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ACKNOWLEDGMENTSACKNOWLEDGMENTS
SCMT is grateful to the Chemistry Department of the University of Reading, Xenova Plc. and the Portuguese Foundation for Science and Technology for funding, as well as the Portuguese Gulbenkian Foundation for travel support. BCG would like to thank the Association of International Cancer Research for their essential funding.
CRYSTALLOGRAPHIC STUDIES OF COMPLEXES OF DNA WITH POTENTIAL ANTI-CANCER DRUGS
S.C.M. Teixeira, J.H. Thorpe, B.C. Gale, C.J. CardinChemistry Department, University of Reading, UK.
d[CGTACG]2
DACA3DACA3
Mg
cavity
CrystallisationConditions(VapourDiffusion,285K)
10 L sitting drops containing sodiumcacodylate buffer pH 6.5, 0.5 mM duplexDNA, 0.25 mM drug, magnesium acetate,cobalt chloride, spermine and MPD.
Data collectionand Processing(1996, Cardinet al.)
= 0.979Å, DESY X31, Hamburg.T=100K. Resolution 13.5-2.35Å. Overallcompleteness 92%. Space group C222: a= 29.507Å, b = 53.738Å, c = 40.061Å.HKL package used (DENZO andSCALEPACK).
REFINEMENTStructure solved by Molecular Replacement with model
from structure as described in [2].Resolution range (Å) 9-2.4R-factor ( % for all data) 23.8Free R-factor ( % for all data) 30.6Last resolution shell (Å) Unique reflections Completeness (%) R-factor (% for all reflections) R-factor (%, reflections with I>2(I)) <I/(I)>
2.55-2.4213
94.730.825.4
6.1Data/Parameter ratio 1971/844Goodness of Fit
2.45Average B-value for DNAresidues (Å2)
34.2
Drug B-values Chromophore & Linker (Å2) Side chain (Å2)
58.760.5
Drug Occupancy Chromophore & Linker (Å2) Side chain (Å2)
1.00.5
Obtained from SHELXL. GooF = {[w(F2
obs – F2calc]
2/(n-p)}1/2, where w isthe weighting scheme, n is the number of reflections and p the number ofparameters.
Orientation/position of the drug (BISDACA) relative to the neighbouring base-pairs and the junction. In green a van der Waals surface of the drug is represented. Different from previously observed bis-intercalation modes (shown bellow; from [1]).
STRUCTURE HIGHLIGHTS
Cobalt ions stabilise the flipped-out bases (see Figures in DACA3).
Novel intercalation mode: although the size of the linker chain should allow for intercalation conforming with the nearest-neighbour exclusion principle, one single drug intercalates between two duplexes.
This work has been submitted for publication (Teixeira et al., 2002).
BBIISSDDAACCAA
Cobalt ion bridging flipped-out based with a
symmetry related cytosine
Penta-hydrated cobalt ion
forming H-bonds to phosphate of disordered 5’
cytosine
Sugar preceding 2001 disordered
Cytosine (base not modelled; see figure for
9AMINO-DACA complex)
DATACOLLECTIONANDPROCESSING(Adams et al.)
In-house facilities (Department ofBiochemistry, The University of Sydney,Australia). = 1.5418Å, T=100K,.Resolution: 50-1.7Å. Space group C222: a =29.137Å, b = 52.584Å , c = 40.480Å. HKLpackage used (DENZO and SCALEPACK).
STRUCTURE HIGHLIGHTS
Disordered Mg ion found in the pseudo-Holliday junction (see Figure in 9AMINO-DACA)
Drug shows alternate conformations. Carboxamide side-chain for one of the drugs is not observed (higher disorder).
Carboxamide side-chain of one of the drug’s conformations (orange) H-bonding to a phosphate oxygen on the DNA backbone. (2Fo-Fc) maps: 1contours are shown in pink, 0.8in blue.
REFINEMENTStructure Solved by Molecular Replacement withmodel from structure as described in [2].Atoms (assymetric unit)
Nucleic AcidHeterogenSolvent
2305218
Resolution range (Å) 30-1.9Completeness(%) 94 (93)I/(I) 11.3 (6.0)Total number of Unique reflections 2631Data (F > 4(F) ) / Parameter 2494/1084R-factor (% for F > 4(F) ) 23.4Free R-factor (% for F > 4(F) ) 30.8Goodness of Fit 3.6R.m.s. deviations for Bond lengths (Å) 0.025Average B values (Å2) Nucleic Acid Chain Drug conformation A (0.48 occupancy) Drug conformation B (0.52 occupancy) Whole structure, Isot. Beq.
35.453.3
57.438.2
DDAACCAA33
REFINEMENT STATISTICSData collected in 1997 by Cardin et al. inSincrotrone Elettra, Trieste. Structure Solved byMolecular Replacement with model fromstructure as described in [2].Atoms (assymetric unit)
Nucleic AcidHeterogenSolvent
2425317
Resolution range (Å) 50-1.9Completeness (%) 92.5 (95.7)Number of Unique reflections 2429Data (F > 4(F) ) / Parameter 2169/1130R-factor (% for F > 4(F) ) 23.3 (31.2)Free R-factor (% for F > 4(F) ) 34.3Goodness of Fit 6.1I/(I) 13.8 (5.3)R.m.s. deviations (Bond lengths, Å) 0.063Average B values (Å2)
Nucleic Acid Chain ANucleic Acid Chain BDrug conf. A (0.31 occupancy)Drug conf. B (0.69 occupancy)Whole structure
36.135.173.549.939.2
(Alternate conformations)
Mg cavity
2001A2001B
(2Fo-Fc) electron density maps
contoured at 1level
99AAMMIINNOO--DDAACCAA
d[CGTACG]2
5Br-9amino-5Br-9amino-DACADACA
Spacer
Intercalator
55BBr-r-99AAMMIINNOO--DDAACCAA
REFINEMENT STATISTICSStructure Solved by Molecular Replacement with model fromstructure as described in [3]. Anisotropic refinement withriding hydrogens added.Atoms (assymetric unit)
Nucleic AcidHeterogenSolvent
1408038
Resolution range (Å) 10-1.1Completeness(%) 94.7 (95.8)I/(I) 27.1 (6.0)Total number of Unique reflections 8308Data (F > 4(F) ) / Parameter 7900/1969R-factor (% for F > 4(F) ) 15.6 (18.4)Free R-factor (% for F > 4(F) ) 20.7Goodness of Fit 2.1R.m.s. deviations for Bond lengths (Å) 0.042Average B values (Å2) Nucleic Acid Chain Spacer Drug (0.5 occupancy) Intercalating Drug : conf. A (0.68 occupancy) Conf. B (0.32 occupancy) Whole structure, Isot. Beq.
16.619.424.920.618.7
Intercalating drug (two alternate conformations)
CRYSTALLISATION AND DATA COLLECTIONCrystallisation
conditions (VapourDiffusion, sittingdrops) T=17°C
11l drop containing Ammonium Acetate,Mg Acetate, Na Cacodilate (pH 6.5), PEG8K and 3:1 Drug/DNA. Yellow ~0.3x0.6mmcrystals grew after 6 months.
Data CollectionDESY, Hamburg,
beamline X11.
= 0.8068Å, T=100K. MARCCD scanner.Data was processed and scaled with the XDSpackage in space group P64: a = 30.186Å, b =30.186Å, c = 39.443Å. Resolution up to 1.1Å.
Spacer drug (4 alternate conformations)
Intercalating drug
STRUCTURE HIGHLIGHTS
Anisotropic refinement to atomic resolution reveals structural details: a disordered thymine and the disordered drugs intercalating in a pseudo-infinite helical stacking.
The carboxamide side-chains show high displacements.
The N1-CD1=OD1 atoms in the carboxamide side-chain show non-planar geometry for both drugs. Although the standard deviations of the CD1-OD1 bond lengths were not small enough to confirm this, it is possible that this is due to steric effects, as has been observed before for other structures (see for example [7]).
REFERENCESREFERENCES
[1][1] Blackburn, G. M. and Gait, M.J. (Editors), (1996). “Nucleic Acids in Chemistry and Biology”, 2nd Edition, Oxford University Press. [2][2] Thorpe, J.H., Hobbs, J.R., Todd, A., Denny, W.A., Charlton, P., Cardin, C.J., (2000). Biochemistry, 39, 15055-15061. [3][3] Todd, A. K., Adams, A., Thorpe, J. H., Denny, W. A., Wakelin, L. P. G., Cardin, C. J. (1999). J.Med. Chem., 42, 536-540. [4][4] Wakelin, L.P.G., Atwell, G.J., Rewcastle, G. W., Denny. W.A. (1987). J. Med.Chem., 30, 855-861. [5][5] Hurley, L.H. (2002). Nature Reviews Cancer, Vol. 2, No.3, 188-200.[6] [6] West, K. L. and Austin, C. A. (1999). Nucleic Acids Res., 27, 984-992. [7][7] Dodson, E. , (1998). Acta Cryst. , D54, 1109-118.
NH+
N NH+O
DACA35-Br-9AMINO
-DACA
STRUCTURE HIGHLIGHTS The drug shows alternate conformations. For one of the conformations the carboxamide side chain is not visible in the electron density maps (as happened with the DACA3-DNA complex). For the other conformation the side chain H-binds to the N7 of a neighbour Guanine base.
2001 Cytosine seems to show less disorder up to the sugar ring of the nucleotide, that has alternate conformations. Although the base cannot be seen in the maps, it should be present in the same cavity as the disordered carboxamide side-chain (also not possible to model into the maps) of one of the drug’s conformations.
Disordered Thymine
RESULTS AND DISCUSSIONRESULTS AND DISCUSSION
The drugs in the studies here described come as the result of many studies on structure/activity relationships as well as DNA-binding kinetics (see for example [4]), through which it has been determined that these compounds bind selectively to CG-rich sequences and the 4-position of the carboxamide chain is optimal to increase cytotoxicity. With the exception of DACA3, all drugs here mentioned have shown cytotoxicity in vivo. BISDACA has shown cytotoxicity against a wide range of tumour cells in culture (Wakelin, L.P.G. and Denny, W.A., unpublished observations) and it seems to form a more stable complex with DNA than the parental monomer. It is thought that the cytotoxicity of these drugs is due to their role in stabilising the transient complex of topoisomerases (I and/or II) with DNA: the so-called cleavable complex. This complex is reversible once the drug is dissociated [5], so the drug residence time is an important factor.
A characteristic of all the structures here shown is the disorder of the drug in the intercalation cavity. The atomic resolution structure of the complex with 5Br-9amino-DACA shows that even with high data quality it is very difficult to fully resolve the drug positions, particularly the carboxamide side-chains. In the structures with DACA3, 9amino-DACA and BISDACA the DNA forms a pseudo-Holliday junction (see 9amino-DACA’s picture). DNA junctions play important roles in the normal physiology of cells during DNA replication, DNA repair, recombination, and viral integration, for example, making them potential targets for the development of novel anti-tumor, antiviral and antibacterial agents. Importantly, it has been demonstrated that human DNA topoisomerase II binds to four-way junction DNA [6], strengthening the proposal that in vivo topoisomerases preferentially interact with DNA crossovers, cruciforms and hairpins.
XENOVA
The pseudo-Holliday junctions observed in the structures obtained are stabilised by the drug. The length of the side chains seems sufficient to allow for many possible interactions with the oligonucleotides, which may be the reason behind the high disorder observed. The structure with BISDACA shows less disorder in the drug cavity despite the high displacements observed. In this case the drug mobility is restricted (both by the linker chain between the two chromophores and by the carboxamide side-chain) to form a structure with a novel intercalation mode. It is likely that less disordered structures are more stable and have higher residence times of the drug in the DNA (as happens with BISDACA). However, the role of kinetics and the drug geometry within the complex with DNA cannot be ignored. The complexity and correlation of the factors that can influence the activity of these compounds require further work to support the design of optimised drugs with high potency and specificity in vivo. For this reason structural studies are currently being done on intercalators and several different oligonucleotide sequences.
d[CGTACG]2
BISDACABISDACA
NH+
NONH+
NH2 9-AMINO-DACA
O
N
NH+
NH+NH+ NH
(CH2)8
NH
O
N
NH+
BISDACA
DNA-targeting agents are being extensively studied for their cytotoxic properties and potential medical applications. The structures here described are binary complexes of DNA with intercalators. These compounds are acridine derivatives containing a fused tricyclic aromatic moiety and a cationic carboxamide side-chain that interacts with the DNA through H-bonds. Although many studies have been and are being done on these compounds, a lot is yet unknown about the mechanisms involved. The studies described here aim at providing a structural basis for the interpretation of the different cytotoxic activities shown by the drugs, as well as at supporting drug design.
d[CGTACG]2
9AMINO-DACA9AMINO-DACA
(pseudo-Holliday junction)
NH+
BrNO
NH+
NH2