial A-site
A-site - aminoacyl tRNA biding site
AT - Ataxia telangiectasia
CB - Carbonate buffer
CD - Circular dichroism
CE - Capillary electrophoresis
CF - Cystic fibrosis
CFTR - Cystic fibrosis transmembrane conductance regulator
Co-Hex - Cobalt Hexammine
DCC - N'-N'-dicyclohexylcarbodiimide
DDW - Double distilled water
DMD - Duchenne muscular dystrophy
DMF - Dimethylformamide
DMSO - Dimethyl sulfoxide
EF - Elongation factor
ELISA - Enzyme-Linked Immunosorbent Assay
ESI - Electrospray ionization
E-site - Exit tRNA binding site
ESRF - European Synchrotron Radiation Facility
FBS - Fetal bovine serum
FIA - Flouroimmunoassays
FRET - Fluorescence resonance energy transfer
G418 - Geneticin
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G418-Leish - Geneticin bound to leishmanial A-site
G418-Bact - Geneticin bound to bacterial A-site
GAG - Glycosaminoglycan
GC - Gas chromatography
GTA - Glutaraldehyde
H34 - helix 34
H44 - helix 44
H69 - Helix 69
HPLC - High-performance liquid chromatography
HRP - Horseradish Peroxidase
HS - Hürler syndrome
I50 - Sensitivity level (50% Antibody inhibition)
I20 - Limit of detection (20% Antibody inhibition)
IC50 - 50% Growth inhibition values
IP - Intraperitoneal
ITC - Isothermal titration calorimetry
KLH - Keyhole Limpet Hemocyanin
LC50 - 50% Cell growth inhibition values
LD50 - lethal toxicity values
MPD - 2,4-methyl-pentadiol
MPS I-H - Mucopolysaccharidosis type-I-Hurler
MS - Mass spectra
NHS - N-hydroxysuccinimide
NMR - Nuclear magnetic resonance
Os-Hex - Osmiun Hexammine
OVA - Ovalbumin
PAGE - Polyacrylamide gel electrophoresis
PBS - Phosphate buffered saline
PBST - PBS containing Tween-20
PDB - Protein Data Bank
PDR - Physicians Desk Reference
PEG - Polyethylene glycol
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PMe3 - Trimethylphosphine
PSI - Paul Scherrer Institue
P-site - peptidyl tRNA binding site
PTC - premature termination codon
RF - Release factors
RIA - Radioimmunoassays
ROS - Reactive oxygen species
RRE - Rev responsive element
S.E.M - Standard error mean
SLS - Swiss Light Source
SMA - Spinal muscular atrophy
SMN - Survival motor neuron 1
TAR - Tat responsive element
THF - Tetrahydrofuran
TLC - Thin layer chromatography
TMB - 3,3',5,5'-tetramethyl benzidine
USH - Usher syndrome
VPA - Valoparic acid
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1. Introduction
1.1 Aminoglycosides
Aminoglycosides (AG) were first established as antibiotics in the 1940s with the
discovery of Streptomycin and are still widely used worldwide for the treatment of
various infectious diseases 1, 2
. Chemically AG are cationic oligosaccharides composed of
between two and five amino sugar rings. At physiological pH, the amino groups are
nearly all protonated, giving the AG a net positive charge 3, 4
. AG can be categorized
structurally into three groups based upon the identity of conserved aminocyclitol-2-
deoxystreptamine (2-DOS) ring around which they are built. The amino sugar moieties
are distributed about the 2-DOS ring in three major substitution patterns: 4-
monosubstituted - Apramycin; 4,5-disubtituted - Neomycin and Paromomycin; and 4,6-
disubsituted - Gentamicins and Kanamycins (Figure 1.1).
The bacterial ribosome is thought to be the primary target of AG containing a 2-
DOS ring, in which they selectively bind to some highly conserved rRNA residues
composing the ribosomal aminoacyl-tRNA binding site (A-site) where tRNA selection
occurs. AG binding modifies the ribosome's ability to effectively select tRNAs; therefore
severely enhance the abundance of miscoding events, resulting in the accumulation of
mistranslated proteins and eventually leading to cell death. In addition to bacterial
ribosomes, AG have been shown to bind numerous RNA constructs such as ribozymes 5,
introns 6, HIV-1 Tat-responsive element (TAR)
7 and HIV-1 Rev-responsive element
(RRE) 8 and modulate their structure and function. Recent studies, demonstrating the
potential of some AG to influence translational processes of eukaryotes also highlighted
these compounds as potential therapeutic candidates for the treatment of a wide variety of
parasite related infections 9 and some genetic disorders caused by nonsense mutations
10-
12. However, despite these promising clinical results, the mechanistic and structural
information regarding AG action in eukaryotes is currently very limited.
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OH OH
NH2 H
OH
OH
R1 R2 R3
Kanamycin A
Tobramycin
NH2 OH
NH2 OH
R1 R2
NeamineRibostamycin
O
OH
NH2
AHB =
IV
OHHO
NH2NHR4
O
O
O
NH2
R3R2
R1
OHO
H2NOH
OH
II
III
I III
R3
H
H
AHBOH
OH OH OH
R4
H
H
AHB
O
ONH2
OH
OH
H2N
OHO
O
OH
NH2
NHR3
OH
O
O
R1
HOR2
H2N
III
NH2 H H AHBHNH2 H H
H
HNH2
OH
OH
OH
Paromomycin
Neamine
Ribostamycin
Neomycin B
6' 6'
5
4
5
Butirosin A
ButirosinAmikacin
4
ArbekacinDibekacin
Neomycin B
Paromomycin
HNH2 OH OHKanamycin B
NH2
NHCH3 CH3
NH2 CH3
H
R1 R2
Gentamicin C1
Gentamicin C2
Gentamicin C1A
I
HO
NH2
NH2
O
O
OR1
H2N
OHO
HN OH
R2
II
III
6'
4
6
R3
R4 R3 R4
H H
HH
H H
Geneticin (G418) OH CH3 OH OH
I
HO
NH2
NH2
OH
O
O
H2N
IIOO
O
OH
H2NHO
OHHN
CH3
OH
4Apramycin
Figure 1.1: Chemical structures of natural AG derivatives. The 2-DOS conserved ring is highlighted in
blue.
1.2 Aminoglycosides Mechanism of Action in Bacteria
The antibacterial mechanism of action of AG has been well characterized and it
was discovered in the late 1980s that their molecular target is the 16S rRNA which is
located in the small ribosomal subunit (30S) 13
. The ribosome is a large macromolecular
enzyme that catalyzes the sequential addition of amino acids to a growing polypeptide
chain, using mRNA as a template and aminoacylated tRNAs (aa-tRNA) as substrates. In
all organisms, the ribosome consists of a large and a small subunit, which are referred
according to their rate of sedimentation. During the subsequent process of translation,
both subunits act in concert, which involves the movement of mRNA and all three tRNAs
by precisely one codon with respect to the ribosome. The entire process also depends on
several extrinsic protein factors and the hydrolysis of GTP. The ribosome catalytic site
consists of three tRNA-binding sites, designated A (aminoacyl), P (peptidyl) and E (exit),
after their respective tRNA substrates. Recent structural evidence indicate that these three
binding sites are all composed of RNA elements from at least two different ribosomal
domains (Figure 1.2).
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During decoding, a critical step in aminoacyl-tRNA selection is based on the
formation of a mini-helix between the codon of the mRNA and the anti-codon of the
cognate aa-tRNA. This step is believed to occur within the ribosomal A-site, located in
the small ribosomal subunit. Data from chemical protection 14
, cross linking 15
, and
genetic analysis 16
suggest that the decoding site is a region around the ribosomal A-site
that includes helix 44 (H44), the 530 loop, and helix 34 (H34) of 16S RNA. The correct
recognition of each A-site codon by the anticodon of the corresponding aa-tRNA requires
delicate discrimination against non-cognate and near-cognate tRNAs. The high level of
accuracy of translation by the ribosome is thought to be a combination of the initial
codon-anticodon interactions and a very sophisticated proof-reading ability.
Following the determination of the bacterial ribosome structure by X-ray
crystallography, Ramakrishnan and co-workers 17
suggested an atomic level mechanism
of tRNA recognition by the ribosome. In general, the binding of mRNA and cognate
tRNA in the A-site induces two highly conserved bases, A1492 and A1493, to flip out
from the internal loop of H44 (Figure 1.3A). In their new conformations, these residues
tightly interact with the first two bases of the codon-anticodon helix and are therefore
able to monitor and discriminate between Watson-Crick base pairing and mismatches. In
the absence of cognate codon-anticodon mini-helix the two conserved adenines can
occupy one of 2 possible conformations in which they could either both be folded back
within H44 (Figure 1.3B) or only one residue is flipped out (A1493) where the other
(A1492) is directed inside the helical core held by stacking interactions with an adjacent
adenine residue located at the large ribosomal subunit (Figure 1.3C) 18
. When one of
these two conformations occurs the ribosome occupies a rather open conformation and
the translation process is hindered. These possible conformational states were later been
denoted by Westhof and co-workers 19
as 'OFF' (hindered) and 'ON' states and could be
seen as a molecular switch that irreversibly decides whether translation will continue.
The binding of AG to the bacterial A-site seem to stabilize the 'ON' conformation even in
the absence of cognate tRNA-mRNA complex (Figure 1.3D). Thus, the affinity of the A-
site for a near-cognate mRNA-tRNA complex is increased upon AG binding, preventing
the ribosome from efficiently discriminating between near-cognate and cognate
complexes. These interactions reduce the fidelity of normal translational processes
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leading to the accumulation of mistranslated non-functional proteins in bacterial cells,
eventually leading to cell death.
Figure 1.2: Molecular glance at the bacterial decoding site. The 30S ribosomal subunit is showed at the left
side with three tRNA molecules bound (A-site – green; P-site – Yellow; E-site – Blue) and mRNA
highlighted in black. The A-site tRNA is bound to an EF (elongation factor – light pink) which is an
intrinsic protein participating in the translation elongation process. The actual decoding site is highlighted
in red, and is enlarged in the right side of the figure. The mRNA in the enlarged view is highlighted in
yellow; A-site bound tRNA in green; and the two conserved adenine residues, A1492 and A1493, are
highlighted in blue and red, respectively. The PDB entry for the presented structure is 2WRQ.
Figure 1.3: The molecular basis of aminoglycosides
action in the bacterial ribosome, as visualized from
recent X-ray crystal structures of T.thermophilus and
E.coli. The relevant part of H44 is presented in
cartoon representation (grey); A1492 (blue), A1493
(red), and the conserved A1408 (orange) are
represented in filled ring ball and stick
representation. The various conformational
corresponding to A. 'ON' state conformation, where
A1492 and A1493 are flipped out from the helical
core (PDB ID: 1IBM); B. 'OFF' state conformation,
where both adenines are stacked within the interior
of the rRNA helix (PDB ID: 2B9O); and C.
Alternative 'OFF' state conformation where A1492 is
directed towards the helical core and A1493 is fully
bulged out (PDB ID: 3I9B). D. The 30S particle in
complex with Paromomycin. The unique H-bond
pattern is indicated in blue dashed lines. The A1492
and A1493 bases are flipped out, thus indicating the
'ON' state conformation. Paromomycin is
represented in yellow ball and stick representation
(PDB ID: 1IBL).
A. B.
C. D.
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1.3. Aminoglycosides and Genetic Diseases
AG antibacterial activities as miscoding agents have been extensively investigated
over the last 7 decades. However, nearly 2 decades after the discovery of streptomycin, it
was discovered that AG are also able to suppress premature termination codons (PTCs),
restoring full-length protein production in Escherichia coli (E. coli) 20
. PTCs are genetic
code mutations usually occurring due to base pair insertions, deletions, or substitutions
that result in the replacement of an amino-acid codon by one of the three universal stop
codons (UAA, UGA or UAG). These mutations generally lead to the production of
truncated, non-functional proteins (Figure 1.4). In humans, PTCs have been linked to
nearly 2,000 genetic disorders, such as Cystic fibrosis (CF), Duchenne muscular
dystrophy (DMD), ataxia-telangiectasia (AT), Hurler syndrome (HS), Rett syndrome,
Usher syndrome (USH), Hemophilia A, Hemophilia B and Tay-Sachs 21, 22
. For many of
these diseases there is presently no effective treatment.
In the last several years, a new therapeutic strategy has suggested the use of PTCs
suppression in the treatment of several PTC induced genetic disorders 23
. Recent
scientific evidence have demonstrated the ability of some natural AG, such as
Gentamicin, G418 and Paromomycin to induce PTC read-through in various eukaryotic
systems 24-26
, via the selective insertion of a near cognate tRNA at the PTC position;
restoring the production of full length functional proteins (Figure 1.4C). These proof-of-
principle studies have suggested the use of such AG as a possible treatment for human
genetic diseases cause by PTCs, and indeed, recent clinical studies performed in Cystic
fibrosis patients carrying one of the CFTR stop mutations indicated Gentamicin's ability
to improve patients' symptoms 27, 28
.
These encoureging results were further exploited for the establishment of several
clinical trials in DMD patients 29
altogether with massive experiments performed in
various in-vitro, ex-vivo and in-vivo systems on DMD 30, 31
, CF 32
, Rett syndrome 33, 34
,
HS 35
, nephrogenic diabetes insipidus 36
, nephropathic cystinosis 37
, retinitis pigmentosa
38, ataxia-telegiectasia
39, spinal muscular atrophy (SMA)
40 and several genetically
induced cancer types 41-43
. The resulting data supported the previous findings and
highlighted AG as possible candidates for PTC therapy. However, it also emphasized the
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complexity of the mechanisms in which these agents induce their therapeutic effects. For
example, it has been noticed that the sequence context of the PTC affects the efficacy of
mutation suppression (UGA > UAG > UAA). In addition, the nucleotides located
immediately downstream of the stop codon (+4 position), also seem to determine the
read-through potential (C > U > A ≥ G) as well as nucleotides located one residue
upstream to the codon triplet (-1 position), where U seemed to induce the higher read-
through levels. The local sequence context around the PTC and its position within the
gene sequence where also shown to affect the induced read-through levels 26, 44, 45
.
The chemical structure of AG also seems to play a major role in their ability to
interfere with translation termination. In fact, not all AG are capable of inducing
suppression termination, and the explanation for these facts yet remains rather obscure.
In general, AG containing a 6'-OH group on their first ring (such as G418 and
Paromomycin) are superior to those enclosing an NH2 moiety at the same position 26, 46
.
These observations are usually subjected to one of the main differences between their
putative binding sites in eukaryotic vs. prokaryotic systems: the 1408 position (E. coli
numbering). It has been well documented that an A1408G mutation confers resistance to
AG, and that higher levels of resistance are usually observed towards AG containing an
amino moiety in their 6' position 47, 48
. The fact that eukaryotic ribosomes contain a
guanine residue in the corresponding position might explain the higher activity observed
for the 6'-OH derivatives. However, no structural evidence were presented to support
such biochemical observations.
Figure 1.4: A comparison of the A. normal
translation process leading to functional protein
production, B. translation which is interrupted by
a premature termination codon (PTC) leading to a
truncated non-functional protein production, and
C. translation process which was restored by a
read-through inducing compound such as an
aminoglycoside. Terminal codons and PTCs are
highlighted in yellow, aminoglycoside in green.
A.
B.
C.
Stop codon
Stop codon
Stop codon AG
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1.4. The Read-Through Mechanism
In general, a read-through event is defined as the encoding of a PTC by a near
cognate aa-tRNA. Read-through events spontaneously occur in basal levels of less than
1% of translating PTC containing transcripts 49
. Therapeutic compounds inducing higher
read-through activity levels are believed to be able to enhance the possibility of near-
cognate aa-tRNAs to bind PTCs; therefore preferentially enhance their binding over the
binding of release factors (RFs), enabling the synthesis of full length transcripts. These
events can rarely occur on actual stop codons as extensively explained in Bedwell and
coworkers 49
, and are only limited to PTCs.
Recent progress in structural exploration of prokaryotic ribosomes deciphered the
mechanisms of which translation and translation termination occurs at the atomic level.
These findings altogether with the elucidation of AG mechanism of action in bacterial
cells might help in understanding how these therapeutic agents can induce read-through
of PTCs.
Overall, the termination of translation greatly differs from translating sense
codons. The translation of sense codons is performed by a careful selection of cognate aa-
tRNAs containing an appropriate anticodon sequence that can form Watson-Crick pairs
with all 3 codon nucleotides located in the mRNA template 50, 51
. Translation termination,
on the other hand, is encoded by only 3 consensus nucleotide triplets that can only be
recognized by proteins termed class I RFs 50-54
. Structural studies indicated an overall
structural similarity between the structures of RFs and aa-tRNAs. These findings
indicated that both factors occupy an ‘L’ shape conformation while bound to the
ribosome 50-54
. Moreover, both factors have been shown to be rather flexible. These
features are believed to enable the simultaneous interaction with both the ribosomal
decoding center - the A-site, located in the small ribosomal subunit, and the peptidyl
transferase center, located in the large ribosomal subunit.
At the A-site, both factors are able to recognize the relevant codon triplet (sense
codon/termination codon). tRNAs recognition is performed by tight monitoring of
specific hydrogen bonds between the anti-codon triplet located in the tRNA and the
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codon triplet embedded in the mRNA template. RFs recognition is performed by specific
interactions of evolutionary conserved amino-acid (aa) motifs in the RF that tightly
interact with the 3 stop codon nucleotides. Upon recognition of the relevant codon
triplets, conformational changes occur in both the decoding center and the recognition
domain itself. These changes enable the direction of relevant domains of either the tRNA
or RF towards the peptidyl transferase center, where the synthesis of emerging
polypeptide chain occurs. These changes are irreversible and encompass the GTP to GDP
transfer of several extrinsic protein factors such as elongation factors (EFs) or additional
RFs (e.g. eRF3). In the peptidyl transferase center tRNAs catalyze the formation of
peptide bond between the relevant aa and the nascent polypeptide chain. RFs, on the
other hand, catalyze the hydrolysis of peptide chain, followed by its release, and the
beginning of ribosome recycling towards the next translational round (Figure 1.5).
At the atomic level, the above mentioned structural rearrangements upon codon
recognition greatly differ while comparing the two translational factors. The binding of a
cognate tRNA to the decoding center in bacterial ribosomes induces the flip out of the
two evolutionary conserved adenine residues, A1492 and A1493 (Figure 1.2). However,
structural studies on RF binding to bacterial ribosomes 50-54
, demonstrated that unlike in
the sense codon case, in the termination complex only A1492 flips out of H44, leaving
A1493 inside the helical core to establish stacking interactions with an adenine residue,
A1913, located at Helix69 (H69) in the large ribosomal subunit (Figure 1.5C). These
interactions are considered to be highly important for the proper termination of
translation process.
AG were long shown to stabilize the flipping out of the two conserved adenine
residues upon binding to bacterial ribosomes (Figure 1.3D). These interactions were
shown to affect ribosomal accuracy and lead to the production of mistranslated proteins
(Chapter 1.2). AG have also been shown to interfere with RF based peptide release and
induce read-through activity in prokaryotes quite long ago 20, 55
. However, only recent
kinetic indications have linked these interactions to RF inhibition in a competitive
manner 56
. Using the accumulated structural evidence presented here we are now capable
of understanding how such inhibition is possible at molecular levels. As indicated above,
AG binding to the A-site induces the flipping out of A1492 and A1493. The induced
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conformation results in the steric clash of A1493 with RF, therefore, it might inhibit the
actual binding of RFs to the A-site (Figure 1.6). This conformation interferes with RF
binding to the A-site but also promotes the binding of a near cognate tRNAs (Figure
1.6); therefore enabling the resulting read-through activity. In addition, the flipping out of
A1493 prevents the stacking interactions with A1913 of H69 18
. Since these interactions
have been highlighted as important components of RF recognition by the ribosome, it
might add an additional explanation for their induced inhibition.
Figure 1.5 - Schematic view of translation (A) and translation termination (B,C).The left side in object C is
a representation of the 30S ribosomal subunit as obtained from the crystal structure coordinates (PDB ID:
2X9R), with mRNA highlighted in black; P-site and E-site tRNA molecules highlighted in green and blue,
respectively; and RF highlighted in red. The decoding site was highlighted in yellow and an enlarged
representation is presented in the right side of the figure. In the enlarged object RF is highlighted in red;
mRNA in gold; and the conserved adenine residue A1492 and A1493 in blue and purple, respectively.
A.
B.
C.
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Figure 1.6 - A. A molecular glance at the decoding site conformation upon RF binding to the bacterial A-
site. RF is highlighted in green; A1492 (blue) is flipping out from the helical core; A1493 (red) is directed
towards the inner part of the helical core stacking upon A1913 (purple) belonging to H69 at the large
ribosomal subunit (50S). Structures are derived from coordinations taken from PDB entry 2X9R. B.
superposition of the bacterial ribosome structure with the AG paromomycin (yellow sticks) bound to the A-
site (PDB ID: 3IBL) and RF bound to the bacterial ribosome (PDB ID: 2X9R). The structure demonstrate
the clashing of A1493 (red) with the Cα backbone of the RF (green), therefore suggests for a sterical
hindrance of RF to the A-site upon the induced conformation when AG is bound to the bacterial A-site.
These recent findings highlight the possibility that enhanced read-through events
occur due to steric interference of ribosomes with RFs, limiting their binding affinities to
stop codons while enhancing the binding of near-cognate tRNAs to PTCs. These findings
have only been partially demonstrated experimentally in bacteria, however, up to this
date, no such information regarding the read-through mechanism in eukaryotes is yet
available.
A. B.
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1.5 Aminoglycosides Activity against Parasitic Protozoa - Leishmania
AG activities in eukaryotes were extensively investigated over the last few
decades and as such it has been found that some AG could be used as highly potent
agents for the treatment of infections caused by parasitic protozoa such as
trypanosomiasis, giardiasis, amoebiasis and leishmaniasis 57-61
. Very recently,
Paromomycin, a clinically approved AG for the treatment of bacterial infections was
suggested as an alternative treatment of Leishmaniasis.
Leishmaniasis is a spectrum of diseases caused by parasitic protozoa belonging to
the genus Leishmania. It affects millions of people worldwide, appearing mainly in
tropical and subtropical areas 61
. The parasite is transmitted by sandflies, and depending
on the type of disease can be fatal if untreated. The current state-of-the-art in treating
leishmaniasis is based on chemotherapy using a limited array of drugs such as antimony
containing agents, amphotericin B, and Miltefosine 61
. However, due to the emergence of
pronounced parasite drug resistance in some regions, relatively high costs, and/or severe
toxic effects, there has been an extensive search over the last few years for new
therapeutic agents. As such, Paromomycin has been highlighted as a promising candidate
which is now tested in various clinical trials worldwide 61
. Paromomycin was found to be
effective against a wide range of Leishmania parasites and was suggested for the
treatment of both cutaneous and visceral leishmaniasis, which is the fatal form of this
disease 62
. Paromomycin is already being clinically used as the major component of a
topical ointment (Leishcutan) used to treat cutaneous leishmaniasis, and it is registered in
India and Nepal for the treatment of visceral leishmaniasis. Clinical trials using
Paromomycin in combination with other anti-leishmanial drugs are underway in order to
prevent development of parasite resistance 63
.
A very recent paper has demonstrated Paromomycin's ability to inhibit
leishmanial ribosome synthesis in-vitro, and cause translational misreading events in-
vitro. However, despite the great potential in the use of AG as anti-protozoal therapeutic
agents, very little information is available regarding their molecular mechanisms of
action in eukaryotes, and to our knowledge, no comprehensive study has been designed
to test the susceptibility of such parasites to various AG derivatives.
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1.6 Aminoglycosides Toxicity
Despite the promising results indicating the great potential of using AG to treat
eukaryote related disorders, their use for these therapeutic purposes is quite restricted
nowadays. These limitations are mainly due to the relatively high toxicity associated with
their prolonged administration in eukaryotic systems. The mechanisms of which AG
induce their toxic effects in eukaryotes are not fully deciphered yet; the current
information in this field is very little, and is mainly attributed to their positively charged
nature that makes them prone to interact with various negatively charged cellular
components such as phospholipids, phospholipases and various metal ions. These
interactions are believed to eventually lead to the generation of reactive oxygen species
(ROS) 64
which are known to enhance cell toxicity.
AG attribution to the inhibition of eukaryotic translation machinery is also
considered to play a major part in their induced toxicity. However, recent publications
highlighted the mitoribosome as a main target responsible for AG toxic effects. These
papers demonstrated the ability of some natural derivatives to inhibit mitochondrial
protein synthesis; apparently, due to the relatively high similarity the mitochondrial
ribosome shares with their primary target site, the bacterial ribosome 65, 66
. Bacterial and
mitochondrial A-sites 2-dimensional structures are presented in Figure 1.7.
Over the last few years, many efforts have been directed in an attempt to
overcome the toxicity associated with AG administration. Those included co-
administration with a variety of compounds including several antioxidants such as
vitamin E 67
and salicylic acid 68-71
, iron chelators 70, 72
, and some negatively charged
agents such as poly-L-aspartate 73, 74
and daptomycin 75, 76
, which are considered to limit
their unspecific interactions with
negatively charged cellular components.
Recent papers have suggested various
synthetic derivatives with a relatively
reduced toxicity when compared to the
natural compounds.
A. B.
Figure 1.7: Bacterial (A) vs. Human
mithochondrial A-site (B).
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A more recent paper demonstrated the ability to diminish the induced toxicity by the
development of synthetic moieties that selectively target the cytoplasmic eukaryotic
ribosome, avoiding mitochondrial inhibition. These derivatives will further be discussed
in Chapter 1.7.
1.7 Challenges in the Design of Novel Drugs Targeting the Eukaryotic Ribosome
Synthetic derivatives caring an aminoglycosidic scaffold have been extensively
investigated over the last few decades as potential therapeutic agents to be used for the
treatment of general bacterial infections. These efforts resulted in the development of
improved derivatives with reduced toxicity, and enhanced ability to delay the emergence
of resistance among bacterial species. These compounds were extensively investigated in
both biochemical and structural manners, and along with the molecular understanding of
AG mechanisms of action and resistance in bacterial species, opened a new avenue in the
field of antibacterial therapy. However, due to the limited information regarding their
mechanism of action in eukaryotes as well as the higher complexity of these systems,
when compared to the prokaryotic ones, only a few novel semi-synthetic derivatives have
been suggested for the treatment of eukaryote related diseases (Figure 1.8).
1.7.1 Development of Read-Through Inducers
In 2006, Lorson and coworkers 77
demonstrated the ability of 6 semi-synthetic
Neamine and Kanamycin derivatives, such as compound 1 (Figure 1.8), also named
TC007, to promote read-through of PTC in survival motor neuron-1 (SMN) in fibroblasts
derived from SMA patients. These agents have shown to induce an up to a 30 fold
increase in normal protein production compared to the untreated baseline in a dose
dependent manner. Some compounds have shown to induce better activity than the well
documented histone deacetylase inhibitor, valoparic acid (VPA), which is known to
induce SMN expression in mutant cells 78, 79
. TC007 has later been reported to induce
beneficial effects on muscle fiber size and gross motor function in an SMN mice model
77, 80. However, the synthetic derivatives tested within this assay were initially designed
as antibacterial agents 81
therefore contained an amino group in their 6' position.
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Unfortunately, no direct comparison to the natural aminoglycosidic scaffold has been
reported in this manuscript. Comparison of the reported results with earlier work
presented by the same authors indicated similar ability of natural derivatives such as
Tobramycin and Amikacin to induce read-through in SMA patient fibroblasts 40
.
In 2006, Baasov and coworkers first reported on the development of the semi-
synthetic Paromomycin derivative, NB30 (compound 2) 82
(Figure 1.8) to be used as a
prototype read-through inducer. NB30 has been shown to induce PTC suppression in
vitro and ex-vivo against DNA fragments that mimic genes with disease causing nonsense
mutations such as DMD, HS, USH and CF 82, 83
, and recently on mutant retinal explants
derived from mice 84
. Comparison of the obtained results with those obtained for natural
derivatives such as G418, Paromomycin and Gentamicin, indicated lower activity of the
synthetic derivative. However, cell toxicity assays 83
, altogether with ototoxicity assays
performed in cochlear explants and acute toxicity experiments in mice 85
indicated
reduced toxicity of the synthetic derivative when compared to the natural inducers.
Encouraged by these results these researchers further reported in 2009 the
development of the new and improved Paromomycin based synthetic derivate, NB54
(compound 3), that contained an (s)-4-amino-2-hydroxybutanoyl (AHB) moiety at N-1
position (Figure 1.8) 85
. The new structure was designed to have lower toxicity values
comparing to its prototype based on previously documented results indicating decreased
lethal toxicity values (LD50) in mice treated with natural derivatives containing AHB in
their N-1 positions 86
. The resulting structure indeed exhibited a much reduced toxicity
values when compared to its prototype, NB30 85
, and has also been shown to induce an
enhanced read-through activity in-vitro 85
, ex-vivo 33, 34
and in-vivo 87
. NB54 activity
exceeded Paromomycin and Gentamicin activities.
Approximately one year later, the group reported the development of two new
synthetic derivatives in which the first two rings of G418 were used as the
aminoglycosidic scaffold (Figure 1.8; compounds 4,5, designated NB74 and NB84) 88
.
These derivatives were designed to contain a 6'-(R) methyl that we hypothesized to act as
a pharmacophore in G418, but nevertheless, did not contain the garosamine 3rd
ring,
which has been shown to enhance toxicity in our previous studies. Evaluation of the
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resulting compounds indicated better read-through activity in vitro and ex-vivo, along
with significantly reduced cell toxicity. Compound 5 has later been shown to induce read-
through activity in an HS mice model 89
.
Continuous research in our lab has recently led to the development of new
generations of synthetic derivatives which have not only been shown to be potent read-
through inducers with relatively low toxicity, but also to be highly selective towards the
eukaryotic ribosome (Figure 1.8; compounds 6-13, NB118-119, NB 122-125 and NB
127-128) 90, 91
. These derivatives were introduced with a new pharmacophore - 5'' (R/S)
methyl. Compounds 12 and 13 were shown to have read-through activity similar to G418
in both in-vitro and ex-vivo systems, but exhibited a much reduced cell toxicity.
The above mentioned progress in designing synthetic derivatives with potent
read-through potential and low toxicity emerged from a very careful inspection of the
structural elements which are highlighted as important for the biological activity and
toxicity of AG. These recent results are indeed very encouraging, but unfortunately,
rational design of synthetic AG is still far from being well established mainly due to the
lack of detailed information in regarding to the mechanisms involved in AG activity and
toxicity in eukaryotic systems. Our recent publications demonstrated the importance of
high selectivity towards the eukaryotic cytoplasmic ribosomes for the development of
therapeutic agents with reduced toxicity 91
. However, compounds that exhibit an
enhanced selectivity towards eukaryotic systems do not always exceed better read-
through activity. The natural AG Apramycin and the synthetic derivative NB33 (Figure
1.9) where shown to exhibit a rather high selectivity towards eukaryotic systems 92
;
nevertheless, their measured read-through activity appeared to be rather negligible 92
.
Over the last several years a few more AG based synthetic derivatives have been
evaluated as selective potent binders of eukaryotic systems 92
. However, the estimation of
their read-through abilities is yet to be determined.
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O
NH2
NH2
OH
O
O
NH2
HOHO
H2N
6'
5
4
O
HOH2NHO
HO
1 (TC007)
O
NH2
NHR
OH
O
O
OH
HOHO
H2N
6'
5
4
2 (NB30): R=H
O
HO OH
H2N
3 (NB54): R=AHB
5''
O
OH
NH2
AHB =
O
NH2
NHR
OH
O
O
OH
HOHO
H2N
6'
5
4
4 (NB74): R=H
O
HO OH
H2N
5 (NB84): R=AHB
5''
Me
O
NH2
NHR
OH
O
O
OH
HOHO
H2N
6'
5
4
6 (NB119): R=H; *(R)
O
HO OH
NH2
7 (NB123): R=AHB; *(R)
5''
Me *
8 (NB118): R=H; *(S) 9 (NB122): R=AHB; *(S)
O
NH2
NHR
OH
O
O
OH
HOHO
H2N
6'
5
4
10 (NB125): R=H; *(R)
O
HO OH
NH2
11 (NB128): R=AHB; *(R)
5''
Me *
13 (NB127): R=AHB; *(S)12 (NB124): R=H; *(S)
Me
OH
H2NH2N
HO
O
O
HO
OHO
NH2
HO
NH2
NH2
OH
O
O
OH
HOO
H2N
NB33
ApramycinHO
NH2
NH2
OH
O
O
OH
H2N
OO
O
HO
OH
H2NHO HN
Figure 1.8: Structures of novel semi-synthetic AG that induce translational read-through
of premature stop codons (compounds 1-12) and compounds exhibiting some high
selectivity towards eukaryotic systems but lacking any read-through activity (Apramycin
and NB33).
1.7.2 Development of Anti-Protozoal Derivatives
AG anti-protozoal activity has been discovered in the 70s 60
, however, only in
recent years Paromomycin has been suggested clinically for the treatment of such
protozoa related infections 61
. As a result, only recently, there have been evidence for the
development of new and improved derivatives for the treatment of leishmaniasis,
giardiasis and trypanosomiasis 59
. In these recent publications, Hanessian and coworkers
describe the preparation and evaluation of two synthetic therapeutic derivatives that
contain an unsaturated ring I (compound 1a and 1b, Figure 1.9). The synthetic
derivatives are 6'-OH derivatizations of Sisomycin, which is an unsaturated natural AG,
exhibiting some excellent antibacterial activities with relatively low toxicity values 59
.
Derivative 1a, also known as 6'-OH Sisomycin, is nearly identical to Sisomycin,
containing only a single modification in regards to Sisomycin (6'-OH group instead of a
6'-NH2 group). Derivative 1b, also known as Paromo-Siso-hybrid, is a simple
hybridization of the 6'-OH Sisomycin's rings I and II with rings III and IV of
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Paromomycin (Figure 1.9). The presented in-vitro susceptibility assays, performed in a
few strains of pathogenic parasites, indicated IC50 values for derivative 1a at the mid-low
μM range 59
. These values were around 1 order of magnitude lower than the ones
observed for Paromomycin, which is currently under clinical trials worldwide. The IC50
values indicated for compound 1b, were similar to those reported for paromomycin. Cell
toxicity values (LC50) performed in rat skeletal muscle (L6) cells indicated similar
toxicity values for both synthetic derivatives and Paromomycin 59
.
IV
II
III
I
O
ONH2
OH
OH
H2N
OHO
O
OH
NH2
NH2
OH
O
O
OH
H2N
6'
5
4I
HO
NH2
NH2
O
O
OOH
H2N
OHO
HN OH
II
III
6'
4
6
1a (6'-Hydroxysisomycin) 1b (Paromo-siso hybrid)
I
HO
NH2
NH2
O
O
ONH2
H2N
OHO
HN OH
II
III
6'
4
6
Sisomycin
Figure 1.9: Structures of novel semi-synthetic AG exhibiting some anti-protozoal properties.
1.8 Aminoglycosides and the Eukaryotic Ribosome - Current Knowledge and
Alternative Model Systems
The elucidation of x-ray crystal structures of bacterial ribosomes at the turn of the
century enabled a "ribosomal renaissance", allowing relationships between structure and
function to be studied at the atomic level 93-96
. However, the structural information
regarding eukaryotic ribosomes was limited until very recently to medium resolution cryo-
EM single particle reconstructions, due to the more complex nature of eukaryotic systems
97, 98. Recent structural work revealed the three dimensional structures of the 80S yeast
ribosome 99, 100
and the 40S, 60S subunits of the protozoa Tetrahymena thermophila 101, 102
at relatively high resolutions. This recent structural information opens a variety of
opportunities underlying the variance in eukaryotic vs. prokaryotic translation mechanisms
together with unlimited possibilities to explore the interactions of small molecules, such as
AG, with eukaryotic ribosomes. Nevertheless, one mustn't forget that this field is still very
young and the study of complex molecules such as ribosomes still remains very
complicated.
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As an alternative path, over the last several years attempts to provide a molecular
basis for AG activity in mammalian ribosomes have resulted in the development of
various eukaryotic rRNA constructs (Figure 1.10) 103-109
, that mimic putative AG binding
sites in eukaryotic systems. Such constructs (which hence will be called models)
contained only a minimal A-site stabilized with additional non-natural Watson-Crick
pairs. Similar constructs were extensively used for the exploration of AG activity in
prokaryotes and were proven to be highly efficient in mimicking the bacterial A-site.
Superimposition of crystal structures of Paromomycin bound to the 70S apparatus and a
minimal bacterial A-site model is presented in Figure 1.11. The demonstration of a rather
high similarity between the obtained conformation in the minimal model and the one
obtained upon binding to the whole ribosomal apparatus altogether with the simplicity of
using such minimal models for crystallization experiments resulted in a rather extensive
use of such models for the exploration of AG activities in prokaryotes and the rational
development of new and improved antibacterial derivatives 4, 110-114
. The suggested
eukaryotic constructs were based on the high level of rRNA binding site conservation
among prokaryotic and eukaryotic systems (Figure 1.10 A,B); therefore they are believed
to properly represent the ribosomal eukaryotic binding sites.
The resulting constructs were extensively used as models to explore the nature of
various AG interactions with their eukaryotic binding sites. Investigations of the physical
properties involving AG interactions with their putative binding sites have been
thoroughly demonstrated by the use of methods such as isothermal titration calorimetry
(ITC) 107, 115, 116
, UV melting experiments 107, 115, 116
, circular dichroism (CD) 115, 116
,
nuclear magnetic resonance (NMR) 105
, and various fluorescence based studies including
fluorescence resonance energy transfer (FRET) 107-109
. The obtained results indicated
binding affinities at the millimolar range, approximately 3 orders of magnitude higher than
their binding to the corresponding prokaryotic binding sites. These values are well
correlated with in-vitro, ex-vivo and in-vivo studies that evaluated AG ability to induce
read-through activity in eukaryotic systems 82, 85, 90-92, 117
. The obtained affinity gap has
been linked to the minor sequential differences between the eukaryotic and the prokaryotic
binding sites which are located at positions that are considered to be important for AG
binding, such as A1408G and G1491A (Figure 1.10).
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Three of the suggested models were exploited for structural exploration and their
three dimensional structures have been determined by X-ray crystallography 92, 103, 104, 118
or NMR 105, 106, 119
. The structure of a hairpin model suggested by Puglisi and coworkers
has been solved in complex with Paromomycin by NMR spectroscopy 105
(Figure 1.10C).
The global RNA structure in the resulting model was rather similar to the one obtained
from the NMR structure of a corresponding bacterial A-site model in complex with
Paromomycin 120
. However, in the eukaryotic model, the paromomycin binding pocket
was slightly shifted, and its binding did not induce the flipping out of the two conserved
adenine resides as usually observed in the corresponding prokaryotic binding site (Figure
1.12). Nevertheless, while analyzing these data, two important issues should be taken
under consideration: i. the mobility of A1492 and A1493 in the bacterial NMR model was
also limited in comparison to the one observed in X-ray experiments 120
. ii. the RNA
construct used in this study corresponds to the Tetrahymena thermophila decoding site,
and therefore is quite remote from the sequence of the Homo sapiens putative binding site.
In 2006 an RNA construct, that contained two minimal eukaryotic A-site cores
separated by four G=C pairs, was suggested by Westhof and coworkers 121
(Figure
1.10G). The construct was based on a similar previous prokaryotic model that has been
shown to properly mimic the AG prokaryotic binding site and corresponded to the Homo
sapiens A-site sequence 113
. The suggested model has been used for the exploration of
both A-site structure and AG mechanism of action in eukaryotic systems. X-ray structure
determination of a "ligand free" construct indicated two different conformations of the two
binding sites present within the model. These two conformations were suggested to
correspond to an 'ON' state, with the two conserved adenine residues A1492 and A1493
fully bulged-out, and an 'OFF' state, where A1491 fully bulged-out and A1493 directed
toward the helical core (Figure 1.12B-D). The same 'OFF' state conformation was later
found in a crystal structure of a different RNA construct that was used for the exploration
of AG interaction with their putative binding site 104
(Figure 1.10F).
Recently, Westhof and co-workers solved the crystal structures of their eukaryotic
A-site model in the presence of two AG with high selectivity toward the eukaryotic
ribosome 92, 118
: the natural AG Apramycin and the synthetic derivative NB33 that has
been developed in our lab (Figure 1.7). Interestingly, these AG have been shown to
stabilize the 'OFF' state conformation (Figure 1.12E,F). Since these two agents have also
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been shown to be effective inhibitors of the eukaryotic ribosome and lack any read-
through activity 92
these recent achievements may provide indirect evidence for the
similarity of AG mechanism of action in eukaryotic systems to their mechanism of action
in the bacterial ribosome. However, to date, no crystal structure has been solved in the
presence of an AG with read-through ability, and direct evidence is currently unavailable.
Figure 1.10: Two dimensional representation of the bacterial (A.) vs. eukaryotic cytoplasmic (B.)
decoding sites (A-sites). The two conserved adenine residues A1492 and A1493 are highlighted in blue and
red, respectively. G/A1491 in green, and A/G1408 in orange. C-G. Synthetic models of the eukaryotic A-
site used for the exploration of aminoglycosides interactions with their putative binding site. A-site
sequence is highlighted in a red dotted square. Capital letters represent a natural sequence. Small letters
represent synthetic sequences added to stabilize the model or generate a loop. The two hairpin models (C.
and D.) were suggested by Puglisi 105, 106
, Tor 108
and Pilch 107
. Model C. was used for NMR experiments;
Model D. was used for fluorescence spectroscopy experiments. The position marked in * was tagged in a
fluorescence acceptor for FRET experiments. Model E. was suggested by Vourloumis 109
and was also
used for fluorescence spectroscopy exploration. Apart from FRET experiments performed by Tor and
collogues A1492-3 were replaced by 2-aminopurine (2-AP) residues in the fluorescence experiments.
Models F. and G. were suggested by Hermann 104
and Westhof 103
, and were used for X-ray
crystallography.
A. B. C. D. E. F. G.
Bacterial
E. coli Human
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Figure 1.12: The eukaryotic A-site models used for the structural exploration of aminoglycoside binding site. A.
NMR structure of the hairpin model suggested by Puglisi and coworkers in complex with paromomycin (PDB
ID: 1FYP). Paromomycin is shown in green ball-and-sticks representation, two conserved adenins 1492, 1493
are highlighted in blue and red respectively. B. X-ray crystal structure of the RNA duplex containing two
minimal eukaryotic A-sites suggested by Westhof and colleagues (PDB ID: 2FQN). Two different
conformational states were demonstrated within the same construct: C. 'ON' state conformation with adenine
residues A1492 (blue) and A1493 (red) fully bulged-out, and A1491(green) directed toward the helical core. D.
'OFF' state conformation with A1491 (green) and A1493 (red) bulged out and A1492 (blue) directed inside the
helical core. E,F. 'OFF' state conformation induced by apramycin (E.) (PDB ID: 2J5K); and the synthetic
derivative NB33 (F.) (PDB ID: 2O3V).
A. B. C.
D.
E.
F.
A 1493
A 1492
Paromomycin
Figure 1.11: Superposition of Paromomycin
bound to a minimal A-site model (blue, PDB ID:
1J7T) and to the 70S ribosome apparatus (orange,
PDB ID: 1IBL). RMSD 0.69Å.
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2. Research Goals and Perspectives
AG hold great potential to serve as therapeutic agents for the treatment of PTC
induced genetic disorders and parasite related infections. This potential has been already
demonstrated dozens of years ago, and has recently been dulled due to their induced toxic
effects in eukaryotic species. Recent efforts in drug design have resulted in the
development of new and improved derivatives that might overcome these drawbacks and
makes them feasible candidates for such therapeutic purposes. These encouraging recent
achievements have also demonstrated the current need in understanding the molecular
mechanisms by which AG exert their biological activity in eukaryotic cells for rational
development of new therapeutic agents.
To date, no structures of the eukaryotic A-site in complex with any of the AG
exhibiting the relevant biological activity are yet available, and the elucidation of the read-
through mechanism in eukaryotic systems as well as killing mechanisms in parasites are
yet to be discovered. Nevertheless, the relatively high similarity of AG binding sites in
bacterial ribosomes to their putative binding sites in the eukaryotic ribosomes, altogether
with the recent development in eukaryotic ribosome structures makes it likely to assume
that these mechanisms share great similarity. However, further detailed investigations are
needed in order to establish their mechanism of action in eukaryotic system making it
practicable for rational drug design.
The main goals of this study were to ascertain the molecular mechanisms of AG-
induced read-through in human cells as well as to elucidate their mechanisms of action in
eukaryotic parasites. To explore such mechanisms we chose to use X-ray crystallography
as a tool to investigate the unique binding pattern of AG with their putative binding sites
in eukaryotes. The structural studies were correlated with biochemical investigations
performed in-vitro and ex-vivo in order to measure the biological activities of the tested
compounds in the relevant eukaryotic systems, as well as to the chemical synthesis of new
and improved therapeutic derivatives. We truly believe that such interdisciplinary studies
performed under the same roof can give rise to the rational development and improvement
of AG as therapeutics for eukaryote related diseases.
In addition to the structural studies, the present study was aimed at developing a
highly sensitive assay for the determination and quantification of AG which are highly
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selective to eukaryotic systems in biological samples. Such assay will be of a great use in
the further development of these therapeutic compounds, allowing us to monitor their
activities in-vivo. To achieve these goals we chose to develop some highly sensitive
polyclonal antibodies (Abs) that will be able to detect some highly conserved epitopes
present in the majority of therapeutic compounds. Such Abs could be used for the
development of a highly sensitive immunoassay for the determination of AG in various
biological matrices. Such tools could also be exploited for testing some additional
mechanistic aspects of AG activities in eukaryotes such as their permeability
characteristics and could be of a great help improving their therapeutic indexes.
The specific goals of the present study included: (a) Co-crystallization and
determination of the three-dimensional structures of natural and synthetic lead AG with
RNA fragments that reproduce their eukaryotic putative binding sites (A-site); (b)
determination of the molecular basis that governs the AG selectivity towards bacterial
versus eukaryotic ribosomal machineries. (c) Performance of an in-depth chemical
analysis of the modes of structural recognition to elucidate the mechanism by which AG
induce stop-codon read-through and anti-protozoal activities. (d) Performance of docking
and modeling experiments using the resulting molecular coordinates for the rational
design of new derivatives. (e) Preparation of highly selective polyclonal Abs that will be
able to recognize 6'-OH AG derivatives. (f) Development and employment of a highly
selective immunoassay for the detection of such derivatives in various biological matrices.
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3. Materials and Methods
3.1 Materials
Geneticin (G418) and Apramycin were purchased from Apollo Scientific Limited,
UK as sulfate salts. Gentamicin sulfate was purchased from Molekula, UK. All the
synthetic derivatives including neamine, paromamine, NB82, NB74, NB84, NB122,
NB123, NB124 and NB125 were prepared as described previously by Baasov and
coworkers 85, 88, 90, 91
and were used as their sulfate salts. All other chemicals and
biochemicals, unless otherwise stated, were obtained from Merck or Sigma.
3.2. General Techniques
3.2.1 X-ray Analysis
X-ray measurements were performed using synchrotron radiation at a wave length
of ~0.9Å, or by using Rigaku Raxis IV++ and NarµX X-ray System machineries with a
fixed wavelength of 1.54Å (Tel-Aviv and Ben-Gurion Universities, respectively).
Synchrotron radiation experiments were performed either in the Swiss Light Source
(SLS) facility located at the Paul Scherrer Institut (PSI) in Villigen, Switzerland (PXI) or
at the European Synchrotron Radiation Facility (ESRF) located in Grenoble, France
(ID14-1, ID14-4, ID23-1, ID23-2 and ID29). Measurements were performed on frozen
crystal samples that were mounted on nylon cryo-loops (Hampton research) prior to flash
freezing in liquid nitrogen.
3.2.2 NMR, MS and TLC
1HNMR (Nuclear Magnetic Resonance) spectra was recorded on a Bruker Avance™ 500
spectrometer at 500 MHz. Chemical shifts reported (in ppm) were relative to the internal
standard Me4Si (δ = 0.0) with CDCl3 as the solvent. 13
CNMR spectra was recorded on a
Bruker Avance™ 500 spectrometer at 125.8 MHz, and the chemical shifts reported (in
ppm) were relative to the residual solvent signal for CDCl3 (δ = 77.00). Mass spectra
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(MS) analysis was obtained on a Bruker Daltonix Apex 3 mass spectrometer under
electrospray ionization (ESI). Reactions were monitored by Thin Layer Chromatography
(TLC) on Silica Gel 60 F254 (0.25 mm, Merck), and spots were visualized by charring
with a yellow solution containing (NH4)Mo7O24•4H2O (120 g) and (NH4)2Ce(NO3)6 (5 g)
in 10% H2SO4 (800 mL). Flash column chromatography was performed on Silica Gel 60
(70–230 mesh).
3.3 RNA Purification and Crystallization
3.3.1 RNA Purification
RNA constructs corresponding to the human and leishmanial A-sites were
chemically synthesized by Dharmacon (Boulder, CO, USA). RNA sequences used were
as follows: Human A-site used for the double A-site model experiments was composed of
two identical RNA sequences: 5'-UUGCGUCGCUCCGGAAAAGUCGC-3'; Human A-
site used for the single A-site experiments was composed of two different RNA
sequences: (a) 5'-GGGCGUCGCUAGUACCG-3', (b) 5'-GGUACUAAAAGUCGCCCC-
3'. Leishmanial double A-site model was composed of a single RNA sequence
corresponding to the leishmanial A-site: 5'-UUGCGUCGUUCCGGAAAAGUCGC-3'.
In order to avoid RNA degradation during shipment the RNA oligos arrive with
an acid-labile orthoester protecting group on the 2'-hydroxyl (2’ACE). Prior to
crystallization these protection groups had been removed in order to “activate” the RNA
oligos. Deprotection procedure has been performed at pH 3.8, 60ºC for 30 minutes.
Following deprotection the oligo RNAs were purified by anion exchange purification
under denaturating conditions (8M urea, 60ºC) on AKTA FPLC device connected to a
UV detector. Elution was performed by gradually increasing the NaClO4 concentration
within the buffer solution (1-400 mM). Fractions of 1 ml were automatically collected
and the purity level of RNA containing samples has been analyzed by analytical PAGE
(Figure 4.2). Samples with high purity were pooled for desalting. Desalting was
performed by using Sep-Pac C-18 column (Waters) that was preconditioned by a wash
with 10 ml of absolute ethanol, followed by a wash with 10 ml of MilliQ water. RNA
samples were loaded on the columns, which were then washed with 10 ml MilliQ water.
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Elution was carried out with 5 ml of 80% ACN. The samples underwent vacuum
evaporation and dissolved in MilliQ water to final concentration of 4mM. The RNA
concentration was determined by spectroscopic measurement of absorption at 260nm.
3.3.2 RNA Annealing
In order to avoid heterogeneity in the RNA constructs' conformations, purification
was followed by additional steps of denaturation (at 90˚C) and annealing (by slow
cooling to 37˚C, ~2h). These stages ensure the formation of thermodynamically stable
RNA constructs with maximal base-pairing. In experiments were ligand interactions with
the RNA construct were examined, an additional step of ligand introduction was added.
Generally, the ligand will be mixed with the RNA constructs at 37˚C for 10 min in
various ligand:RNA ratios prior to crystallization. The RNA solution used in the X-ray
experimentation was usually composed of 2 mM RNA in 100 mM sodium cacodylate pH
5.0-7.5 and 25 mM NaCl.
3.3.3 Crystallization
Crystallization experiments were performed at 20°C using the hanging-drop vapor
diffusion method. Droplets were prepared by mixing 1 μL of RNA/AG solutions and 1
μL of crystallization solutions as indicated in Tables I, II (Appendix). In general, most
crystallization experiments were performed in 24 wells plates (Hampton reaserch) that
were sealed with appropriate rounded siliconized glasses (Hampton research). In most
experiments 500 μL of 40% 2-4-methyl-pentadiol (MPD) was used as a reservoir
solution. Crystals usually emerged after 4-6 days.
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3.4. RNA-models: Crystal Handling, Data Collection, Structure Determination and
Refinement
3.4.1 Standard Crystallization Procedures and Data Handling
Normally, crystals were soaked in 40% MPD and flash frozen in liquid nitrogen.
Frozen crystals were transferred to the SLS or ESRF where X-ray data were collected.
Data processing has been performed with XDS 122
or Mosflm 123
. Initial phases were
determined by molecular replacement using Phaser MR, CCP4 suite 124
, using the
coordinates of previously solved X-ray structures of similar models as a search model.
The molecular structures were constructed and manipulated with COOT 125
. Data
refinement has been performed using Refmac5, (CCP4 suite), Phenix Refine 126
, and
CNS 127, 128
. Graphical representations were made using PyMOL 129
. The atomic
coordinates and structure factors of G418 and Apramycin A-site complexes with the
leishmanial binding site were deposited in the Protein Data Bank (PDB) with the
accession codes 4K32 and 4K31, respectively.
3.4.2 Additional Crystallization Techniques (Seeding, Soaking)
Since some of our crystallization trials resulted in poorly diffracting crystals or
structures exhibiting only a mere indication of ligands presence some of our
crystallization experiments and crystal handling techniques were performed using some
additional crystallization techniques such as seeding and soaking. Seeding and soaking
are highly acceptable methodologies in crystallography that are being used extensively in
crystallographic researches 130
.
For crystals that diffracted well to high resolution but did not exhibit a strong
indication for ligand's presence we applied the soaking technique where we added high
concentrations of the relevant ligand to the cryo-solution prior to freezing the crystal in
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liquid nitrogen. In these experiments we soaked the crystals for 2-24 hours in a 40%
MPD solution containing high ligand concentrations (10 mM and up) altogether with
polyamines and salts compositions at similar concentrations to the ones used in the
crystallization conditions.
For small crystals that gave a poor diffraction we used both macro- and micro-
seeding techniques. In our macro-seeding experiments we transferred the small occurring
crystals as is to a droplet containing 40% MPD for a few minutes (this procedure has
been done in order to wash the outer shell of the crystals) and then transferred them to a
new crystallization droplet containing RNA, AG and all relevant crystallization
conditions. The droplets have been equilibrated over a 40% MPD reservoir solution at
20˚C. Our micro-seeding experiments included the use of a cat mustache hair as a rough
micro surface that was transferred over the crystals a few times (this action should have
resulted in the clinging of micro crystals on the hair's jAG; transferring the hair in a
freshly prepared crystallization solution containing all relevant reagents introduced the
newly prepared droplet with the micro crystals that could be used as nucleation surfaces
for the emergence of new crystals.
3.5 Yeast 80S Ribosomes Crystallization and Crystal Structure Determination
Our crystallization trials of yeast 80S ribosomes complexes with various AG were
conducted in Prof. Marat Yusupov's lab in Strasbourg University, France. The
purification process of 80S ribosomes from JD1370 yeasts has been performed as
previously described by Yusupov and coworkers 100
. In brief, yeast cells were grown in
flasks to an OD600 of 1.45 in YPD at 30˚C. Cells were pelleted by centrifugation, re-
suspended with YP and incubated for 10 min at 30˚. Cells were incubated on ice and
were precipitated and washed three times in a buffer containing 30mM Hepes-K pH 7.5,
50 mM KCl, 10 mM MgCl2, 8.5% mannitol, 2mM DTT, 0.5 mM EDTA). For 4.5 gr of
cells, the pellet was resuspended in 6.5 ml of the same buffer solution and supplemented
with additional 600 μL from a solution of one complete protease inhibitor tablet (without
EDTA, Roche) dissolved in 2 ml buffer solution, 100 μL RNasin (Promega), 120 μL
Pefablock 100 mM and 56 μL freshly prepared Naheparin 100 mg/ml. Lysis has been
performed by adding 425-600 μm glass-beads (Sigma) into the the suspended tube and
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maintaining 5 cycles of 1 min vortexing followed by equal time of incubation at 4˚C.
Beads were removed by short centrifugation (20,000g * 2 min) and the lysate was further
clarified by a longer centrifugation (31,000g * 9 min). PEG 20,000 was then added from
a 30% w/v stock (Hampton Research) to a final concentration of 4.5% w/v and the
solution was left to stand for 5 minutes on ice. The solution was clarified by
centrifugation (20,000g * 5 min) and the supernatant was decanted to a new tube. The
KCl concentration was then adjusted to 130 mM. After 5 min on ice PEG 20,000
concentrations were adjusted to 8.5% and the solution was left to stand for 10 min on ice.
Ribosomes were precipitated (17,500g * 10 min), the supernatant was discarded and
residual solution was removed by a short spin of the pellet (14,500g * 1 min). Ribosomes
were suspended (6.5-7 mg/ml) in a buffer solution similar to the buffer solution used for
cells lysis of which the KCl concentration was adjusted to 150 mM and supplemented
with protease inhibitors and heparin. Ribosomes were further purified by a 15-30%
sucrose gradient in buffer A (20 mM Hepes-K pH 7.5, 120 mM KCl, 8.3 mM MgCl2, 2
mM DTT, 0.3 mM EDTA) using the SW28 rotor (18,000 rpm * 15h). The appropriate
fractions were collected, KCl and MgCl2 concentrations were adjusted to 150 mM and 10
mM respectively, PEG 20% was then added to a final concentration of 7% w/v and the
solution was left to stand 10 min on ice. Ribosomes were precipitated (17,500g * 10
min), the supernatant was discarded and residual solution was removed by a short spin of
the pellet (14,500g * 1 min). Ribosomes were suspended (20 mg/ml) in buffer G (10 mM
Hepes-K pH 7.5, 50 mM KOAc, 10 mM NH4Cl, 2 mM DTT, 5 mM Mg(OAc)2).
Prior to crystallization, the ribosome stock solution was filtered (0.22μm
centrifugal filters, Millipore) and a ribosome solution was prepared containing 5 mg/ml
ribosomes, 2.5 mM Hepes-K pH 7.5, 2.5 mM NH4Cl, 3.33 mM Mg(OAc)2, 1.6 mM DTT,
0.055 mM EDTA, 2.8 mM Deoxy Big Chap, 40 mM KOAc, 5.5 mM NH4OAc, 5.5 mM
Tris-Acetate pH 7.0. The ribosome solution was incubated in the presence or absence of
AG at 30˚C for 10 min and left to cool down for an hour at 4˚C. Ribosomes were
crystallized by the hanging drop method at 4˚C by mixing 2-2.4 μL of ribosome solution
with 1.6 μL of well solution (95 mM Tris-Acetate pH 7.0, 95 mM KSCN, 3 mM
Mg(OAc)2, 19% glycerol, 4-4.5% w/v PEG 20,000 and 4.75 mM spermidine). Crystals
appeared within 7-10 days and reached their full size after additional two weeks.
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Our post-crystallization treatments included a few dehydration steps of which the
ligands were introduced at high concentrations (soaking of vacant ribosomes has been
performed for 24h and included the addition of high ligand concentrations dissolved in
the initial crystallization conditions to the resulting crystallization droplets). The
dehydration steps included the replacement of crystallization mother liqueur by a solution
containing higher concentrations of PEG 6,000 (80mM Tris-Acetate pH 7.0, 70 mM
KSCN, 10mM Mg(OAc)2, 20% v/v Glycerol, 5% w/v PEG 20,000, 6.5 mM spermidine,
7.5 mM NH4OAc, 1.4 mM Deoxy Big Chap and 2mM DTT). This solution was then
replaced step-wise, with 15 minutes breaks between steps, by solutions with increasing
concentrations of PEG 6000, reaching finally after five steps to 20% (80 mM Tris-
Acetate pH 7.0, 70 mM KSCN, 10 mM Mg(OAc)2, 18% v/v Glycerol, 5% w/v PEG
20,000, 6.5 mM spermidine, 7.5 mM NH4OAc, 20% w/v PEG 6000, without DTT,
without detergent). The final step included the introduction of dehydrated crystals for 30
min with 2 mM osmium hexamine which has been dissolved in a solution containing
20% PEG 6000. Crystals were frozen directly on the goniometer under the stream of cold
nitrogen.
Data collection was performed at the PX beamlines at the SLS, PSI facility and
crystal structure determination has been performed by using the molecular replacement
methodology with the 4.15Å solved crystal structure as a template 99
.
3.6 Leishmania Cell Culture and Promastigote Viability Assay (IC50).
The leishmanial susceptibility assay has been performed in Prof. Charles L. Jaffe's
lab at the Hebrew University in Jerusalem. In general, two strains of Leishmania were
used in the present work: L. donovani (MHOM/SD/1962/1S-Cl2d) and L. major
(MHOM/IL/2003LRC-L1025). Promastigotes were grown in complete Schneider’s
Drosophila medium containing 20% fetal calf serum and antibiotics at 26°C. Compounds
were screened for leishmanicidal activity using an alamarBlue (AbD Serotec) viability
assay essentially as previously described by Jaffe and coworkers 131
. Compounds to be
assayed were diluted in serial concentrations ranging from 0.7-400 μM (0.15-40 μM for
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G418) in a complete promastigote medium, containing 1% dimethyl sulfoxide (DMSO),
and were aliquoted in triplicate (125 μL/well) into 96-well flat-bottom plates (Nunc).
Promastigotes (2.0 × 106 cells/mL; 125 μL/well) were added to each well and incubated
for 72 h at 26 °C. The alamarBlue viability indicator was added (25 μL/well) and the
plates were incubated for an additional 5 h, at which time the fluorescence (λex = 544
nm; λem = 590 nm) was measured in a microplate reader (Fluoroskan Ascent FL).
Complete medium both with and without DMSO was used as negative controls (0%
inhibition of promastigote growth). Amphotericin B (1 M, Sigma-Aldrich), a drug used
to treat visceral leishmaniasis, was included as a positive control in each plate.
3.7 The Development of a Highly Sensitive Immunoassay for 6'-OH AG Detection
3.7.1 Preparation of Polyclonal Abs
The preparation of polyclonal Abs included the conjugation of the relevant
aminoglicosidic scaffold to an immune-system-activating protein carrier, followed by
rabbits immunization and serum purification. A general scheme of immunization
conjugate preparation is described in Figure 3.1.
Figure 3.1: Synthetic scheme for the preparation of immunization\coating conjugates.
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3.7.1.1 Preparation of 6'-(R)-methyl-2',3-diazido-1-N-[5-oxopentanoic acid]-
paromamine (compound B, Figure 3.1).
A mixture of compound A 88
(500 mg, 1.2 mmol) and glutaric anhydride (0.29
mg, 2.5 mmol) in 20 mL anhydrous methanol was stirred under argon for 6 h at room
temperature. The reaction was monitored by TLC (EtOAc/MeOH 60:40) which indicated
completion after 6 h. The reaction mixture was evaporated under vacuum to dryness and
purified by flash chromatography, resulting in 400 mg (62% yield) of pure compound B
(Figure 3.1). The purity of the product was determined by NMR and MALDI-TOF Mass
Spectra analysis. 1HNMR (CDCl3, 500 MHz): ‘Ring I’ δH 1.26 (d, 3H, J=6.0 Hz, CH3),
3.10 (dd, 1H, J1=4.2 Hz, J2=10.5 Hz, H-2'), 3.38 (t, 1H, J=9.7 Hz, H-4'), 3.92-3.98 (m,
2H, H-5' and H3'), 4.04 (m, 1H, H-6'), 5.74 (d, 1H, J=3.1 Hz, H-1'); ‘Ring II’ δH 1.43
(ddd, 1H, J1= J2= J3=12.5 Hz, H-2ax), 2.20 (td, 1H, J1=4.5, J2=12.5 Hz, H-2eq), 3.26 (t,
1H, J=9.3 Hz, H-4), 3.52 (m, 3H, H-3, H-5, H-6), 3.78 (m, 1H, H-1). Linker peaks
appeared in the spectrum as follows: δH 1.92 (t, 2H, J=7.2 Hz), 2.29 (m, 2H), 2.36 (t, 2H,
J=7.5 Hz). 13
CNMR(CDCl3, 125.8 MHz): δC 18.0, 22.2, 33.9, 34.2, 36.2, 50.4, 61.4,
64.7, 69.3, 72.3, 74.2, 75.2, 76.2, 78.7, 80.3, 98.7 (C-1'), 175.5 (C=O), 177.1 (C=O). MS:
Calculated mass for C18H30N7O10 ([M+H]+) m/z: 504.20; measured m/z: 504.20.
3.7.1.2 Preparation of Immunization Conjugate (compound E, Figure 3.1).
A mixture of compound B (100 mg, 0.2 mmol) , N-hydroxysuccinimide (NHS)
(23 mg, 0.2 mmol) and N',N'-dicyclohexylcarbodiimide (DCC) (41.2 mg, 0.2 mmol) in
10 mL dimethylformamide (DMF) was stirred for 4h at room temperature followed by a
12 h incubation at 4˚C. The mixture was then centrifuged for 15 min at 2,500 x g at room
temperature. 1 mL of the supernatant containing 10 mg of the compound C were added
to 10 mg KLH dissolved in 4 mL of 0.13 M NaHCO3 at pH 9.2. The reaction was
incubated 12 h at 4˚C, followed by size exclusion product purification for 25 min at 3,000
x g at room temperature using Centricon 30 (Amicon, Millipore, Billerica, MA) to afford
compound D. The concentrated product D (Figure 3.1) was washed 3 times with 4 mL
of NaHCO3 solution. The final volume was adjusted to 4 mL by adding NaHCO3
solution. The reduction of azido-groups was performed by adding 100 μL
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trimethylphosphine (PMe3) solution (1M solution in tetrahydrofuran - THF) and the
mixture was allowed to react 12 h at 4˚C. The final product E (referred as a compound
E, Figure 3.1) was further purified using size exclusion purification as described above,
followed by 2 washes with 5 mL NaHCO3 solution. The final volume was adjusted to 5
mL by adding double distilled water (DDW). Conjugate was stored at -20˚C pending to
use.
3.7.1.3 Immunization and Antiserum Production.
0.5 mL of immunization conjugate (compound E from the above section) were
emulsified with either Complete Freund's adjuvant (1st immunization), or incomplete
adjuvant (2nd-4th boosts) prior to immunization. The emulsion was injected
subcutaneously to two rabbits 8244 and 8245 (~1 mg conjugate were injected per rabbit
at each time point). Bleeds were collected after each boost and were tested for anti-
antigen activity using the checkerboard assay. The 3rd bleed from Rabbit 8245 has been
shown to have the highest anti-antigen activity, and was therefore used for further
experimentations.
3.7.2 The Development of a Highly Sensitive Immunoassay
The development of a highly sensitive immunoassay included the preparation of 4
coating antigens (1 similar to the immunization conjugate that was used for the
homologues assay development and 3 coating antigens for the development of 3
heterologuos assays). The coating antigens altogether with the predeveloped Abs were
used for the development of 4 indirect immunoassays. In addition, cross reactivity
experiments were designed to test the immunoassays reactivity to the various AG
derivatives used in the study and to determine the Abs affinity to various epitopes.
3.7.2.1 Preparation of NB82-OVA Coating Antigen.
NB82-OVA conjugates were prepared similarly to immunization conjugates, as
described above, except that 5 mg ovalbumin (OVA), that was dissolved in 2.5 mL
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NaHCO3 solution, and then conjugated to compound B at molar ratios of 1:1, 1:2, 1:5
and 1:10. The final volume of the resulted NB82-OVA conjugate was adjusted to 1 mL
by adding 0.13 M NaHCO3, and the pH of the solution was adjusted to 9.6 by adding
dilute solution (0.01 M) of NaOH. Protein content has been determined to be 5 mg mL-1
using the Bradford assay (BioRad protein assay, BioRad, Hercules, CA) and the resulting
conjugates antigen activity was evaluated towards 3rd bleed antiserum from Rabbits 8244
and 8245 using the checkerboard assay. The 1:1 NB82-OVA conjugate crossed with 3rd
bleed antiserum derived from Rabbit 8245 has been shown to have the most sensitive
activity, and was therefore used for further experimentation. The conjugates were stored
in aliquots at -20˚C.
3.7.2.2 Preparation of NB82/84/124-GTA-OVA Coating Antigens
To prepare coating antigens for heterologous-cross reactivity assays we used
glutaraldehyde (GTA) coupling method as described by Chen at al. 132
. In brief, 20 mg
OVA was dissolved in 10 mL of 0.01 M phosphate-buffered saline (PBS) at pH 6.5 (the
pH adjustment was performed prior to the addition of OVA by adding dilute solution,
0.01 M, of HCl). 20 mg of each compound (NB82/84/124 sulfate) was added to the
solutions and stirred for a few min at r.t. 1.5 mL of freshly prepared 1% glutaraldehyde
solution was added in a drop-wise manner using a 1 mL syringe equipped with a 0.5 mm
x 16 mm needle. The reaction mixture was allowed to be stirred at r.t for 15 min, of
which yellow color indicated the imine formation. Reduction of imines to amines has
been performed by adding 115 mg sodium borohydride, followed by 12 h incubation at
4˚C. The final product has been purified using Centricon 30 for 30 min at 3,000x g,
followed by 2 washes with 1 mL 0.01 M PBS pH 7.2. The final volume of conjugates has
been adjusted to 1 mL using PBS pH 7.2, and their protein content has been determined
to be 17.5-19 mg mL-1
using the Bradford methodology. Conjugate antigen reactivity was
tested against 3rd bleed antiserum from Rabbits 8244 and 8245 using the checkerboard
assay. The conjugates were stored in aliquots at -20˚C pending to use.
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3.7.2.3 NB82 Indirect Competitive ELISA
The assay developed was an indirect competitive ELISA in which tested samples
or standard solutions were allowed to compete with an immobilized coating antigen for
antiserum binding. The assay has developed by using microtiter 96 well plates (F96
Maxisorp, Nunc Immuno Plate, Roskilde, Denmark). Wells were coated with 100 μL
NB82-OVA (1:1) conjugate, diluted 1:8,000 (62.5 ng conjugate per well) in 0.5 M
carbonate buffer, pH 9.6 (CB). After an overnight incubation at 4˚C, wells were washed 3
times with PBST (defined as PBS pH 7.2 containing 0.3% [v/v] Tween-20). 50 μL of
unknown samples or standard solutions (in PBS, mice serum or Fetal bovine serum -
FBS, Biological Industries, Israel) were added to the wells, followed by the addition of
50 μL of antiserum (Rabbit 8245) diluted 1:80,000 in PBST. The standard samples were
comprised of 12 serial dilutions of NB82, ranging from 10-0.0049 μg mL-1
dissolved in
PBS or serum. Plates were incubated overnight at 4˚C, and washed 3 times with PBST.
100 μL secondary antibody (2nd Ab) conjugated to horseradish peroxidase (HRP) (anti-
rabbit HRP conjugated, Sigma), diluted 1:20,000 in PBST were added to each well.
Plates were incubated for 2 h at r.t, rinsed with PBST, and tested for HRP activity by the
addition of 100 μL substrate solution - 3,3',5,5'-tetramethyl benzidine (TMB substrate
chromagen, Dako, Glostrup, Denmark). The reaction was stopped after 20 min by adding
50 μL of 4 N sulfuric acid, and the absorbance was measured with ELISA reader (Spectra
Max M2, Molecular devices, Sunnyvale, CA) at 450 nm. In all experiments a 5 mg mL-1
OVA solution in PBS has been used as a negative control to evaluate and normalize the
non-specific binding of antisera. Non-specific binding values obtained were almost
negligible in all experiments. Wells coated with coating antigen that were not introduced
with a free antigen sample were used as a positive control and determined the 100%
binding of antisera.
3.7.2.4 Cross Reactivity Experiments
Cross reactivity values of antisera with various AG were determined using the
heterologous indirect ELISA as described above by adding the subjected compounds
(instead of NB82) at 24 serial dilutions ranging from 10 mg mL-1
to 1.19 ng mL-1
, except
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for NB84, of which serial dilution ranged from 1 mg mL-1
to 0.119 ng mL-1
. All AG
tested were in their sulfated form and were dissolved and diluted in PBS or serum. The
standard solutions of the compounds NB82, NB84 and NB124 were shown to be stable in
PBS and standard serum (FBS) solutions that were frozen (-20˚C) for a few months and
demonstrated similar values using at least 5 repeated freezing/thawing cycles.
3.7.2.5 NB82/84/124 Indirect Competitive ELISA
In order to enhance antiserum sensitivity towards hetero-compounds that share
similar epitopes with the derivate of which antiserum antibodies has been produced
against, an indirect heterologous-assay has been developed. The assay has been
performed similarly to the indirect assay for NB82 as described in section 2.8, except for
the coating antigens of which for the NB82 heterologous-test, NB82-GTA-OVA
conjugate diluted 1:128,000 in CB was used as the coating antigen; for the NB84
heterologous-test, NB84-GTA-OVA conjugate diluted 1:64,000 in CB was used as the
coating antigen; and for the NB124 heterologous-test, NB124-GTA-OVA conjugate
diluted 1:16,000 in CB was used as the coating antigen. Solutions that contained 17.5-19
mg mL-1
OVA in PBS were used for coating negative control wells. Cross reactivity
experiments have been performed as indicated in section 3.7.2.4.
3.7.2.6 Data Analysis
The sensitivity (I50) and limit of detection (I20) values were obtained from fitting
concentration vs. % binding curves to a log-logit plot using the Origin software, version
8.5 (Mirocal Software, North Hampton, MA, USA).
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3.7.3 Determination of NB Compounds Levels in Mice Serum
Determination of NB compounds serum levels included an intraperitoneal (IP)
injection of NB84/NB124 to mice and collection of blood samples at various time points.
The protocol included the IP injection of 28.5 μg/gr body weight of the relevant NB
compound to 7 male mice (C57/BL, 2.5-3.5 months old), followed by collection of fresh
blood samples (~200 μL each) at 7 time points (5, 10, 20, 30, 40, 50, 60 min). Blood was
collected to sterile tubes containing 20 μL of 50 mM EDTA pH 8.0. Serum was further
extracted by centrifugation at 2,000x g at 4˚C for 10 min. NB compounds content in
serum samples was determined by using the pre-developed microtiter assay, as described
above, when compared to a linearized calibration curve transformed to a log-logit plot
using the Origin software, version 8.5. Each sample was tested in duplicates at five
dilutions. Serum recovery ratios were determined by spiking experiments of which whole
blood samples derived from untreated mice were spiked with known amounts of the
relevant aminoglycosidic derivatives. Blood samples were incubated for 1 h at 4˚C
followed by serum preparation by centrifugation as described above.
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4. Results and Discussion
4.1 Deciphering Aminoglycosides Mechanisms of Action in Eukaryotes
To explore the interactions of AG within their binding site in eukaryotes we used
complexes of various AG representatives with double stranded rRNA constructs
mimicking their putative binding site. The rRNA constructs contained one or two binding
sites corresponding to the sequences of the eukaryotic A-sites (Figure 4.1) and were
similar to previously reported constructs used for the exploration of AG interactions in
bacterial and human systems by crystallographic means 104, 113, 118
.
Figure 4.1 The rRNA constructs used to explore AG interactions: A. Human double A-site; B. Protozoa
double A-site; C. Human single A-site. Sequences corresponding to the natural A-site are highlighted in
dashed red squares. A/G1408 - orange, A1491 - green, A1492 - blue and A1493 - red. The rRNA residues
are numbered according to the numbering used in E. coli 16S rRNA.
Human
Double A-site model
Protozoa
Double A-site model
Human
Single A-site model
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The crystallographic studies included RNA purification, complex generation,
crystallization and structure determination and are fully described in the materials and
methods section.
4.1.1 rRNA Purification, Complex Generation and Crystallization
The RNA oligomers used in this study were chemically synthesized. Preparation
of active RNA-AG complexes included: RNA deprotection, anion exchange purification,
SPE desalting, RNA denturation/annealing cycle and finally complexation with AG.
These steps are fully described in the materials and methods section and a representative
example of the RNA purification steps is illustrated in Figure 4.2.
Figure 4.2. RNA deprotection and purification. A. Deprotection scheme. B. FPLC diagram. C. PAGE
analysis of FPLC RNA containing RNAs. Lanes highlighted in red are the ones used for further desalting
and complexation procedures.
Our initial crystallization trials were performed using a complex of the human
double A-site model (Figure 4.1A) with one of the lead read-through inducing
compounds developed in our lab, NB54 (Figure 1.7). An initial crystallization screen
was designed to test various mono-valent and di-valent ions as well as 2 different
polyamines and precipitating agents known to mediate RNA crystal growth Table I
(Appendix). Screening was done by an automatic robot screen using a 96 well sitting
drop crystallization plate, in the presence of 2 mM RNA and 4 mM AG solutions at pH
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7.0. Crystals appeared after 3-4 days in 9 out of 96 conditions (Figure 4.3). The best
crystals were obtained in the presence of 250 mM LiCl and 100-250 mM KCl over a 75
μLreservoir containing 40% MPD or 20% PEG3350 (Table I, Appendix). Additional
optimization steps were designed to test a wider range of monovalent salt concentrations
as well as combinations of high concentrations of mono-valent salts with lower
concentrations of di-valent ions (Mg+2
) (Table II, Appendix). The optimization screen
was performed in a wide pH range used to test pH influence on crystal growth
(experiments were performed in the range of pH 5.0-7.0). The optimization steps were
performed in 24 well plates using the hanging drop crystallization method, and resulted in
the emergence of large crystals (100-250 μm) which were highly suitable to X-ray
analysis (Figure 4.4). Our optimization results indicated that crystals grown from the
KCl conditions had better morphologies than the ones that emerged in the presence LiCl
or NH4Cl conditions. In addition MPD was found to be a better precipitant than PEG.
Figure 4.3. Crystals emerged from an initial screen of eukaryotic rRNA duplex in complex with NB54.
The crystallization conditions were A. 250 mM LiCl over a 20% PEG3350 reservoir (condition A6); B. 100
mM KCl over a 40% MPD reservoir (condition C2); C. 250 mM KCl over a 20% PEG3350 reservoir
(condition C6); D. 250 mM KCl over a 40% MPD reservoir (condition C9); E. 100 mM KCl over a 20%
PEG3350 reservoir (condition C11); F. 250 mM NH4Cl over a 20% PEG3350 reservoir (condition D12).
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Figure 4.4. Crystals obtained from an optimization screen of eukaryotic rRNA duplex in complex with
NB54. A. 50 mM KCl. B. 100 mM KCl. C. 150 mM KCl. D. 200 mM KCl. E. Frozen crystal of 200 mM
KCl on a nylon cryo-loop. F. Diffraction pattern of the frozen crystal as obtained in ID14-1 ESRF.
4.1.2 Crystal Structure Determination of Human A-site rRNA Complexes with AG
4.1.2.1 The Double A-site Model
An X-ray analysis of dozens of crystals indicated that best diffraction was
obtained from crystals containing higher salt concentrations (150-200 mM KCl). These
crystals diffracted up to 1.96 Å in the presence or absence of 40% MPD as a cryo-
protecting solution. Crystal data of 3 representatives (CYT1, CYT2 and CYT3) is
summarized in Table 4.1. The resulting crystal structures indicated two distinct
conformations of the two A-sites present in the rRNA model (Figure 4.5). One such
conformation was earlier described as an ‘ON’ state 121
(upper binding site, Figure 4.5 A-
C) where the two conserved adenine residues, A1492 and A1493, are flipped out from
the A-site helix; The conformation observed within the second binding site was denoted
as an ‘OFF’ state, where A1493 and A1491 are flipped out and A1492 is directed towards
the helical groove (Figure 4.5 A,B,D). The two observed conformations are believed to
represent the two possible conformational states in the ribosomes decoding site mediating
translation continuation and arrest in the presence of cognate or near/non-cognate tRNA
molecules, respectively.
A. B. C.
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Table 4.1. Crystal data, data collection and data refinement statistics – NB54
NB54-cyt 3 NB54-cyt 2 NB54-cyt 1
150 mM KCl 200 mM KCl 150 mM KCl Crystallization Conditions
- 40% MPD 40% MPD Cryo-Protectant
Crystal Data P21212 P21212 P21212 Space group
a=45.36
b=47.16
c=56.62
α=90°
β=90°
γ=90°
a=45.7
b=47.1
c=55.6
α=90°
β=90°
γ=90°
a=40.93
b=45.37
c=54.27
α=90°
β=90°
γ=90°
Unit cell parameters (Å/°)
1 1 1 Za
Data Collection
ID14-1 ESRF
Grenoble, France
ID14-1 ESRF
Grenoble, France
Rigaku Raxis IV++
Tel Aviv university
Beamline
0.934 0.934 1.541 Wavelength (Å)
35-2.5 36-1.96 30-2.7 Resolution (Å)
28,162 58,386 11,080 Observed reflections
4,341 8,852 3,033 Unique reflections
99.9 99.9 99.9 Completeness (%)
100 100 100 in outer shell (%)
5.7 7.5 5.4 Rmergeb (%)
13.5 35.5 38 in outer shell (%)
20.1 13.3 14.2 Mean ((I)/sd(I))
10.5 4.7 2.5 in outer shell (%)
Structure Refinement
35-2.5 36-1.96 30-2.7 Resolution range (Å)
4,124 8,831 3,021 Reflections
22.5/26.3 21.2/26.4 21.9/22.9 R-factorc / Rfree
d (%)
920 920 912 Number of RNA atoms
- - 2 Number of ligand molecules
0 (*) 113 3 Number of water molecules
R.M.S.D
0.014 0.005 0.01 Bond length (Å)
2.65 0.896 2.25 Bond angles (°)
a Number of RNA molecules in the asymmetric unit.
b Rmerge = 100 x Σhklj|Ihklj-<Ihklj>|/ Σhklj<Ihklj>.
c R-factor = 100 x Σ||Fo| - |Fc|| / Σ|Fo|, where |Fo| and |Fc| are the observed and calculated structure factor
amplitudes, respectively. d Calculated using a random set containing 5% of observations that were not included in refinement.
* The structure was not fully refined due to the lack of ligand in the electron density.
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Figure 4.5. Structures of rRNA crystals that were crystallized in the presence of a potent read-through
inducer – NB54. A. A 2D representation of the human cytoplasmic double A-site constructs as obtained in
the X-ray crystal structures. B. The double A-site construct 3D structure demonstrating two remote
conformational states of the putative binding sites: an ‘ON’ (upper site) vs. an ‘OFF’ (lower site) state
conformation. C. A closer look into the ‘ON’ state conformation with the two conserved adenine residues
(A1492 - blue and A1493 - red) budging out from the helical core. D. The ‘OFF’ state conformation with
A1491 (green) and A1493 (red) flipping out and A1492 (blue) directed towards the inner part of the helical
core. E. A 3D representation of CYT1 structure with some possible binding sites of NB54. F. Binding of
NB54 2 rings to the ‘ON’ state, Fo-Fc electron density maps are contoured at 2.0σ around the AG. G.
Binding of NB54 to the models GC pairs region, Fo-Fc electron density maps are contoured at 2.0σ around
the AG. H. Superposition of rRNA residues of CYT1 (green), CYT2 (blue), CYT3 (yellow) and an empty
rRNA duplex (PDB ID 2FQN – red). R.M.S.D values for CYT1, CYT2, CYT3 and 2FQN are 0.90Å,
0.40Å and 0.34Å, respectively.
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Unfortunately, an indication of the ligand’s presence was observed in only one of
the three structures solved (CYT1). The extra electron density obtained in this structure
indicated the presence of two NB54 molecules in the double A-site construct. With one
ligand molecule bound to the deep/major groove of the RNA duplex stabilizing the 'ON'
state conformation, and the second bound between the G=C pairs separating the two A-
sites (Figure 4.5 E-G). However, the extra electron densities for both ligands exhibited
some rather low sigma values (σ=2.0 in Fo-Fc maps) indicating low occupancy of the
ligand molecules within the crystal structure, and making it almost impossible to
determine the exact location of the 3 sugar rings within the structure. In addition,
superposition of the three solved crystal structures with a similar rRNA model
crystallized in the absence of an AG molecule 121
indicated that all four structures share a
great conformational and packing similarity (Figure 4.5 H); Implying a rather low
occupancy of ligands’ actual presence in the CYT1 structure.
The AG:A-site stoichiometric ratio used in our experiments was 1:1. Similar
ratios have previously been used for the complex generation of various AG with bacterial
A-sites. However, taking under considerations that AG affinity towards human ribosomes
is 3 orders of magnitude lower than in bacteria 133
we suggested that the low occupancy
values obtained in our structures were as a result of low ligand affinity towards the
human A-site. To further explore this notion, we generated rRNA-NB54 complexes at
higher AG:A-site ratios. Crystal obtained up to 10:1 ratios and were found suitable for X-
ray analysis (higher ratios resulted in the immediate appearance of a thick precipitate in
all tested conditions). In addition to the increase in ligand’s concentration we added the
ligand at as high a concentration as possible to the cryo solution to make sure that the
ligand does not leach out from the crystal during cryo-protection steps. Four examples of
crystal data are presented in Table 4.2. As can be seen, all crystals shared similar
symmetry and unit cell parameters. Unfortunately, the increase in ligands concentration
did not result in any ligand’s indication at the obtained electron density maps and the
resulting structures were identical to the ones reported with lower ligand concentrations.
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Table 4.2. Crystal data, data collection and data refinement statistics
NB54-cyt 7 NB54-cyt 6 NB54-cyt 5 NB54-cyt 4
A-site-NB54 1:10 A-site-NB54 1:8 A-site-NB54 1:4 A-site-NB54 1:2 Crystallization Conditions
40% MPD + 8mM
NB54 soak (2 h)
40% MPD + 8mM
NB54 soak (2 h)
40% MPD + 8mM
NB54 soak (2 h)
40% MPD + 8mM
NB54 soak (2 h) Cryo-Protectant
Crystal Data P21212 P21212 P21212 P21212 Space group
a=45.36
b=47.16
c=56.62
α=90°
β=90°
γ=90°
a=40.93
b=45.37
c=54.27
α=90°
β=90°
γ=90°
a=45.7
b=47.1
c=55.6
α=90°
β=90°
γ=90°
a=45.8
b=47.44
c=57.75
α=90°
β=90°
γ=90°
Unit cell parameters (Å/°)
1 1 1 1 Za
Data Collection
ID23-2 ESRF
Grenoble, France
ID14-4 ESRF
Grenoble, France
ID23-2 ESRF
Grenoble, France
ID14-4 ESRF
Grenoble, France
Beamline
0.873 0.9394 0.873 0.9394 Wavelength (Å)
36-2.5 56.7-2.8 47-2.8 57.75-3.0 Resolution (Å)
13,030 8,588 16,018 12,002 Observed reflections
4,504 2,874 3,375 2,592 Unique reflections
92.7 91.2 99.4 99.4 Completeness (%)
93.6 93.7 99.9 99.6 in outer shell (%)
7.9 8.9 10 8.8 Rmergeb (%)
12.8 11.3 59.4 12.5 in outer shell (%)
9.7 10 10.5 10.2 Mean ((I)/sd(I))
3.9 4.8 3.4 5.7 in outer shell (%)
Structure Refinement
36-2.5 56.7-2.8 57.6-2.8 57.75-3.0 Resolution range (Å)
4,206 2,719 3,214 2,465 Reflections
25.6/26.1 25.1/27 24.1/26 20/22 R-factorc / Rfree
d (%)
920 920 920 920 Number of RNA atoms
1 - - 1 Number of ligand molecules
1 - - - Number of water molecules
R.M.S.D
0.011 0.012 0.012 0.013 Bond length (Å)
2.27 2.22 2.43 2.452 Bond angles (°)
a Number of RNA molecules in the asymmetric unit.
b Rmerge = 100 x Σhklj|Ihklj-<Ihklj>|/ Σhklj<Ihklj>.
c R-factor = 100 x Σ||Fo| - |Fc|| / Σ|Fo|, where |Fo| and |Fc| are the observed and calculated structure factor
amplitudes, respectively. d Calculated using a random set containing 5% of observations that were not included in refinement.
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In parallel to NB54 we crystallized complexes of two additional synthetic
derivatives, NB74 and NB124 (Figure 1.7). These derivatives were synthesized in our
lab and were marked as highly potent read-through inducers 88, 91
. Our crystallization
trials resulted in dozens of crystals suitable for X-ray analysis. Crystals morphologies
were similar to the one present in Figure 4.4 C, as well as the unit cell parameters and
crystal’s symmetry (Table 4.5). Out of 10 solved structures of NB74-A-site complexes in
various rRNA:AG ratios, an indication of ligand's presence appeared only in 2 crystals:
one crystal with two ligands bound (one at the deep/major groove of the RNA duplex
stabilizing the 'ON' state conformation, and the other bound between the G-C pairs
separating the two A-sites, as can be seen in Figure 4.6); and a second crystal with only a
single ligand bound in the G-C pairs region. The ligand's electron density was stronger
than the one obtained for NB54; therefore presenting a higher probability to the ligands
presence. 2Fo-Fc maps (σ=1.0) for the NB74-cyt2 complex, potentially containing two
bound ligands, are presented in Figure 4.6 C,D. As can be seen electron density was
much stronger for the ligand bound at the G-C pairs vicinity, which does not consist a
part of the natural A-site sequence; therefore defined as non-specific binding. Such non-
specific interactions with the non-natural constructed G-C pair region were previously
reported in the studies of both natural and semi-synthetic AG derivatives in complex with
human and bacterial A-site constructs 92, 110, 112
. The electron density for the ‘ON’ state
ligand was much lower than the one present for the G-C pairs bound ligand, and
encountered similar problems as previously explained for the NB54 structure (Figure
4.7). For the NB124 complexes, no ligands were observed in the structures’ electron
density. Samples of crystals data are presented in Table 4.3.
With only a vague indication for ligand’s presence we wondered if due to the
minimal binding site available in our model system the binding site conformation as well
as the ligand’s binding pattern is affected by crystal packing interactions. An in depth
analysis of the crystal packing indeed indicated a stabilization of the obtained adenine
conformations by - stacking interactions with adenine residues bulging out from
adjacent molecules located within the crystal Figure 4.8. These indications suggest that
the two distinct conformations observed result from crystal packing interactions and are
not mediated by the ligand's presence.
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Table 4.3. Crystal data, data collection and data refinement statistics
NB124-cyt 1 NB74-cyt 2 NB74-cyt 1
A-site-NB124 1:1 A-site-NB74 1:4 A-site-NB74 1:1 Crystallization Conditions
40% MPD 40% MPD +
NB74 soak (2 h)
40% MPD Cryo-Protectant
Crystal Data P21212 P21212 P21212 Space group
a=47.36
b=47.63
c=56.86
α=90°
β=90°
γ=90°
a=47.66
b=47.68
c=57.5
α=90°
β=90°
γ=90°
a=48.03
b=48.77
c=58.13
α=90°
β=90°
γ=90°
Unit cell parameters (Å/°)
1 1 1 Za
Data Collection
NarµX X-ray System
Ben-Gurion university
ID14-4 ESRF
Grenoble, France
ID23-2 ESRF
Grenoble, France
Beamline
1.541 0.9394 0.873 Wavelength (Å)
56.8-3.0 57.5-2.77 37.03-2.8 Resolution (Å)
6,778 10,742 6,953 Observed reflections
2,540 4,989 3,248 Unique reflections
92.2 94.8 83.0 Completeness (%)
94.3 96.8 86.3 in outer shell (%)
11.3 7.5 12.9 Rmergeb (%)
27.5 43.4 29.2 in outer shell (%)
6.1 7.3 5.5 Mean ((I)/sd(I))
2.8 2.5 2.7 in outer shell (%)
Structure Refinement
56.8-3.0 57.5-2.77 37.035-2.7 Resolution range (Å)
2,406 4,206 3,098 Reflections
21/24.8 24.9/27.5 22.1/23.06 R-factorc / Rfree
d (%)
920 920 920 Number of RNA atoms
- 2 1 Number of ligand molecules
- 12 - Number of water molecules
R.M.S.D
0.013 0.012 0.011 Bond length (Å)
2.58 2.34 2.24 Bond angles (°)
a Number of RNA molecules in the asymmetric unit.
b Rmerge = 100 x Σhklj|Ihklj-<Ihklj>|/ Σhklj<Ihklj>.
c R-factor = 100 x Σ||Fo| - |Fc|| / Σ|Fo|, where |Fo| and |Fc| are the observed and calculated structure factor
amplitudes, respectively. d Calculated using a random set containing 5% of observations that were not included in refinement.
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Figure 4.6. NB74-human cytoplasmic A-site complex. A. A 2D representation of the human cytoplasmic
double A-site constructs as obtained in the X-ray crystal structure in the presence of NB74. Putative
binding sites for the two ligands are marked as Lig1, Lig2, respectively. Each A-site occupies a different
conformation: An ‘ON’ (upper) vs. an ‘OFF’ state (lower). B. The double A-site construct with two NB74
molecules (yellow) bound: one ligand is bound within the upper binding site demonstrating an ‘ON’ state
conformation; while the other ligand (lower) is bound to the G-C pairs region. A1491 is highlighted in
green, A1492 in blue and A1493 – red. C. 2Fo-Fc electron density maps (contoured at 1.0σ around the AG)
of lig1. D. 2Fo-Fc electron density maps (contoured at 1.0σ around the AG) of lig2. A1491 is highlighted in
green, A1492 in blue and A1493 – red.
Figure 4.7. B-factor analysis of NB54-
Cyt1(A) and NB74-Cyt2 (B). Residues
colored in red/orange/yellow indicate high
B-factor values; thus implying for low
electron density or high residual flexibility.
Residues colored in blue/green indicate low
B-factor values; implying for a rather high
electron density or low flexibility.
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Figure 4.8. Crystal packing. A. Stabilization of a flipped out A1491 (green) by A1492 (blue) belonging to
an adjacent rRNA molecule in the crystal packing by - stacking interactions. Stacking interactions are
marked in black arrow (distance = 3.9Å). B. Stabilization of a flipped out A1491 (green) by A1493 (red)
of an adjacent molecule in the crystal packing by - stacking interactions.
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Previous studies exploring the interactions of strong inhibitors of human
ribosomes, exhibiting no read-through activity 92
, used a similar double A-site construct
and resulted in a different crystal packing with a strong indication for ligand’s presence.
As a positive control to our experimentations we chose Apramycin, an efficient anti-
bacterial agent that is known to bind the A-site but exhibits no read-through activity in
eukaryotes 92
. Generation of a 1:1 ratio complex of Apramycin with the human double A-
site model resulted in the emergence of diamond shaped crystals (Figure 4.9 A) that
diffracted up to 2.9Å, and exhibited crystal symmetry and unit cell parameters which are
different than the ones present for the rRNA read-through inducing derivatives
complexes (Table 4.4). As can be seen in Figure 4.9, the two model A-sites occupied the
same conformational state, the ‘OFF’ state, with two Apramycin molecules bound, one to
each A-site and two cobalt-hexammine (Co-Hex) molecules bound at the G-C pairs
region. Similar Co-hex interactions were previously found in various crystal structures of
human and bacterial models crystallized in the presence of Co-Hex 19, 118, 121
.
Figure 4.9. A. Crystals Apramycin-eukaryotic-rRNA duplex. B. A 2D representation of the human
cytoplasmic double A-site constructs as obtained in the X-ray crystal structure with both A-site at the
‘OFF’ state conformation. C. The double A-site construct 3D structure with two Apramycin molecules
(yellow) bound, one ligand molecule to each A-site. Both A-sites occupy the ‘OFF’ state conformation
where A1491 (green) and A1493 (red) are flipped out and A1492 (blue) is directed towards the helical
core. Cobalt hexamine molecules are highlighted in pink. D. Apramycin bound to a single ‘OFF’ state.
2Fo-Fc electron density maps are contoured at 1.0σ around the AG.
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Table 4.4. Crystal data, data collection and data refinement statistics for the Apra-cyt crystal
Apra-cyt
Crystal Data P31 Space group
a=32.56; b=32.56; c=111.09 ; α=90°; β=90° γ=120° Unit cell parameters (Å/°)
1 Za
Data Collection
ID29 ESRF
Grenoble, France
Beamline
0.9763 Wavelength (Å)
37.0-2.9 Resolution (Å)
5,988 Observed reflections
2,056 Unique reflections
93.9 Completeness (%)
95.4 in outer shell (%)
17.2 Rmergeb (%)
51.1 in outer shell (%)
4.7 Mean ((I)/sd(I))
2.6 in outer shell (%)
Structure Refinement
37.0-2.9 Resolution range (Å)
1,945 Reflections
24.0/26.0 R-factorc / Rfree
d (%)
940 Number of RNA atoms
2 Number of ligand molecules
- Number of water molecules
R.M.S.D
0.021 Bond length (Å)
3.0 Bond angles (°)
a Number of RNA molecules in the asymmetric unit.
b Rmerge = 100 x Σhklj|Ihklj-<Ihklj>|/ Σhklj<Ihklj>.
c R-factor = 100 x Σ||Fo| - |Fc|| / Σ|Fo|, where |Fo| and |Fc| are the
observed and calculated structure factor amplitudes, respectively.
d Calculated using a random set containing 5% of observations
that were not included in refinement.
Figure 4.10. Crystal packing of
the Apra-cyt complex, showing a
stabilization of a flipped out
A1491 (green) by A1493 (green)
belonging to an adjacent rRNA
molecule in the crystal packing by
- stacking interactions.
Stacking interactions are marked
in black arrow (distance = 4Å).
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The electron densities for both Apramycin molecules bound were well ordered
and indicated the binding of the two ligand molecules at similar conformations in both A-
sites (Figure 4.9). The Fo-Fc maps prior to ligand's introduction indicated clearly the
ligand’s presence and could easily be used for the determination of ligand’s configuration
within the crystal structure. Crystal packing analysis of the resulting structure
demonstrated the stabilization of the flipped out adenine residues, A1491 and A1493, by
the same residues bulging out from adjacent rRNA molecules; indicating that these
residues might be more flexible and occupy additional conformations when in solution.
In-vitro experiments previously performed in our group indicated that Apramycin
as well as several read-through inducers such as Paromomycin and various NB
compounds are similarly capable of inhibiting eukaryotic ribosomes at the low micro-
molar range 92
. Similar experiments performed in prokaryotes demonstrated that AG such
as Paromomycin and Apramycin have a 3 order of magnitude higher capability to inhibit
prokaryotic ribosomes when compared to eukaryotes 92
. Combining this knowledge with
our indefinite indication for the presence of the 3 tested NB compounds within the crystal
structure, altogether with packing interference and the positive control demonstrating a
strong binding of Apramycin to the models A-sites we assumed that in order to get a
definite indication for read-through inducers binding to the A-site we will need to either
use extremely different crystallization conditions that will induce a different crystal
packing or try and search for a new model.
Our search for new crystallization conditions included the use of commercial
screening kits for RNA crystallization such as Natrix and Nucleic Acid Mini Screen by
Hampton research. Unfortunately, no new crystal forms emerged from the crystal screen,
and crystals emerged only in conditions which were similar to the ones that resulted in
crystal growth in our previous crystallization experiments. A few crystallization
experiments performed in conditions using extreme pH values (pH 5) (Table II,
appendix) resulted in the emergence of two different crystal morphologies at the same
crystallization droplet (Figure 4.11 A). The crystals emerged from complexation of the
natural read-through inducer, G418 (Figure 1.1), with the human double A-site construct
and appearance of crystals with the new morphology was after two month of incubation
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at 20°C. X-ray analysis of both crystal types revealed that indeed crystals that
demonstrated the new morphology had different unit cell parameters than the previously
obtained ones (Table 4.5); whereas crystals that demonstrated a similar morphology to
the previously reported ones emerged after 4 days and were found to be identical to the
previous one and ligand free. The structure of the new crystal form could be easily
determined by using molecular replacement with bacterial A-site models complexed with
G418, and both the Fo-Fc maps prior to ligand’s introduction and 2Fo-Fc maps after its
addition clearly indicated the ligand’s binding to one of the human A-sites (Figure 4.11).
However, the relatively small crystal size (~20 μm) resulted in a fast deterioration of
tested crystals upon X-ray exposure; therefore resulting in the need to merge a large
number of data sets for data interpretation (data from 5 crystals were merged to build a
single structure). The resulting resolution was poor (up to 3.2Å) and the electron density
for the unpaired adenine residues (A1491-3) at the additional unoccupied binding site
was disordered; therefore making it impossible to determine their orientation and
finalizing the crystal structure’s determination.
Figure 4.11. G418-human
cytoplasmic A-site complex at
pH 5.0. A. Crystals emerged
from crystallization experiments
at pH 5.0. Two crystal
morphologies were obtained in
a single crystallization droplet.
The round crystals highlighted
in red are those with the new
symmetry-unit cells parameters.
The orthorhombic crystals at the
right lower corner are identical
in symmetry and unit cell
parameters to the previously
obtained crystals in pH 7.0.
B. The occupied A-site in the absence of G418. 2Fo-Fc electron density maps are contoured at 1.0σ - blue; Fo-Fc
electron density maps are contoured at 3.0σ – red (maps are presented in COOT). C. The occupied A-site after the
addition of G418. 2Fo-Fc electron density maps are contoured at 1.0σ - blue; Fo-Fc electron density maps are
contoured at 3.0σ – red (maps are presented as calculated in COOT). D. The occupied A-site 3D structure. G418 is
highlighted in yellow, A1491-green, A1492 – blue and A1493 – red.
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Table 4.5. Crystal data, data collection and data refinement statistics
G418-Cyt – pH 5.0
G418-cyt
Na cacodylate pH 5.0 Crystallization Conditions
40% MPD Cryo-Protectant
Crystal Data P21212 Space group
a=30.49
b=85.86
c=45.46
α=90°
β=90°
γ=90°
Unit cell parameters (Å/°)
1 Za
Data Collection
ID23-1 ESRF
Grenoble, France
Beamline
0.9763 Wavelength (Å)
50.0-3.2 Resolution (Å)
5,988 Observed reflections
2,056 Unique reflections
93.9 Completeness (%)
95.4 in outer shell (%)
17.2 Rmergeb (%)
51.1 in outer shell (%)
4.5 Mean ((I)/sd(I))
2.5 in outer shell (%)
Structure Refinement
50.0-3.2 Resolution range (Å)
1,772 Reflections
33/38 R-factorc / Rfree
d (%)
898 Number of RNA atoms
1 Number of ligand molecules
- Number of water molecules
R.M.S.D
0.011 Bond length (Å)
1.797 Bond angles (°)
a Number of RNA molecules in the asymmetric unit.
b Rmerge = 100 x Σhklj|Ihklj-<Ihklj>|/ Σhklj<Ihklj>.
c R-factor = 100 x Σ||Fo| - |Fc|| / Σ|Fo|, where |Fo| and |Fc| are the observed and calculated structure factor
amplitudes, respectively. d Calculated using a random set containing 5% of observations that were not included in refinement.
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Unfortunately, despite our efforts to improve crystal quality we did not obtain
better diffracting crystals. Our failure to improve the resulting crystals could mainly be
attributed to the long crystallization period (at least two months) and the very low
reproducibility of the crystallization method. The optimization trials included the addition
of various additives such as glycerol and PEGs to the crystallization solutions, as well as
the use of a wide range of MPD concentrations at both reservoir and crystallization
solutions. In addition both macro- and micro- seeding experiments were performed (these
trials are fully described in the materials and methods section).
However, using such extreme pH values (5 and below) in RNA crystallization
is usually not recommended due to adenine protonation. Such protonation events might
induce some un-natural base-paring interactions that are rarely possible under
physiological conditions; therefore inducing an experimental artifact (Figure 4.12 D). In
addition, low pH values also induce protonation of additional amino groups in the ligand,
which might also enhance some non-natural interactions with the rRNA construct.
Despite the G418 data incompleteness, the bound A-site electron density is very clear and
therefore we could use it to further explore the resulting structure. Our explorations
indeed indicated the presence of possible interactions of a protonated adenine residue,
A1491, with a cytosine residue located at the opposite rRNA strand (C1409). Such
interactions resulted in the generation of a pseudo-canonical base-pair between two
nucleotides that usually don’t interact in canonical modes, making the A-site geometry
much more similar to the one present in bacterial A-sites (r.m.s.d 0.4 Å; Figure 4.12 A).
Superposition of the C1409:A1491 base pairs obtained in the ‘ON’ state A-sites of the
two structures (pH 5.0 and 7.0) indeed indicate a large difference in base paring
interactions (Figure 4.12 C). As a matter of fact, although the two bound A-sites occupy
the ‘ON’ state conformation where the two conserved adenine residues are bulged out,
their actual geometry is vastly different (r.m.s.d 2.604 Å; Figure 4.12 B). These
geometrical differences might be attributed either by the intermolecular interactions
inside the crystal packing or by the absence of solid base-pair interactions of the
C1409:A1491 pair in the pH 7.0 structure. The significant geometrical similarity and
ligand’s orientation of the pH 5.0 structure with the bacterial binding site, altogether with
its strong indication for ligand’s presence, implicates the importance of such geometry
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for the ligands binding and might explain why the pH 7.0 structure presented only a
vague indication to ligand’s presence, if any.
Figure 4.12. Analysis of the pH 5.0 ‘ON’ state structure. A. Superposition of bacterial A-site (blue) and
human A-site at pH 5.0 (orange) with a bound G418 molecule. B. Superposition of pH 7.0 (green) and pH
5.0 (orange) ‘ON’ states. C. Superposition of the conformation of the C1409:A1491 pair of the pH 5.0
structure (orange) indicating a pseudo Watson-Crick pair, and the pH 7.0 (green) indicating a shifted
conformation. D. Pseudo Watson-Crick pair possible between adenine and cytosine at low pHs.
C1409 A1491
A1492
A1493
A1493
A1493
A1492
A1492
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To further explore the importance of binding site geometry to ligand’s binding
and avoid working in extreme pH conditions and long crystallization periods, we decided
to generate a mutant A-site model containing a single mutation at position 1409, C1409U
(Figure 4.1 B). We hypothesized the such a mutation would induce the generation of a
‘real’ Watson-Crick pair that is not pH dependent, and will result in a geometry that is
similar to the one obtained in pH 5.0 without inducing a major difference in the binding
site sequence or the ligand’s charge. Interestingly, such A-site sequence is identical to the
w.t. A-site of several parasites, such as Leishmania, which are known to induce severe
disease symptoms in mammals. The obtained results will be described in chapter 4.1.4.
4.1.2.2 The Single A-site Model
Additional crystallization trials of human A-site in complex with AG included the
use of an extended single A-site model (Figure 4.1 C). The model is composed of two
rRNA strands with sequences that correspond to an extended sequence of the human A-
site with some additional nucleo-base-pairs used to stabilize the double stranded model.
The addition of the 3’-cytosine and guanine residues was intended to induce “sticky”
ends in order to facilitate crystal growth. A similar model was previously used to explore
the interactions of Apramycin with the human A-site 104
.
Our crystallization trials included similar matrixes as the ones used for the double
A-site model, and resulted in the emergence of large (150-350 μm) crystals from rRNA
complexes with Apramycin (control), NB54, G418, NB84, NB74 and NB124 (Figure
4.13). All complexes were crystallized with a 1:1 A-site:AG ratio. The control
experimentations with Apramycin resulted in the emergence of well-ordered cubic-
shaped crystals that diffracted up to 2.6 Å resolutions in a home-source X-ray machine.
The unit cell parameters as well as crystal symmetry and structure solution statistics are
summarized in Table 4.6. The resulting structure indicated the binding of a single
Apramycin molecule to the A-site, which is in the ‘OFF’ state conformation (similarly to
the conformation observed upon Apramycin to the double A-site model) – Figure 4.14.
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Figure 4.13. Crystals emerged from single eukaryotic A-site rRNA duplex in complex with Apramycin
)A(, NB54 (B), G418 (C), NB84 (D), NB74 (E) and NB124 (F).
Table 3.8. Crystal data, data
collection and data refinement
statistics
Figure 4.14. Apramycin bound to
the extended single A-site model.
A. A-site 2D representation (‘OFF’
state). B. The three dimensional
structure determined with
Apramycin bound to the ‘OFF’
state with the conserved adenine
residues highlighted in green
(A1491), blue (A1492) and red
(A1493) and Apramycin in yellow.
C.2Fo-Fc electron density maps
contoured at 1.0σ around the
ligand and the rRNA residues.
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Table 4.6. Crystal data, data collection and data refinement statistics
a Number of RNA molecules in the asymmetric unit.
b Rmerge = 100 x Σhklj|Ihklj-<Ihklj>|/ Σhklj<Ihklj>.
c R-factor = 100 x Σ||Fo| - |Fc|| / Σ|Fo|, where |Fo| and |Fc| are the observed and calculated structure factor
amplitudes, respectively. d Calculated using a random set containing 5% of observations that were not included in refinement.
Apra-single-cyt
Single A-site model -
Apramycin Crystallization Conditions
- Cryo-Protectant
Crystal Data P212121 Space group
a=28.62
b=36.94
c=87.64
α=90°
β=90°
γ=90°
Unit cell parameters (Å/°)
1 Za
Data Collection
Rigaku Raxis IV++
Tel Aviv university
Beamline
1.541 Wavelength (Å)
43.8-2.6 Resolution (Å)
14,526 Observed reflections
3,143 Unique reflections
100 Completeness (%)
100 in outer shell (%)
6 Rmergeb (%)
10 in outer shell (%)
14 Mean ((I)/sd(I))
2.5 in outer shell (%)
Structure Refinement
43.8-2.6 Resolution range (Å)
1,545 Reflections
19.15/23.25 R-factorc / Rfree
d (%)
698 Number of RNA atoms
1 Number of ligand molecules
- Number of water molecules
R.M.S.D
0.005 Bond length (Å)
0.879 Bond angles (°)
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Nevertheless, the crystals emerged from complexation of the rRNA model with
the 5 read-through inducers poorly diffracted to 3.5 Å at best. Crystal analysis revealed
that all crystals had a P65 symmetry with unit cell parameters of a=49.19, b=49.19,
c=127.33, α=β=90°, γ=120°. Structure phases were determined using molecular
replacement with the rRNA residues of the Apramycin structure. Two molecules were
observed in the asymmetric unit. The molecules in an asymmetric unit were rotated in
60° facing one another (Figure 4.15 A), with the “sticky” ends resulting in the generation
of long rRNA fibrils that fabricated an ‘X’ shaped crystal packing (Figure 4.15 B).
Unfortunately due to the low resolution and the high flexibility of the conserved adenine
residues (A1491-3) it was impossible to model these residues and the absolute A-site
conformation could not be determined. In addition no clear indication was observed in
the electron density for the ligand’s presence. The R/Rfree values for the semi refined
structure were 30/35%, respectively.
Figure 4.15. Asymmetric unit )A( and crystal packing (B) of crystals emerged of complexes of the read-
through inducers NB54, NB74, NB84, NB124 and G418 with the single A-site model. The two molecules
present in the asymmetric unit were colored in blue and red for molecule 1 and 2, respectively.
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4.1.3 Crystal Structure Determination of 80S Yeast Ribosomes in Complex with AG
In addition to the single human A-site model used in our experimentations,
crystallization trials were also performed using complete 80S yeast ribosomes. Yeast 80S
ribosomes share great similarity with human ribosomes 99, 100
, with 100% sequence
similarity of the ribosomal decoding site. Ribosome purification and crystallization
techniques are fully described in the materials and methods section. In general, two
methods were used for complex generation: co-crystallization with up to 20 fold AG
excess and soaking of vacant ribosomes with up to 100 fold AG excess. Two synthetic
derivatives were used for complex generation: NB33, a strong inhibitor of human
ribosome translation that lacks any read-through activity 92
and NB127, a lead compound
developed in our lab that exhibited highest read-through activity thus far and has a great
selectivity to eukaryotic ribosomes 91
(Figure 1.7).
Our crystallization experiments resulted in the emergence of hundreds of
medium-large sized crystals (~100 μm). The initial crystals diffracted to ~12 Å (Figure
4.16 A, B). Crystals' quality was improved by systematic dehydration process that
resulted in resolution enhancement of up to 3 Å. Out of dozens of crystals scanned, we
collected 4 full data sets at 3.5-6 Å resolutions, two data sets for each compound, one
soaked and one co-crystallized (Table 4.7). Due to high radiation damage observed upon
crystals X-ray exposure, each data set was composed of a few merged data sets collected
from several crystals or several spots on the same crystal (Table 4.7). The asymmetric
unit contained two ribosomes with unit cell parameters that are identical to the ones
obtained in previously reported vacant yeast 80S ribosome structures 99, 100
.
Unfortunately, no density indicating the presence of bound AG could be identified.
A thorough investigation of the resulting crystal structures indicated two major
issues that might give an explanation to the lack of AG presence within their putative
binding site. One such topic was that in order to achieve structural homogeneity among
empty ribosomes purified for crystallization, yeasts were grown under glucose starvation.
Such conditions were previously reported to result in highly homogenous 80S ribosomes,
all exhibiting the same translational stage 134
. Apparently, such stress conditions resulted
in the expression of STM1, a protein that causes ribosome arrest upon stress conditions
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135. Our X-ray results indeed indicated the presence of a large extra electron density in the
crystal structure. Mass Spectra experiments performed by Ben-Shem at al. 100
indeed
indicate the present of a non-ribosomal protein within the crystals. The protein was later
identified as STM1 and localization of its amino-acids within the extra electron density
revealed a rather tight interactions of STM1 with both ribosomal subunits (Figure 4.16
C); blocking the m-RNA entrance tunnel and forcing the ribosome into a ratcheted form,
preventing it from binding its substrates. This inhibition of dynamics appears to be the
source of translational arrest. STM1 presence also seemed to affect the A-site
conformation, resulting in a distorted conformation that changes the A-site’s geometry,
therefore preventing the AG molecules from binding to their putative binding site
(Figure 4.16 D, E).
Figure 4.16. Crystallization of yeast 80S ribosomes. A. Apo crystals emerged from crystallization trials. B.
Frozen crystal on a cryo-loop during X-ray analysis (SLS). C. 80S ribosome structure with STM1 highlighted in
red and the A-site in yellow. D.A closer glance at the ribosomal A-site, with conserved residues highlighted in
green (A1491), blue (A1492), red (A1493) and orange (G1408). Non canonical interactions between A1493 and
G1408 are highlighted in dashed blue lines. E. Superposition of eukaryotic A-sites from 80S yeast ribosome
structure (yellow) and 40S tetrahymena structure (cyan). F. Binding of Os-Hex to the ribosomal A-site. The
conserved Adenine residues are highlighted as in D.
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Table 4.7. Crystal data, data collection and data refinement statistics
a Number of RNA molecules in the asymmetric unit.
b Rmerge = 100 x Σhklj|Ihklj-<Ihklj>|/ Σhklj<Ihklj>.
c R-factor = 100 x Σ||Fo| - |Fc|| / Σ|Fo|, where |Fo| and |Fc| are the observed and calculated structure factor amplitudes, respectively.
d Calculated using a random set containing 2% of observations that were not included in refinement.
NB127- Ribo-cox NB127- Ribo-soak NB33-Ribo-cox NB33-Ribo-soak
Ribosome:NB127 (1:20) Vacant ribosomes Ribosome:NB33 (1:20) Vacant ribosomes Crystallization Conditions
- NB127 100 fold (24h) - NB33 20 fold (24 h) Soak
Crystal Data P21 P21 P21 P21 Space group a=285.45
b=302.47
c=433.32
α=90°
β=98.85°
γ=90°
a=287.82
b=306.99
c=437.54
α=90°
β=99.07°
γ=90°
a=285.78
b=303.84
c=434.74
α=90°
β=99.07°
γ=90°
a=297.68
b=283.06
c=432.44
α=90°
β=97.87°
γ=90°
Unit cell parameters (Å/°)
2 2 2 2 Za
Data Collection PXI - X06SA, SLS
Villigen, Switzerland
PXI - X06SA, SLS
Villigen, Switzerland
PXI - X06SA, SLS
Villigen, Switzerland
PXI - X06SA, SLS
Villigen, Switzerland Beamline
1.00 1.00 1.00 1.00 Wavelength (Å) 7 3 3 5 No. of datasets merged 200-3.5 300-4.2 300-4.0 400-6.0 Resolution (Å) 4,647,856 1,510,820 792,785 888,919 Observed reflections 939,466 464,259 191,313 175,94 Unique reflections 99.8 99.9 99.1 99.9 Completeness (%) 100 100 98.7 99.9 in outer shell (%) 18.9 18.3 20.5 21.6 Rmerge
b (%)
51.8 42.8 57.6 46.4 in outer shell (%) 4.8 6.1 5.93 5.5 Mean ((I)/sd(I)) 2.97 2.85 2.67 2.6 in outer shell (%)
Structure Refinement 56.7-2.8 300-4.2 300-4.0 293-6.0 Resolution range (Å) 2,719 4,206 3,214 173,340 Reflections 18.1/22.8 19.1/22.9 18.2/22.3 20.5/23.7 R-factor
c / Rfree
d (%)
R.M.S.D 0.013 0.015 0.015 0.016 Bond length (Å) 1.512 1.672 1.651 1.713 Bond angles (°)
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In addition, as indicated, the initial crystals obtained were soft and fragile, and
therefore poorly diffracted to 12 Å and less. These parameters indicated that the water
content within the crystals was too high; therefore several dehydration steps were used to
lower water content by replacing the water molecules with organic solvent (PEG) and the
addition of poly-amines (Os-hex/Co-hex) to decrease RNA repulsion and tighten crystal
packing; thus improving crystals diffraction properties. However, despite the large excess
of AG in the crystallization and dehydration steps, 2 Os-Hex molecules were shown to
occupy the ribosomal A-sites in all tested crystals; therefore preventing the AG from
binding their putative binding site (Figure 4.16 F).
4.1.4 Leishmania A-site Crystal Structure Determination
In the absence of a solid evidence for AG binding to models mimicking their
binding sites in human and in continuation of our experimentations with the double A-
site model, we generated a double A-site construct containing a single mutation in
comparison to the human A-site. Such mutation was meant to limit the flexibility of
A1491 by generating a Watson-Crick pair with position 1409 (C1409U). Such
interactions were meant to stabilize the geometrical conformation promoting ligand
binding; thus increasing our chances to get strong indications for ligand binding under
physiological conditions. Interestingly, introduction of this single mutation resulted in an
A-site that is identical to the A-site of the eukaryotic parasite, Leishmania, (Figure 4.17)
known to cause mild to severe symptoms in humans, as well as dogs; affecting as many
as 12 million people worldwide, with 1.5–2 million new cases each year.
Figure 4.17. Secondary structures of the
bacterial (A), leishmanial (B) and human
(C) cytoplasmic A-sites. A/G1408 -
orange, A1491 - green, A1492 - blue and
A1493 - red. The rRNA residues are
numbered according to the numbering
used in E.coli 16S rRNA.
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Our structural investigations resulted in the crystal emergence and determination
of the three dimensional structures of two AG representatives, G418 and Apramycin
(Figure 1.1), bound to leishmanian rRNA double A-site duplexes model at 2.65 Å and
1.4 Å resolutions, respectively (G418-Leish, Apra-Leish; Figures 4.18, 4.19, 4.20 Table
4.8). The resulting structures indeed demonstrated a high geometrical similarity between
the leishmanial A-site and the bacterial A-site, despite the 33% dissimilarity in their
nucleotides sequence (5 out of 15 nucleotides located in positions 1408-1410 and 1490-
1491 are different; Figure 4.17). These geometric similarities do not come as a surprise,
since both sites are believed to be an important molecular switch dictating the tRNA
recognition at the ribosomal A-site. However, since AG mostly interact with the
nucleotide bases, and there are significant differences in sequence, we hypothesized that
the obtained interaction patterns and conformational changes induced by different AG
might differ in Leishmania from the ones present in bacteria.
Overall, in both the G418-Leish and Apra-Leish structures a single AG molecule
was found to specifically interact with the deep/major groove of each A-site (Figure
4.19). In the Apra-Leish structure 2 additional Apramycin molecules were found to
interact with the G-C pairs region connecting the two putative binding sites present in the
rRNA model (Figure 4.19 B, Figure 4.21). Similar non-specific interactions with the
non-natural constructed G-C pair region were previously reported in our experiments.
However, despite the fact that both ligands (G418 and Apramycin) were shown to bind
the same region of their primary binding site, the two ligands demonstrated a
significantly different interaction pattern with the rRNA construct, and thus each AG
induced a different conformational change to the A-site. The conformational change
induced by G418 is similar to that already observed for the bacterial A-site, where two
conserved adenine residues, A1492 and A1493, are fully bulged out from the helical core
(Figure 4.22). In contrast, Apramycin induces a rather different conformation, in which
the A1492 base moiety is sequestered within the helical core, forming a pair with G1408,
while A1493 is bulged out (Figure 4.19, Figure 4.22).
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Figure 4.19. 2D (right panels) and 3D (left panels) representations of the double A-site complexes as
obtained in the X-ray crystal structures. A. The double A-site construct in complex with G418 (PDB ID:
4K32): each A-site binds one molecule of G418 (yellow). Both A-sites are in the 'ON' state conformation
where A1492 (blue) and A1493 (red) are bulged out from the helical core. B. The double A-site construct
in complex with apramycin (PDB ID: 4K31): each A-site binds one molecule of apramycin (yellow). Two
additional apramycin molecules can be observed in the G-C pairs region separating between the two A-sites
(marked in black arrows). Both A-sites are in the 'OFF' state conformation where A1493 (red) is bulged out
from the helical core and A1492 (blue) interacts with G1408 (orange) in a non-canonical manner. A1491
(green) forms a Watson-Crick pair with U1409 in both complexes.
Figure 4.18. Crystals emerged from
crystallization experiments of complexes of
the leishmanial A-site model with G418 (A)
and Apramycin (B).
A. B.
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Figure 4.20. Electron density maps of the Apra-Leish (A) and G418-Leish (B) structures. 2Fo-Fc maps are
contoured at 1.0σ around rRNA molecules (cyan) and 1.5σ around ligand molecules (dark blue).
Figure 4.21. Surface representation of apramycin bound to the double A-site rRNA construct corresponding to its
putative binding site in leishmanial ribosomes. Apramycin molecules are represented as yellow sticks. The surface
colors indicate residue conservation among prokaryotic and eukaryotic systems, where residues marked in cyan
are highly conserved among all kingdoms, residues marked in light pink are rather diverse and residues marked in
light yellow are non-A-site nucleotides that were used to stabilize the model. Double helices (left to right) are
rotated by 90° around the helical core. Apramycin molecules are numbered I-IV (top to bottom).
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Table 4.8. Crystal data, data collection and data refinement statistics
a Number of RNA molecules in the asymmetric unit.
b Rmerge = 100 x Σhklj|Ihklj-<Ihklj>|/ Σhklj<Ihklj>.
c R-factor = 100 x Σ||Fo| - |Fc|| / Σ|Fo|, where |Fo| and |Fc| are the observed and calculated structure factor
amplitudes, respectively. d Calculated using a random set containing 5% of observations that were not included in refinement.
Apra-Leish G418-Leish
4K31 4K32 PDB code
Crystal Data P31 P21212 Space group
a=b=32.79
c=107.40
a=33.08
b=90.76
c=47.01
Unit cell parameters (Å)
1 1 Za
Data Collection
ID14-4 ESRF ID14-4 ESRF Beamline
0.9394 0.9394 Wavelength (Å)
35.8-1.4 23.5-2.65 Resolution (Å)
67,545 18,136 Observed reflections
23,324 3,615 Unique reflections
94.5 80.9 Completeness (%)
98.0 84.5 in outer shell (%)
4.8 4.8 Rmergeb (%)
13.7 30.8 in outer shell (%)
14.1 25.11 Mean ((I)/sd(I))
5.7 5.15 in outer shell (%)
Structure Refinement
35.8-1.4 23.5-2.65 Resolution range (Å)
22,089 3,612 Unique reflections
22.6/26.4 19.5/23.9 R-factorc / Rfree
d (%)
940 955 Number of RNA atoms
4 2 Number of ligand molecules
209 0 Number of water molecules
R.M.S.D
0.025 0.013 Bond length (Å)
2.47 2.162 Bond angles (°)
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Figure 4.22. Crystal structure visualization of (A) G418 or (B) Apramycin (yellow) - leishmanial A-site
rRNA complexes. Electron density 2Fo-Fc maps are contoured at 1.0σ around the AG. (C) A representation
of Apramycin bound to the human A-site construct (PDB code 2G5K). G1491 is highlighted in green,
A1492 in blue, A1493 in red and U1409 in purple. (D) Superimposition of G418 bound to the bacterial A-
site (orange) and the leishmanial A-site (blue). G418 is highlighted in ball-and stick representation.
Superimposition was performed using the PyMol software align algorithm using all identical atoms
(r.m.s.d. 0.9 Å). PDB codes are 1MWL and 4K32 for the bacterial and Leishmania structures, respectively.
(E) Superimposition of Apramycin (ball and stick) bound to the bacterial (orange) and leishmanial (blue)
A-sites (r.m.s.d. 2.2 Å). PDB codes are 1YRJ and 4K31 for the bacterial and Leishmania structures,
respectively. (F) Superimposition of Apramycin (ball and stick) bound to the human cytoplasmic (green)
and leishmanial (blue) ribosomal A-sites (r.m.s.d. 1.4 Å). PDB codes are 2G5K and 4K31 for the human
and Leishmania structures, respectively. In all figures A/G1491, A1492, A1493 and A/G1408 are
highlighted in the relevant colors.
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4.1.4.1 The Binding Mode of G418 to the Leishmanial A-site - ‘ON’ State.
Upon binding to the deep major groove of the leishmanial A-site, G418 forms 14
hydrogen bonds with the rRNA bases and phosphate oxygen atoms (Figure 4.23). Ring I
forms a pseudo base pair with G1408, by forming 2 hydrogen bonds involving the ring
oxygen and the 6’-OH group (Figure 4.23 B). The planar nature of these interactions is
dominated by stacking interactions of ring I and A1491 (Figure 4.23 C). On the opposite
side of ring I, two OH groups located at the 3’ and 4’ positions make additional contacts
with the oxygen atoms belonging to the phosphate backbone of the two conserved
adenine residues, A1492 and A1493 (Figure 4.23 D). These interactions prevent the two
adenine residues from flipping back inside the helical core. This interaction is therefore
deemed to be highly important for the induction of the flipped out conformation of
A1492 and A1493. The chiral exocyclic methyl group at 6' position, (R)-6'-Me, points in
the middle of the A1491-U1409 pair (Figure 4.23 D); thus adding some hydrophobic
stabilization as the one observed upon G418 ring I interaction with the G1491-C1409 pair
in bacteria 114
. Ring II interacts with three sequential residues A1493, G1494 and U1495
(Figure 4.23 E). Due to the high conservation of these residues, similar interactions were
previously highlighted in the exploration of w.t. and mutant bacterial A-sites in complex
with AG containing a 2-DOS ring 114, 137
. Ring III, forms 6 hydrogen bonds with G1405,
U1406 and C1407, these interaction are typical to 4,6-disubstituted AG containing a
garoseamine ring (ring III) at position 6 (Figure 4.23 F). Similar interactions were
previously reported for the interaction of 4,6- disubstituted garoseamine derivatives such
as Gentamicin C1A and G418 with bacterial A-sites 110, 114
.
Superposition of the G418-Leish structure with the crystal structure of G418 bound to the
bacterial A-site (G418-Bact) indicated a rather similar binding mode with r.m.s.d values
of 0.9 Å (Figure 4.22 D). The overall conformation of the binding site upon G418
binding was nearly identical to the one present in the bacterial A-site, where the two
conserved adenines 1492 and 1493 are flipped out from the helical core prone to interact
with both the mRNA and the tRNA codon:anti-codon complex. This conformational state
was earlier referred to as an ‘ON’ state 19, 121
and is known to mimic the conformational
change resulting from the complexation of a cognate aminoacylated-tRNA molecule and
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the mRNA at the bacterial ribosomal A-site; thus signaling for the continuation of
translation process. The induced conformational change upon AG binding to bacterial
ribosomes is believed to play a major role in their ability to induce translational
miscoding 110
. Although no direct evidence is currently available showing that G418
induces miscoding by leishmanial ribosomes, G418 has long been marked as a potent
miscoding agent in eukaryotes 24
. In addition, recent work has demonstrated the ability of
a structurally related derivative, Paromomycin, to induce miscoding in Leishmania 138
.
The structural similarity between the two structures (G418-Leish and G418-bact) together
with documented evidence for AG such as Paromomycin enhancing miscoding events in
leishmanial ribosomes might suggest a similar mechanism of action in Leishmania.
Figure 4.23. Description of the contacts between G418 and the leishmanial A-site. (A) The 3D structure of
bound G418. Ring numbers (I-III) and atom names are specified. rRNA atoms are numbered according to
the E. coli numbering. Hydrogen bonds and salt bridges are presented as black dashed lines. Bond lengths
are presented in dark green in ångström - Å. (B-F) The atomic details of the contacts involving the rings
with conserved and non-conserved rRNA residues.
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4.1.4.2. The Binding Mode of Apramycin to the Leishmanial A-site - ‘OFF’ State.
In contrast to G418, Apramycin demonstrated a looser interaction network,
maintaining only 6 direct contacts with the leishmanial A-site (Figure 4.24). Four of
these contacts are mediated by ring II (2-DOS) that interacts with the RNA bases and
phosphate backbones of the 3 conserved residues G1405, U1406 and G1494 (Figure 4.24
B). The 2 additional remaining bonds are between the 2’-NH2 group located at the
bicyclic ring I to the O6 atoms of G1494 and G1408 (Figure 4.24 A). As can be seen
from the superposition with the bacterial-A-site-Apramycin complex (Apra-Bact)
(Figure 4.22 E) the binding pattern of Apramycin to the leishmanial A-site is rather
distinct from the one observed in bacteria (r.m.s.d 2.2 Å). As a matter of fact, when only
the two ligand molecules are compared their atoms barely overlap; with the molecules
oriented in a ~180° horizontal flip one to another. As a result Apramycin induces a
distinctive conformational change upon binding to the leishmanial A-site; where only
A1493 is flipped out from the helical core. A1492, which is directed towards the helical
core forms pseudo canonical interactions with G1408 (Figure 4.24 C). A conformation
where one of the two conserved adenines (A1492 and A1493) or both are directed
towards the helical core was earlier referred as an ‘OFF’ state conformation 121
. This
conformation usually occurs when an inappropriate or no tRNA molecule is present at the
ribosomal A-site; therefore it might signal for translational arrest. Despite the great
dissimilarity of the Apramycin structure to the previously reported X-ray structures, our
results are in good agreement with a recent solution NMR structure indicating the lack of
A1492 and A1493 destacking upon Apramycin’s binding to an rRNA bacterial A-site
model 106
. The obtained results also correlate with some recent evidence for Apramycin’s
ability to inhibit translocation in bacterial ribosomes, but not to induce bacterial
miscoding 106, 139
or PTC suppression in eukaryotes 92
.
Interestingly, the binding pattern observed upon Apramycin binding to the
leishmanial A-site was found to be rather similar to the one observed in our previous
complexes of Apramycin with the human cytoplasmic A-site constructs (superposition
r.m.s.d 1.4 Å; Figure 4.22 C, F). The human A-site departs from the leishmanial one by
a single nucleotide located in position 1409 (C1409 human vs. U1409 Leishmania -
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Figure 4.17). However, although these two A-sites share greater sequential similarity
when compared to the bacterial one, the geometry of the two A-sites is more distinct due
to the abrogation of the 1409:1491 Watson-Crick pair in the human A-site; giving its
helical core a more complex conformational arsenal. Nevertheless, despite the noticeable
differences in the orientations of A1491-A1493, Apramycin binding and interaction with
the leishmanial A-site is nearly identical to the one observed in the human A-site (Figure
4.22 F). The obtained results indicate a rather conserved interaction pattern in eukaryotes.
Such similarity could be attributed to the presence of the highly conserved guanine
residue in position 1408, maintaining a preserved interaction with the OH moiety located
in the bicyclic ring I (Figure 4.24 A).
Figure 4.24. Description of the contacts between Apramycin and the leishmanial A-site. Ring numbers (I-
III) and atom names are specified. rRNA atoms are numbered according to the E.coli numbering. Hydrogen
bonds and salt bridges are presented as black dashed lines. Bond lengths are presented in dark green in
angström - Å. The atomic details of the contacts involving ring I (A) and the bicyclic ring II (B) with
conserved and non-conserved rRNA residues. (C) Pseudo Watson-Crick interactions of A1492 and G1408.
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4.1.5. In-vitro Inhibition of L. donovani and L. major Promastigote Growth by AG.
The structural evidence obtained in this study increases our understanding of the
molecular mechanisms of AG interaction with leishmanial ribosomes. However, to date a
comprehensive study comparing susceptibility to different AG derivatives was not carried
out. In order to evaluate the effect of AG on Leishmania growth 5 representative
derivatives were chosen. Paromomycin and Neomycin B were chosen as representatives
of the 4,5-disubstituted 2-DOS group; G418 and Gentamicin as 4,6-disubstituted and
Apramycin as mono-substituted. The representatives of each group differ by the nature of
the substituent group at the 6’-position of ring I (Figure 1.1), which is known to be an
important component of selectivity towards eukaryotic species 140
. Drug susceptibility
was tested using two species, L. major and L. donovani, which induce cutaneous and
visceral leishmaniasis in humans, respectively. The concentrations giving 50% of growth
inhibition (IC50) are listed in Table 4.9. All AG tested inhibited Leishmania growth in a
dose dependant manner.
The IC50’s obtained for the 6’-OH AG (Paromomycin and G418) were in the μM
range, and showed good agreement with previous published values for these compounds
138, 141, 142. The results were also similar to the MIC values reported in bacterial systems
for the two compounds 85, 88, 143
; thus adding additional support to the suggested
similarity in their mechanism of action in bacteria and Leishmania. However, the
obtained results indicate that G418 is ~10 times more potent than Paromomycin against
the two Leishmania strains tested. Although no such potency gap for the two derivatives
has been documented in w.t. bacterial strains, similar tendency has been reported in
A1408G mutant bacteria 114
, sharing greater binding site similarity with Leishmania.
These results also correlate well with the higher miscoding 24
and PTC suppressing
potency reported for G418 over Paromomycin in eukaryotes 91
. The obtained IC50 values
for the 6’-NH2 derivatives (Neomycin and Gentamicin) were higher than those obtained
for the 6’-OH derivatives, and are also in good agreement with previously reported work
demonstrating the lower potency of Neomycin B, when compared to Paromomycin, for
treatment of leishmaniasis 138
. These results are also correlated well with the facts that 6’-
NH2 derivatives are less effective against AG resistant bacterial strains containing an
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A1408G substitution, and have a very limited read-through and misreading activity in
higher eukaryotes.
However, unlike in bacterial systems, where Apramycin is conserved as a potent
antibacterial agent, our IC50 results in Leishmania indicated a rather low activity of the
compound (IC50>2,000 μM; Table 4.9). These results could be explained either by the
distinct A-site conformation observed in X-ray experiments designed to explore
Apramycin’s binding pattern in bacteria 139, 144
(Figure 4.22 E), or by the possibility of
alternative binding sites at the bacterial ribosome. The conformational alternatives
suggested for Apramycin in bacterial systems cannot be obtained in the leishmanial A-
site due to alternations in the rRNA sequence preventing several H-bonds from forming;
therefore limiting the binding affinity of such conformations. The recent indications of
the rather low translation inhibition of human ribosomes by Apramycin 115
, altogether
with the structural similarity in Apramycin’s binding to eukaryotic A-sites further support
our bio-activity results indicating a low susceptibility of eukaryotic parasites to
Apramycin.
Table 4.9. In-vitro inhibition of Leishmania promastigotes by natural AG
IC50 (μM)
L.donovani L.major
48.1 ± 5.8, n=6a 31.4 ± 5.7, n=5
a Paromomycin
>2000 >1000 Neomycin
5.8 ± 0.9, n=4a 1.95 ± 0.05, n=2
a G418
>2000 273.1 Gentamicin
>2000 >1000 Apramycin a Each value represents the mean ± standard error of n repeats.
The obtained susceptibility results for the natural AG highlighted the superiority
of the 6'-OH di-substituted derivatives for the treatment of leishmaniasis. Adding these
results to the similar tendency of the tested AG to induce translational read-through
activities in human encouraged us to further test the leishmanial parasites susceptibility to
our lead NB compounds (NB74, NB 84 and NB 124, Figure 1.7). These compounds
were shown to induce the highest read-through activities in eukaryotes reported for
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synthetic derivatives thus far 88, 91
; and indeed, our susceptibility tests indicate that the
NB compounds were able to inhibit leishmanial growth at the low micromolar range
(Table 4.10); with NB124 exhibiting the highest anti-leishmanial activity values. The
IC50 values were 3-6 fold lower than those obtained for paromomycin, which was used as
a basic scaffold for the design of our NB compounds, but 2.5-5 times higher than those
observed for G418, of which rings I and II are identical. The improved activity of our NB
compounds when compared to Paromomycin might be a direct effect of the 6'-(R)-
Methyl group presence in the NB compounds directly interacting with the π systems of
the A1491-U1409 pair as observed in the G418 structure (Figure 4.23 D). In addition,
the presence of the 5''-NH2 group when compared to the 5''-OH in paromomycin, might
add some additional stabilization with the G1408 residue (Figure 4.25).
Table 4.10. In-vitro inhibition of Leishmania promastigotes by synthetic AG and toxicity measurements in
HEK293 cells (LC50)
Cell Toxicity
LC50 HEK293 (mM)b
Anti-leishmanial activity
IC50 L.major (μM)a
1.31 ± 0.06 1.95 ± 0.05 G418
4.13 ± 0.51 31.4 ± 5.7 Paromomycin
22.7 ± 1.06 8.2 ± 2.1 NB74
5.77 ± 0.68 10.4 ± 1.8 NB84
5.40 ± 0.45 5.6 ± 0.9 NB124 a Each value represents the mean ± standard error of at least 2 repeats performed in triplicates.
b The LC50 values were previously reported [
88, 91].
In addition to exhibiting some excellent anti-leishmanial properties the developed
NB compounds were also demonstrated to be less toxic than both G418 and
Paromomycin, as indicated previously in our cell toxicity assays (Table 4.10) 88, 91
. These
preliminary results are highly encouraging and gave rise to some additional trials
currently ongoing in our lab.
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Figure 4.25. Surface representations of AG binding sites in leishmanial ribosomes. (A) The G418 position
as crystallographically determined and putative binding modes of Paromomycin (B) and NB124 (C) as
obtained by manual docking based on this ligands binding to the bacterial ribosome. AG are represented in
stick representation and highlighted in yellow. The coloring of the surface represent residue conservation
among prokaryotic and eukaryotic systems, where residues marked in cyan are highly conserved among all
kingdoms and residues marked in light pink are rather diverse. The Paromomycin and NB124 positions
were obtained by superimposition of the A-site coordinates from the G418/leishmanial A-site structure
(PDB ID: 4K32) and the paromomycin coordinates from the crystal structure of paromomycin bound to the
bacterial A-site (PDB ID: 1J7T ). Possible H-bonds are represented in dashed yellow lines. H-bonds
alternatives for the 5''-NH2 group of NB124 are highlighted in a red circle. Figure was made using Pymol.
(D) A 2-D representation of the additional contacts of 5''-NH2 group with G1408.
4.1.6. Aminoglycosides Mechanisms of Action in Leishmania
The combination of our structural and biochemical data shows for the first time the
mechanistic parameters governing the interactions of AG in leishmanial ribosomes, and
supplies the molecular explanation for their potent activity against Leishmania. The data
also reveals that in contrast to bacterial systems, the substitution pattern of AG around the
2-DOS ring affects their binding mode and might induce an entirely different
conformational change upon binding. Our data also indicate that the induction of an ‘ON’
A. B.
C.
A1491
A1491
A1491
G1408
G1408
G1408
N
NNH
N
O
N
OH
H
H
H
+H2N
O
H
5''-Ring III
Ring I
6'
ring oxigen
NB compound
G1408
D.
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state conformation is highly important for AG potency as anti-Leishmania agents; thus
implying the significance of miscoding events in the killing mechanism. The data
highlights the importance of 6'-OH ring I for the induction of an 'ON' state conformation.
Based on the obtained data we could suggest a simple diagram for AG mechanism of
action against leishmanial cells (Figure 4.26).
Figure 4.26. General scheme of AG activities in Leishmania. Normal translation is presented in the middle
where cognate tRNA is highlighted in green. The lower part describes mistranslational events in the present
of 6'-OH AG inducing and 'ON' state conformation upon binding (6'-OH AG are highlighted in red). The
upper part describes a simple inhibition of translation upon Apramycin's binding (Apramycin is highlighted
in yellow).
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4.2. The Development and Employment of a Highly Sensitive Immunoassay for the
Detection of AG in Biological Samples
Our crystallization trials increased our understanding of how AG act in
eukaryotes and gave rise to the further development of new and improved derivatives.
The promising results of our pre-developed NB compounds in the treatment of PTC
induced genetic disorder altogether with our recent results in Leishmania necessitated a
fast and effective methodology for the detection and quantification of the developed lead
NB-compounds in various biological derived matrices; such methodology will be of high
importance in further progress in drug development.
Over the last few decades many analytical and bioanalytical assays were
suggested for the qualitative and quantitative analysis of AG that are in clinical,
veterinary and agricultural use to treat bacterial infections. These methods were applied
for the detection of drug residues in a wide range of biological derived matrices such as
cell tissues 132, 145-147
, serum 148-154
, milk 145, 148-152, 154-160
, eggs 147, 157
, and honey 157
.
Chemical methodologies included the application of gas chromatography (GC) 161
, TLC
162, high-performance liquid chromatography (HPLC)
163 and capillary electrophoresis
(CE) 164
. Biological methodologies included the development of microbiological assays
165, radiochemical and radioimmunochemical assays (RIA)
166, enzyme linked- and
flouro- immunoassays (ELISA, FIA) 149, 167, 168
, nano-sensors and nano-particle based
immunoassays 169, 170
.
The main advantages of using chemical methods for the detection of AG in
biological samples is the possibility of detecting a wide variety of therapeutic agents
simultaneously altogether with monitoring their corresponding metabolitic products.
Nevertheless, due to their high polarity and the lack of chromophoric or florophoric
properties AG chemical detection is quite a challenging task. Recent work have
demonstrated the use of various analytical methodologies conjugated to MS-MS detectors
171, 172, whereas other works used various UV/floroscentic derivatization methods that
were further exploited for the detection of the derivatizied products 173
. These methods
were found to be highly sensitive and demonstrated an efficient procedure for the
detection of AG. However, applying these methods on biological derived matrices
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required several pre-purification and sample concentration steps prior to detection
process. Moreover, these methods cannot be readily utilized in all laboratories and
engage the use of specific instrumentation which is highly costly and requires well
trained human resources to operate it and analyze the resulting data.
Immuno-based methodologies such as ELISA, FIA and RIA have shown to be as
sensitive and accurate as the chemical methods 174
. These methods could be easily
applied on a wide variety of biologically derived matrices, are simple to perform and
analyze, and do not require the use of high cost instrumentations. Immuno-based
methodologies are commercially available for the detection of some widely used natural
AG such as gentamicin, streptomycin, dihydrostreptomycin, neomycin and kanamycins
(eg. MaxSignal® gentamicin ELISA Test Kit, AG enzyme immune-assay-EIA kit for the
detection of gentamicin, neomycin, streptomycin and dihydrostreptomycin -
Europroxima, and kanamycins ELISA kit - Wanger). These commercially available kits
can be exploited for highly sensitive detection of AG in various matrices, including body
fluids, cell tissues and food products. Similar immuno-based methodologies are used in
hospitals to monitor the serum levels of clinically used AG such as gentamicin and
amikacin. However, these assays are mostly used for the detection of AG antibiotics that
are based on a neamine core, containing an amino group at their 6' position (ring I,
Figure 4.27). The use of the derived antibodies for the detection of therapeutic
derivatives containing other substituents on the neamine core is quite limited due to low
cross reactivity values. Recent documentation demonstrated the superiority of 6'-
hydroxyl containing AG such as paromomycin and G418 over the 6'-amino derivatives in
the treatment of various genetic disorders 26
. These derivatives are mostly based on either
a paromamine or an NB82 2-ring core (Figure 4.27) and are extensively explored for the
treatment of various genetic diseases 82, 85, 88, 90
. To our knowledge, no immunoassays
exist for the detection of AG containing a 6'-hydroxyl group.
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Figure 4.27: Chemical structures of standard and semi-synthetic AG derivatives used in the study.
4.2.1 Preparation of Immunization Conjugate
The main goal of this part of the present study was to develop an
immunochemical assay for monitoring lead compounds of NB-series in biologically
derived samples. The specific aims included the production of a generic polyclonal
antiserum that will be able to recognize a wide variety of NB compounds (Figure 4.27)
that have previously been shown to exhibit great potential for the treatment of various
nonsense mutations related genetic diseases 34, 82, 83, 85, 87-91
.
6'-(R)-methyl-paromamine (NB82) (Figure 4.27), a semi-synthetic 2-ring
fragment derived from G418, is a common scaffold, appearing in nearly all compounds
of NB-series (Figure 4.27). We assumed that by choosing NB82 as hapten for
immunogen preparation we could obtain polyclonal antibodies that will be able to
recognize a diverse selection of these compounds. Similar methodology was previously
described by Van Amerongen and coworkers 158
by using neamine as an immunogenic
hapten for the generation of polyclonal antiserum capable of recognizing some natural
AG such as neomycin, kanamycin A and gentamicin.
Our next challenge was to choose a proper conjugation strategy for the
preparation of immunization conjugate. Since NB82 contains three primary amino
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groups, it could be easily subjected to either direct conjugation with carrier proteins via
the formation of amide bonds using the EDC method 175
, or to indirect conjugation
methods using linking agents such as glutaraldehyde 132, 175
. These methods were
previously reported as highly efficient for the generation of anti-AG antibodies.
However, due to amino group multiplicity the coupling process usually results in a
spontaneous non-specific linkage to any amino group of the subjected hapten. Some of
our lead compounds, like NB84, contain an (S)-4-amino-2-hydroxybutanoyl (AHB)
moiety at their N-1 position (Figure 4.27). Our earlier studies have demonstrated that the
addition of AHB group selectively at N-1 position significantly enhances the read-
through activity of the resulted derivatives in comparison to that of the parent structure
without the AHB, while its toxicity profile was not significantly affected 85, 88, 90
. These
derivatives are rather bulky, and could interfere with the antibodies binding affinity
towards some of the ligands arsenal; therefore we assumed that the best linking option
would be through the N-1 position. Taking into account that the antibody raised against a
hapten linked to the carrier protein through N-1 position would have similar affinity to
both compounds with and without AHB, further supported our strategy for linking NB82
to a carrier protein selectively at N-1 position.
In order to selectively conjugate the NB82 scaffold to a carrier protein via the N-1
position we first had to selectively protect the additional two amino groups at positions 2’
and 3 to prevent them from reacting with the linking agent. This was done by selective
azidation of NB82 at 2’ and 3 positions to yield 2’,3-diazido-NB82 (compound A,
Figure 3.1). Treatment of compound A with glutaric anhydride afforded compound B in
62% isolated yield. The carboxylic acid function of compound B at its N-1 side-chain
was activated by reaction with N-hydroxysuccinimide in the presence of DCC to yield
compound C. Conjugation of the NHS-activated compound C with the carrier protein
KLH was followed by the conversion of two azido groups to the corresponding amines
using Staudinger reaction (Me3P, pH 9.2) to yield the immunization conjugate NB82-
KLH (compound E, Figure 3.1). The resulting conjugate was used as an immunization
agent and was injected to two rabbits.
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4.2.2 Development of an Indirect Competitive ELISA
The two rabbits immunized with the NB82-KLH conjugate showed high antibody
serum titers, as indicated in checkerboard homologous-assays performed using the NB82-
OVA conjugate as a coating antigen (Figures 4.28, 4.29). These experiments were
further used to determine the optimal concentrations of coating antigen (NB82-OVA,
1:1), rabbit antiserum (8245) and secondary antibody to be 1:8,000, 1:80,000 and
1:20,000, respectively. These conditions were used to determine the I50 value and assay’s
detection limit (I20) of NB82 to be 50±6 ng mL-1
and 8±1 ng mL-1
(n=10), respectively
(Figure 4.30, Table 4.11).
Figure 4.28: Antiserum activity of pre-immune (PI) rabbits, two weeks after 1st immunization with 1 mg
NB82-KLH conjugate (1st bleed), 2 weeks after 1
st boost with additional 1 mg immunogen (2
nd bleed), and
after 2nd
boost (3rd
bleed). Microtitter plate assay performed by using NB82–OVA conjugate 1:1 in 1:8,000
dilution and antisera of rabbits 8244 (A) and 8245 (B) diluted in a range of 1:5,000-1:40,000. Each bar
represents the mean of 3 independent repeats performed in duplicates.
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Figure 4.29: Antigen recognition (Chessboard) assay. Coating antigen (NB82-OVA, prepared in 1:1 molar
ratio of NB82 and OVA) dilutions were 1:8,000-1:64,000 in carbonate buffer pH 9.6; 3rd
bleed antiserum
derived from RB8244 (A) and RB8245 (B) dilutions were 1:5,000-1:160,000. Each bar represents the mean
of at least 3 independent repeats performed in duplicates.
Figure 4.30: Standard curve of NB82 (Homologous assay). NB82-OVA conjugate dilution 1:8,000;
Antisera dilution (Rabbit 8245) 1:80,000. I50 50±6 ng mL-1
; Limits of detection: 8±1 ng mL-1
. Each value
represents the mean±S.E.M. (Standard error mean) of 10 independent repeats performed in duplicates.
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Table 4.11: Concentrations at 50% inhibition (I50) and limits of detection (I20) of various AG using the
homologous ELISA with NB82-OVA as a coating antigena.
Compound I50
(µg/mL) Detection limit (I20) (µg/mL)
NB82 0.05±0.006 0.008±0.001
G418 0.4±0.04 0.06±0.02
NB125 1.25±0.14 0.3±0.03
Paromamine 3.0±0.34 0.6±0.1
NB84 3.1±0.75 0.7±0.1
NB74 3.5±0.15 0.7±0.1
NB124 12±1.1 3±0.7
Gentamicin 130±8.99 20±3
NB123 250±22.6 40±1
Paromomycin 325±56.6 50±2
NB122 400±34.2 80±4
Neomycin 700±71.7 90±4
Kanamycin A 1000±144 120±11
Neamine 2000±251 150±14 a
NB82-OVA conjugate dilution 1:8,000 was used as coating antigen; Antisera dilution (Rabbit 8245) 1:80,000. Each value represents
the mean±S.E.M. of at least 3 experiments performed in duplicates.
In order to evaluate the efficiency of the assay for the detection of various AG, 13
representative members were chosen to be detected for antiserum cross-reactivity via
homologous-cross-reactivity assays, using the NB82-OVA conjugate as a coating
antigen. Five of them including G418, gentamicin, paromomycin, neomycin and
kanamycin A are natural AG that are mostly known for their antibacterial activities
(Figure 4.27). Six compounds including NB74, NB84, NB122, NB123, NB124 and
NB125, are semi-synthetic agents systematically developed in our laboratory as read-
through inducers and have previously shown to bear a great potential of treating various
genetic disorders caused by premature stop codon mutations 33, 34, 82, 83, 85, 87-90
. Three
additional compounds selected in this study included 2-ring structures as common
scaffolds for various natural and semi-synthetic AG: neamine, a common scaffold of
gentamicin, neomycin B and kanamycin A; paromamine, a common scaffold of
paromomycin, NB122 and NB123; NB82, a common scaffold of G418, NB74, NB84,
NB124 and NB125. As can be seen from the data in Figure 4.31, antiserum antibodies
were able to recognize all tested members at various sensitivity ranges, where NB82, as
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expected, demonstrated the lowest I50 value, thus indicating highest antibody affinity
(Table 4.11).
Earlier studies have shown that AG with a 6'-OH group on ring I (such as G418
and paromomycin, Figure 4.27) are more effective read-through inducers than those with
an amine at the same position 26
. The affinities of serum antibodies towards the
structurally related AG containing a secondary (R)-6'-OH on ring I (NB82, G418, NB74,
NB84, NB124, NB125) were higher than those obtained for similar compounds
containing either a primary 6'-OH (NB122, NB123, paromomycin, paromamine) or a 6'-
NH2 (neomycin, neamine, gentamicin, kanamycin A). These results correlate well with
our expectations that the resulting antibodies will show higher specificity towards
compounds that share greater similarity with the parent compound used for
immunization. Using NB82-OVA as the coating agent the antiserum indicated a rather
high cross reactivity value of 12.5% for G418, with I50 of 400 ng mL-1
(Figure 4.27,
Table 4.11). These values can be attributed to the high number of similar epitopes G418
shares with NB82, as can be seen in Figure 4.27.
Cross reactivity values of the structurally related synthetic derivatives NB74,
NB84, NB124 and NB125 where shown to range from 0.4-4%, with I50 values of 1.25-12
μg mL-1
(Table 4.11). These compounds are derived from G418; therefore they all
contain the NB82 scaffold. The observed 1- to 2-orders of magnitude gaps in cross
reactivity values (when compared to G418) can be explained by the difference in their
substitution pattern regarding the 2-deoxy-streptamine (2-DOS) ring (ring II, Figure
4.27). NB compounds are 4,5 di-substituted 2-DOS derivatives, whereas G418 is a 4,6 di-
substituted 2-DOS member. Since immunization conjugate was linked via the N-1
position, it is reasonable to hypothesize that the epitopes detected by the various antibody
populations are located in the 1st sugar ring (ring I) and the glycosidic bond relative
positions of the 2-DOS ring (ring II); as far as possible from the N-1 position. Following
this line of thought, compounds of NB-series substituted with ring III at C5 position will
generate greater steric interference with the antibody than that of G418 in which ring III
is substituted at C6 position. According to this rationale, compounds that differ only in
the substitution pattern at N-1 position should have similar affinities to the antibody.
Indeed, as can be seen from the data in Table 4.11, NB74 and NB84 that differ at their
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N-1 position demonstrated similar cross reactivity values of 3.5±0.15 µg mL-1
and
3.1±0.75 µg mL-1
, respectively. This is the direct result of our design strategy of the
conjugation hapten. Cross reactivity values for compounds that did not contain NB82 as a
main scaffold were shown to be less than 0.04%, with I50 values of 130-2000 μg mL-1
(NB122, NB123, paromomycin, neomycin, gentamicin, kanamycin A and neamine,
Figure 4.31, Table 4.11).
Interestingly, the antibody populations generated were able to differentiate
between the diastereomeric compounds that differ only by the configuration at a single
chiral carbon center. Among the series of compounds tested in this study we had two
such pairs of compounds: NB122 (5"-S) vs NB123 (5"-R), and NB124 (5"-S) vs NB125
(5"-R). The I50 values of NB125 (5"-R) were 1 order of magnitude lower than those
obtained for its 5"-diastereomer, NB124 (5"-S) (Table 4.11). Similarly, the values
obtained for NB123 (5"-R) were twice lower than those obtained for NB122 (5"-S).
These results demonstrate a stereo-selective preference towards the 5"-R isomers. An
opposite tendency was observed for these compounds regarding their readthtough activity
and cytotoxicity values 33, 34, 82, 83, 85, 87-91
. These observations could be attributed to the
different pattern of interactions between the 5''-groups and the antibody binding pocket.
Figure 4.31: Homologous assay cross reactivity. Antiserum cross reactivity of natural and synthetic
representative AG derivatives: NB82 (♦), geneticin (G418) (●), NB125 (◊), paromamine (□), NB84 (■),
NB74 (▲), NB124 ( ), gentamicin (○), NB123 (■), paromomycin (+), NB122 (▲), neomycin (-),
kanamycin A (X), and neamine ( ). NB82-OVA was used as coating antigen (dilution 1:8,000); Antiserum
dilution (Rabbit 8245) 1:80,000. Each value represents the mean of at least 3 experiments performed in
duplicates. Mean I50 values ±S.E.M. are summarized in Table 4.11.
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4.2.3 Development of Heterologous-Assays
The cross reactivity values obtained in the homologous assays demonstrated the
antibodies ability to bind a wide range of synthetic and natural derivatives. However, the
enhanced affinity of the antibodies towards the homologous coating conjugate (NB82-
OVA) used in these assays resulted in a 1-5 orders of magnitude gap between the
affinities to NB82 and the other tested compounds. We assumed that in the homologous
ELISA used, the specific antibodies, induced by the NB82-KLH immunogen conjugate,
might bind the homologous coating conjugate NB82-OVA with such a high affinity that
competition with the free hapten (NB82) or other similar structures will only be visible at
high solution concentrations of the free antigen. Previous studies have demonstrated that
using heterologous conjugates as coating antigens can significantly enhance the
sensitivity of such assays towards different antigens 158
. A heterologous ELISA refers to a
system in which the haptens used for the immunogen and for the coating conjugate have
slightly different chemical structures 158
. In this case, a heterologous coating conjugate
will have a lower affinity to the antibody, and consequently, the free hapten and or
similar structures will be able to displace the antibodies more sensitively. Therefore, in
attempts to enhance the sensitivity of our assay, we developed heterologus ELISAs
towards 2 leading NB compounds, NB84 and NB124, which have shown the greatest
activity in ex-vivo and in-vivo experimentations 89, 176
. Three heterologous-coating
conjugates were prepared using the glutaraldehyde conjugation method: NB82-GTA-
OVA (control), NB84-GTA-OVA and NB124-GTA-OVA. Conjugate preparation was
followed by the development of 3 indirect heterologous-assays to test cross reactivity
values of 3 representative semi-synthetic members. The obtained results indicated a
major improvement in heterologous-antigen recognition, where I50 values for NB82,
NB84 and NB124 were reduced from 50±6 ng mL-1
to 10±3 ng mL-1
, 3.1±0.75 μg mL-1
to 0.5±0.04 μg mL-1
and 12±1 μg mL-1
to 1±0.12 μg mL-1
, respectively (Table 4.12).
Detection limits, while using the heterologous-assays were 0.001±0.0003 μg mL-1
for
NB82, 0.02±0.007 μg mL-1
for NB84 and 0.015±0.008 μg mL-1
for NB124 (Table 4.12).
The values obtained for the NB82-GTA-OVA conjugate indicated no significant
difference.
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Table 4.12: Concentrations at 50% inhibition (I50) and limits of detection (I20) of 3 representative
members (NB82, NB84 and NB124) using the heterologous ELISAsb.
Compound Coating antigen I50
(µg/mL) Detection limit (I20) (µg/mL)
NB82 NB82-OVA 0.05±0.006 0.008±0.001
NB82-GTA-OVA 0.05±0.009 0.007±0.001
NB84-GTA-OVA 0.04±0.006 0.007±0.001
NB124-GTA-OVA 0.01±0.003 0.001±0.0003
NB84 NB82-OVA 3.1±0.75 0.7±0.1
NB82-GTA-OVA 6±0.3 0.5±0.1
NB84-GTA-OVA 0.5±0.04 0.02±0.007
NB124-GTA-OVA 0.8±0.03 0.03±0.009
NB124 NB82-OVA 12±1.1 3±0.7
NB82-GTA-OVA 7±0.5 0.5±0.06
NB84-GTA-OVA 2±0.1 0.03±0.009
NB124-GTA-OVA 1±0.12 0.015±0.008
b NB82-OVA conjugate dilution was 1:8,000; NB82-GTA-OVA conjugate dilution was 1:128,000; NB84-GTA-OVA conjugate dilution was 1:64,000;
NB124-GTA-OVA conjugate dilution was 1:16,000; Antisera dilution (Rabbit 8245) 1:80,000. Each value represents the mean±S.E.M. of at least 3
experiments performed in duplicates.
4.2.4 Determination of Selected NB Compounds Serum Levels in Mice
Our next challenge was to use the pre-developed assay to monitor AG content in
mice serum in-vivo. For this purpose, we initially determined the antibodies affinities and
detection limits to AG in serum. We prepared 24 serial dilutions of 3 representative
members NB82, NB84 and NB124 in PBS, FBS and mice serum and quantified them by
using our pre-developed ELISA. The selection of NB84 in these experiments is based on
our recent results obtained with this designed AG as a potential treatment of
mucopolysaccharidosis type-I-Hurler (MPS I-H) disease caused by nonsense mutation 89
.
In the latter study we investigated the efficiency of a series of different suppression
therapy agents including the conventional AG gentamicin, G418, amikacin, and
paromomycin along with the designed AG NB54 and NB84, to suppress the idua-W392X
nonsense mutation in an MPS I-H mouse model. We found that NB84 suppressed the
idua-W392X nonsense mutation much more efficiently than any of the other compounds
tested. In-vivo administration of NB84 to idua-W392X mice significantly reduced urine
glycosaminoglycan (GAG) excretion and tissue GAG storage. These studies
demonstrated for the first time that NB84-mediated suppression therapy has the potential
to attenuate the MPS I-H disease phenotype. Preliminary tests of NB124, which is the
most lately developed lead compound, on the same disease models showed that it is much
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better than NB84 176
. NB82 was used as a positive control to test serum interference with
ELISA protocol. The measured I50 and limit of detection (I20) values for all three
compounds tested (NB82, NB84 and NB124) in mice serum were similar to those
observed in PBS solutions (Table 4.13). Next, we determined the recovery of AG from
whole mice blood samples by spiking 250 μL blood samples with 10, 25 or 50 μg mL-1
of
the relevant compound, followed by serum extraction. Recovery ratios were determined
to be 97-102% for NB82, 92-105% for NB84 and 100-107% for NB124 (Table 4.14).
Table 4.13: Concentrations at 50% inhibition (I50) and limits of detection (I20) of 3 representative
members (NB82, NB84 and NB124) in mice serumc.
Compound Matrics I50
(µg/mL) Detection limit (I20) (µg/mL)
NB82 PBS 0.01±0.003 0.001±0.0003
Serum 0.008±0.001 0.001±0.0002
NB84 PBS 0.5±0.04 0.02±0.007
Serum 0.4±0.1 0.02±0.009
NB124 PBS 1±0.12 0.015±0.008
Serum 0.8±0.06 0.01±0.005
c For NB82 and NB124 detection: NB124-GTA-OVA conjugate dilution 1:16,000 was used as coating antigen; For NB84 detection: NB84-GTA-OVA
conjugate dilution 1:64,000 was used as coating antigen; Antisera dilution for all experiments was 1:80,000 (Rabbit 8245). Each value represents the
mean±S.E.M. of at least 3 experiments performed in duplicates.
Table 4.14: Recovery of NB82, NB84 and NB124 from whole blood samples d.
Compound Spiked concentration (µg/mL) Detected concentration (µg/mL) % Recovery
NB82 10 10.0±0.0 011
25 24.2±0.5 97
50 50.8±0.8 102
NB84 10 9.9±0.2 99
25 26.3±0.3 105
50 46.1±1.5 92
NB124 10 10.3±0.1 103
25 26.7±0.7 107
50 50.0±0.1 100
d Each value represents the mean±S.E.M. of at least 3 experiments performed in duplicates.
To determine serum levels of the above selected NB compounds in-vivo we
carried out IP injections at therapeutic doses (28.5 mg/kg body weight) to mice, and
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collected blood samples at various time points post injection. Similar doses of NB84
(30.0 mg/kg body weight) were previously shown to enhance therapeutic effect in-vivo in
Idua-W392X model mice for MPS I-H disease without causing any detectable side
effects 89
. ELISA measurements of AG content in serum samples indicated that the serum
concentrations of NB84 and NB124 reached the peak levels of 121.6 μg mL-1
and 140 μg
mL-1
, respectively, within 10 min post injection, and then declined to ~10 μg mL-1
and 28
μg mL-1
by 60 min after injection (Figure 4.32). Remarkably similar kinetics were
previously reported for the AG gentamicin and amikacin detected in mice serum 167, 168
.
The obtained results also correspond to the alpha phase in the 3-compartment
pharmacokinetic model of AG serum availability, of which, a fast deterioration is
observed in the first 60 min after injection 177
.
Figure 4.32: Serum levels of NB84 and NB124 following a single dose IP injection in male mice (C57/BL,
2.5-3.5 months old), Each point represents the AG content (NB84 - black parallelogram, NB124 - white
triangle) in a single blood sample taken from different mice at various time points following the injection of
28 μg/gr body weight NB compound. The concentrations were determined using the pre-developed
heterologous-immunoassays. Each sample was prepared at 5 different concentrations and tested in
duplicates.
Due to the relatively high toxicity values observed upon AG administration,
monitoring the serum levels of these compounds in treated patients is highly important.
According to the Physicians Desk Reference (PDR) 178
, the recommended intravenous
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(IV) doses of gentamicin and amikacin for antibacterial treatment in patients are 6-7.5
mg/kg/day and 15 mg/kg/day, respectively. The maximum peak serum levels allowed for
these compounds are 12 μg mL-1
and 35 μg mL-1
. However, in a recent clinical trial,
performed in France, testing the potential use of gentamicin for the treatment of CF
patients carrying various nonsense mutations, higher gentamicin peak serum levels of 20-
40 μg mL-1
were allowed 179
. These values are 2- to 4-fold higher than those approved for
antibacterial treatment. No toxic effects were reported using the declared doses during the
15 day trial, of which gentamicin was administrated once daily at 10 mg/kg body weight.
Our previous studies have indicated that all the compounds of NB-series used in this
study lack antibacterial activity and exhibit significantly lower toxicity than that of
gentamicin 33, 34, 82, 83, 85, 87-91
. However, whether the observed peak levels of 121.6 μg
mL-1
and 140 μg mL-1
, upon the administration of ~30 mg/kg body weight of NB 84 and
NB 124, are within the safety doses or not for the administration in humans, is not clear
yet and requires further investigation. Nevertheless, the sensitivity and versatility of the
generic immunoassay developed in this study are adequate for the detection of NB84 and
NB124; lowest detection limits of 20 ng mL-1
and 15 ng mL-1
, respectively that are far
below the peak levels of these compounds observed in mice serum.
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5. Summary and Conclusions
The present work aimed at exploring AG mechanisms of action in eukaryotes
towards the rational development of new and improved therapeutic compounds. The
structural work performed together with the obtained biochemical data enabled us to
better understand AG mechanisms of action in eukaryotic protozoa. Due to the high
similarity to human ribosomes, our Leishmania structural data also supplies indirect
evidence regarding AG action in humans. The detailed information obtained at the atomic
level also added some more benefits in our understanding of structure-activity
relationships of AG action in eukaryotes; this information is highly beneficial in the
rational design and development of new derivatives as potential therapeutic agents to
treat leishmaniasis and genetic disorders, and is currently being used in our group in
several ongoing studies.
The development of a highly sensitive methodology for the detection of the potential
therapeutic derivatives in biological samples altogether with its application for in-vivo
monitoring of selected AG in serum has also been demonstrated within the course of the
present work. This methodology is already being in an extensive use in our group and in
collaboration with additional groups working towards the clinical development of the
therapeutic compounds.
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Appendix
Table I Crystal screen and initial crystallization conditions determination a
Salt Precipitant Polyamine Buffer
A1 10 mM LiCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
A2 100 mM LiCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
A3 250 mM LiCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
A4 10 mM LiCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
A5 100 mM LiCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
A6 250 mM LiCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
A7 10 mM LiCl 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
A8 100 mM LiCl 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
A9 250 mM LiCl 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
A10 10 mM LiCl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
A11 100 mM LiCl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
A12 250 mM LiCl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
B1 10 mM NaCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
B2 100 mM NaCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
B3 250 mM NaCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
B4 10 mM NaCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
B5 100 mM NaCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
B6 250 mM NaCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
B7 10 mM NaCl 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
B8 100 mM NaCl 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
B9 250 mM NaCl 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
B10 10 mM NaCl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
B11 100 mM NaCl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
B12 250 mM NaCl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
C1 10 mM KCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
C2 100 mM KCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
C3 250 mM KCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
C4 10 mM KCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
C5 100 mM KCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
C6 250 mM KCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
C7 10 mM KCl 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
C8 100 mM KCl 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
C9 250 mM KCl 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
C10 10 mM KCl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
C11 100 mM KCl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
C12 250 mM KCl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
D1 10 mM NH4Cl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
D2 100 mM NH4Cl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
D3 250 mM NH4Cl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
D4 10 mM NH4Cl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
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D5 100 mM NH4Cl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
D6 250 mM NH4Cl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
D7 10 mM NH4Cl 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
D8 100 mM NH4Cl 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
D9 250 mM NH4Cl 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
D10 10 mM NH4Cl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
D11 100 mM NH4Cl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
D12 250 mM NH4Cl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
E1 10 mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
E2 100 mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
E3 250 mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
E4 10 mM MgCl2 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
E5 100 mM MgCl2 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
E6 250 mM MgCl2 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
E7 10 mM MgCl2 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
E8 100 mM MgCl2 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
E9 250 mM MgCl2 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
E10 10 mM MgCl2 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
E11 100 mM MgCl2 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
E12 250 mM MgCl2 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
F1 10 mM CaCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
F2 100 mM CaCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
F3 250 mM CaCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
F4 10 mM CaCl2 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
F5 100 mM CaCl2 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
F6 250 mM CaCl2 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
F7 10 mM CaCl2 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
F8 100 mM CaCl2 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
F9 250 mM CaCl2 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
F10 10 mM CaCl2 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
F11 100 mM CaCl2 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
F12 250 mM CaCl2 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
G1 10 mM SrCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
G2 100 mM SrCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
G3 250 mM SrCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
G4 10 mM SrCl2 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
G5 100 mM SrCl2 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
G6 250 mM SrCl2 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
G7 10 mM SrCl2 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
G8 100 mM SrCl2 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
G9 250 mM SrCl2 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
G10 10 mM SrCl2 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
G11 100 mM SrCl2 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
G12 250 mM SrCl2 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
H1 10 mM BaCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
H2 100 mM BaCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
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H3 250 mM BaCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
H4 10 mM BaCl2 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
H5 100 mM BaCl2 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
H6 250 mM BaCl2 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 7.0)
H7 10 mM BaCl2 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
H8 100 mM BaCl2 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
H9 250 mM BaCl2 40% MPD 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
H10 10 mM BaCl2 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
H11 100 mM BaCl2 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0)
H12 250 mM BaCl2 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 7.0) a Conditions highlighted in blue were found to enhance crystal growth.
Table II Crystal optimization matrix
Salt Precipitant Polyamine Buffer
1 10 mM LiCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 6.0/7.0)
2 50 mM LiCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 6.0/7.0)
3 100 mM LiCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 6.0/7.0)
4 150 mM LiCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 6.0/7.0)
5 200 mM LiCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 6.0/7.0)
6 250 mM LiCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 6.0/7.0)
7 300 mM LiCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 6.0/7.0)
8 50 mM KCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
9 100 mM KCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
10 150 mM KCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
11 200 mM KCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
12 250 mM KCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
13 300 mM KCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
14 50 mM KCl + 5mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
15 100 mM KCl + 5mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
16 150 mM KCl + 5mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
17 200 mM KCl + 5mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
18 250 mM KCl + 5mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
19 50 mM KCl + 10mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
20 100 mM KCl + 10mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
21 150 mM KCl + 10mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
22 200 mM KCl + 10mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
23 250 mM KCl + 10mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
24 50 mM KCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 6.0/7.0)
25 100 mM KCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 6.0/7.0)
26 150 mM KCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 6.0/7.0)
27 200 mM KCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 6.0/7.0)
28 250 mM KCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 6.0/7.0)
29 300 mM KCl 20% PEG3350 1 mM Spermine 50 mM Na Cacodylate (pH 6.0/7.0)
30 50 mM KCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.0/5.5/6.0/7.0/7.5)
31 100 mM KCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.0/5.5/6.0/7.0/7.5)
32 150 mM KCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.0/5.5/6.0/7.0/7.5)
33 200 mM KCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.0/5.5/6.0/7.0/7.5)
34 250 mM KCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
35 300 mM KCl 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
36 50 mM KCl + 5mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
37 100 mM KCl + 5mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
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38 150 mM KCl + 5mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
39 200 mM KCl + 5mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
40 250 mM KCl + 5mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
41 50 mM KCl + 10mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
42 100 mM KCl + 10mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
43 150 mM KCl + 10mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
44 200 mM KCl + 10mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
45 250 mM KCl + 10mM MgCl2 40% MPD 1 mM Spermine 50 mM Na Cacodylate (pH 5.5/6.0/7.0/7.5)
46 50 mM LiCl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 6.0/7.0)
47 100 mM LiCl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 6.0/7.0)
48 150 mM LiCl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 6.0/7.0)
49 200 mM LiCl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 6.0/7.0)
50 250 mM LiCl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 6.0/7.0)
51 300 mM LiCl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 6.0/7.0)
52 100 mM NH4Cl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 6.0/7.0)
53 150 mM NH4Cl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 6.0/7.0)
54 200 mM NH4Cl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 6.0/7.0)
55 250 mM NH4Cl 20% PEG3350 1 mM CoHex 50 mM Na Cacodylate (pH 6.0/7.0)
a Conditions highlighted in blue were found to result in the best shaped crystals suitable for X-ray analysis.
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תקציר מחקר
י תכונות אנטיבקטריאליות פוטנטיות אמינוגליקוזידים מהווים משפחה של חומרי טבע בעל
אתר הפעולה . המשמשים בקליניקה כאנטיביוטיקות רחבות טווח לטיפול במגוון רחב של מחלות זיהומיות
הקטנה של המצוי בתת היחידה ( A-site-ה)של אמינוגליקוזידים בתא החיידקי הינו האתר המקודד
המובילות אל הריבוזום את ( tRNA)א "באתר זה מתרחשת הסלקציה למולקולות הטרנספר רנ. הריבוזום
כתוצאה מקישור אמינוגליקוזידים לריבוזום . חומצות האמינו המשמשות כאבני בניין לחלבון המתהווה
דבר שמוביל , נפגע מנגנון הסלקציה הריבוזומאלי ונכנסות שגיאות ראנדומליות בתהליך התרגום
חלבונים פגומים אלה גורמים בסופו . להצטברות של חלבונים שגויים ולא פונקציונאליים בתא החיידקי
מנגנון הפעולה של אמינוגליקוזידים בחיידקים נלמד כבר שנים רבות . של למותו של התא החיידקי
וזידים בריבוזום החיידקי כי אתר הקישור של אמינוגליק, למעשה ידוע. ובספרות קיים ידע רב אודותיו
מהווה מעין מתג מולקולארי המחליט על המשך או הפסקת תהליך התרגום לפי התאמת מולקולת
מתג זה מורכב משני שיירי . המקודד ליצירת החלבון( mRNA)א השליח "א לתבנית הרנ"הטרנספר רנ
. 1493-ו 1492אדנין שמורים מאוד אבולוציות הממוספרים
שני העשורים האחרונים הראו כי לאמינוגליקוזידים יש גם מספר אתרי מחקרים שבוצעו ב
מחקרים אלה הראו כי למעשה . בינהם גם ריבוזומים שמקורם ביצורים אאוקריוטים, קישור נוספים בטבע
לחלק מהנגזרות המשוייכות למשפחה יש גם יכולות תרפיוטיות נוספות כגון טיפול במחלות גנטיות
ק וטיפול במחלות זיהומיות שנגרמות כתוצאה מטפילים אאוקריוטים דוגמאת שמקורן במטוציית פס
למרות התוצאות הניסיוניות המבטיחות שהתקבלו (. אמבה טפילית)וגיארדיה ( שושנת יריחו)לישמניה
כמו גם המספר ההולך וגדל של מחקרים , בעבור שימוש באמינוגליקוזידים למטרות תרפיוטיות אלה
מנגנוני הפעולה של , ימוש באמינוגליקוזידים לטיפול במחלות אאוקריוטיות שונותקליניים הבוחנים ש
השימוש באמינוגליקוזים בקליניקה הוא יחסית , בנוסף. אמינוגליקוזידים באאוקריוטים עדיין אינם ידועים
. מוגבל בעיקר בעקבות תופעות הלוואי הקשות ורעילותם למשתמשים
רות סינטטיות של אמינוגליקוזידים לטיפול במחלות גנטיות בשנים האחרונות הוצעו מספר נגז
נגזרות אלה הן בעלות פעילות משופרת ורעילות מופחתת ביחס . י טפילים"ובמחלות זיהומיות הנגרמות ע
נגזרות אלה הינן גם סלקטיביות מאוד כלפי הריבוזום האאוקריוטי ובניגוד . לנגזרות הטבעיות הקיימות
מרבית הנגזרות הללו סונתזו במעבדת המחקר . כולות לשמש גם כאנטיביוטיקותלנגזרות הטבעיות לא י
לאור , עם זאת. שלנו והתוצאות הפרלימינריות שיש ברשותינו בעבור פעילותן ורעילותן הן מבטיחות
העובדה כי הידע הקיים ברשותינו בנוגע למנגנוני הפעילות של חומרים אלה במערכות אאוקריוטיות הוא
ד נוצר צורך לבצע מחקר שיתרום להבנה מעמיקה של מנגנוני הפעולה והאינטראקציות של מוגבל מאו
.אמינוגליקוזידים אלה עם אתרי המטרה האאוקריוטיים שלהם ברמה המולקולארית
י "במחקר שבוצע פענחנו את מנגנונים הפעולה של אמינוגליקוזידם במערכות אאוקריוטיות ע
הטכניקה . פיתרון המבנים התלת מיימדיים של אמינוגליקוזידים שונים קשורים לאתר המטרה שלהם
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כאשר במהלך המחקר גובשו , העיקרית בה השתמשנו לצורך המחקר היתה קריסטלוגרפיה של קרני איקס
עם ( חלקם אגוניסטים לאתר המטרה וחלקם אנטגוניסטים)קסים של אמינוגליקוזידים שונים קומפל
כאשר אתר המטרה הנחקר היה האתר . החלקים הרלוונטיים מריבוזומים של טפילים וריבוזומים אנושיים
במקביל . אנגסטרם 4.1-0.3המבנים הגבישיים נפתרו ברזולוציות אטומיות של . המקודד האאוקריוטי
ניסויים המבנים בוצעו גם מספר ניסויים ביוכימיים בהם נמדדו הפעילויות של אמינוגליקוזידים שונים ל
שילוב זה של מחקר מבני זה עם מחקר ביוכימי תרם לרכישת . ותאים אנושיים( לישמניה)על תאי טפילים
קישור שונה לאתר ידע רב בתחום והראה כי לאמינוגליקוזידים בעלי פעילות ביולוגית שונה יש תבנית
. הם משרים קונפורמציה שונה בעת קישורם, המטרה שלהם וכמו גם
הקרוי , נפתר בפעם הראשונה מבנה של קומפלקס של אמינוגליקוזיד, במסגרת עבודת המחקר
מבנה זה נפתר ברזולוציה של . עם אתר הפעולה שלו בטפיל אאוקריוטי הקרוי לישמניה( G418)נטיצין 'ג
ולמעשה הראה כי מנגנון הפעולה של אמינוגליקוזידים בלישמניה דומה למנגנון הפעולה אנגסטרם 6.2
מושרה בו , במבנה ניכר כי בעת קישור מולקולת האמינוגליקוזיד לאתר המטרה. שלהם בחיידקיים
קונפורמציה זו . קונפורמציה בה שני שיירי האדנין השמורים פונים אל מחוץ לסליל אליו הם משוייכים
א בעלת התאמה מלאה של כל שלושת "מושרית בריבוזום בעת קישור מולקולת טרנספר רנ כ"בד
קודון שעל גביה לשלושת הנוקליאוטידים המשוייכים לרצף הקודון המצוי -הנוקליאוטידים ברצף האנטי
קונפורמציה דומה מתקבלת גם בעת קישור אמינוגליקוזידים לריבוזומים . א השליח"ג מולקולת הרנ"ע
מחקרים מבניים . זאת למרות השינויים הרצפיים שבין אתרי הקישור בחיידק ובלישמניה, קייםחייד
וביוכימיים שבוצעו בחיידקים הראו כי קונפורמציה זו היא שתורמת להכנסת השגיאות הרנדומאליות
א באתר הקישור גם אם רצף האנטי"בתהליך התרגום בחיידקים תוך שהיא מייצבת מולקולות טרנספר רנ
. קודון שלהן אינו תואם לרצף הקודון
( HEK293)התוצאות המבניות שהתקבלו לוו גם בבדיקות עמידות של תרביות תאים אנושיים
וטפילי לישמניה לאמינוגליקוזידים שונים ובמסגרתם נמצא כי לאמינוגליקוזידים בעלי שייר של
יותר לשמש כחומרים תרפיוטיים יש יכולת טובה , הממוקמת בטבעת הראשונה, 2'הידרוקסיל בעמדה
י המבנים שנפתרו שהראו בפעם "תוצאות אלה נתמכו ע. לטיפול במחלות גנטיות ובמחלות טפיליות
הראשונה מדוע נגזרות אלה הינן בעלות פעילות משופרת ביחס לנגזרות המותמרות באופן שונה בעמדה
בעלות פעילות אנטילישמנייטית טובה הן הניסויים הביוכימיים שבוצעו הראו כי נגזרות שהן, בנוסף. זו
הדימיון הרב שבין אתר הקישור . י מוטציית פסק"גם יעילות יותר לטיפול במחלות גנטיות הנגרמות ע
מצביעות על כך כי מנגנון G418האנושי לזה של הטפילים כמו גם התוצאות המבניות שהתקבלו בעבור
יות המבוססות על קודוני פסק גם הוא דומה למנגנון הפעולה של אמינוגליקוזידים בתיקון מחלות גנט
. הכנסת השגיאות בחיידקים ובטפילים אאוקריוטיים
תוצאה מעניינת נוספת שהתקבלה במחקר היא שאמינוגליקוזידים שאינם משרים פעילות תיקון
, אאוקריוטים גנטי באדם או גורמים להכנסת שגיאות ראנדומאליות בתהליך התרגום בחיידקים וביצורים
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אך ידועים כמעכבים חזקים של תהליך התרגום בכל מערכות החיים השרו קונפורמציה שונה לחלוטין
תוצאה זו למעשה מהווה הוכחה עקיפה למנגנוני הפעילות של . באתר הקישור בעת קישורם
ישור היא מכיוון שהיא מעידה שהקונפורמציה המושרית באתר הק, אמינוגליקוזידים בכל מערכות החיים
. זו שרלוונטית לצורך הפעילות הביולוגית ולאו דווקא עצם הקישור או חוזקו
, בנוסף להבנת המנגנונים המולקולאריים של פעילות אמינוגליקוזידים במערכות אאוקריוטיות
התוצאות הניסיוניות הביאו להבנה מעמיקה של תבניות הקישור של אמינוגליקוזידים שונים באתר
החשיבות של קבוצות פונקציונאליות בעמדות שונות לקיום הפעילות ולצורך הסלקטיביות הקישור ושל
אספקטים אלה של המחקר חשובים מאוד לפיתוח מושכל של נגזרות . של אמינוגליקוזידים למערכות הללו
.עתידיות של אמינוגליקוזידים בעלי פעילות משופרת ביחס לנגזרות האם
פותחה שיטה אימונית , יוכימיים שבוצעו במסגרת מחקר זהבנוסף למחקרים המבניים והב
י יצירה של נוגדנים "השיטה פותחה ע. לקביעה וכימות של אמינוגליקוזידים בדוגמאות ביולוגיות
פוליקלונאליים שהיו מסוגלים לזהות באפיניות גבוהה אמינוגליקוזידים סינטטיים שפותחו לצורך טיפול
שהופיע בכלל הנגזרות , י הזרקת הפטן גנרי"הנוגדנים יוצרו ע. ותבמחלות גנטיות ובמחלות טפילי
הנוגדנים שבודדו מהסרום של הארנבות שומשו ליצירת מבחן . לארנבות, הסנטטיות וצומד לחלבון נשא
היעילות והחזרתיות של השיטה הודגמה במספר מטריצות ביולוגיות . באריות 62דיאגנוסטי בצלחת של
שיטות . יושם בהצלחה על דגימות דם שנלקחו מעכברים שטופלו באמינוגליקוזידיםשונות וכמו גם המבחן
מכיוון שהן , אנליטיות מסוג הינן בעלות חשיבות רבה מאוד בפיתוח עתידי נוסף של הנגזרות התרפיוטיות
, בנוסף. מאפשרות ניטור יעיל ומהיר של הנגזרות בניסויים המבוצעים לבדיקת הנגזרות על בעלי חיים
לים שפותחו ניתן להשתמש גם להערכת אספקטים נוספים של פעילות הנגזרות כדוגמאת הערכת בכ
אספקטים אלה גם הם חשובים לפיתוח . חדירותן לתאים אנימאליים ומעקב אחרי פיזורם בתאים וברקמות
.ושיפור נגזרות עתידיות
. ערכות אאוקריוטיותבמחקר שבוצעו נחקרו מנגנונים הפעולה של אמינוגליקוזידים במ, לסיכום
תוצאות המחקר תרמו להבנת המרכיבים המולקולאריים הקשורים בסלקטיביות של אמינוגליקוזידים
מסויימים למערכות אלה ופתחו אפיקים חדשים ביכולת שלנו לפתח באופן מושכל נגזרות חדשות
יטה לניטור כמו גם פיתוח הש, הניסויים הביוכמיים שבוצעו במקביל למחקר המבני. ומשופרות
אמינוגליקוזידים ממטריצות ביולוגיות שונות תרמו רבות להבנה שלנו לגבי מנגנונים הפעולה של
תובנות אלה יוכלו לשמש בעתיד למחקר מקיף יותר ולפיתוח יעיל יותר . אמינוגליקוזידים באאוקריוטים
.של נגזרות חדשות למטרות תרפיוטיות אלה
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נעם אדיר בפקולטה לכימיה' טימור באזוב ופרופ' פרופהמחקר בוצע בהנחיית
ברצוני להודות לטכניון ולמר סימור שוליך על התמיכה הכספית הנדיבה בהשתלמותי
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הפעולה של אמינוגליקוזידים חקר מנגנוני
ככלי לפיתוח תרופות חדשות באאוקריוטים
חיבור על עבודת גמר
התואר לקבלת הדרישות של חלקי מילוי לשם
לפילוסופיה דוקטור
עמי-מורן שלו בן
לישראל טכנולוגי מכון - הטכניון לסנט הוגש
6340ג חיפה יוני "תמוז תשע
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הפעולה של אמינוגליקוזידים חקר מנגנוני
ככלי לפיתוח תרופות חדשות באאוקריוטים
עמי-מורן שלו בן
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![NOAM YEFET, arXiv:1910.07517v4 [cs.LG] 27 May 2020 · 2020. 5. 28. · NOAM YEFET, Technion, Israel URI ALON, Technion, Israel ERAN YAHAV, Technion, Israel Neural models of code have](https://img.pdfslide.us/doc/110x75/60b9bfd293a6b42b89172c79/noam-yefet-arxiv191007517v4-cslg-27-may-2020-2020-5-28-noam-yefet-technion.jpg)
