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Chemical Approaches to theDisruption of Telomerase Function
Chemical Approaches to theDisruption of Telomerase Function
Joseph StringerBlackwell Group 1.25.07
2
Cancer
- 1,444,000 predicted new cases diagnosed in 2007 (U.S.)
- 559,650 expected deaths from cancer in 2007 (U.S)
- 2nd leading cause of death (U.S.)- $206 billion cancer costs in 2006 (U.S)
- Emotional aspect
www.cancer.org
3
Traditional Cancer TreatmentsRadiotherapy – damaging DNA by ionization
not selective/highly toxic
Surgery – removal of malignant tumordifficult to
remove/invasive
Chemotherapy – use of drugsside effects
Telomerase inhibitors – selective/minimal side effects
4
Biology Basics
Human body
Systems
Organs
Tissue Cells
Nucleus
Chromosomes
DNA
5
DNA Basics
5'
3'
3'
5'
6
End Replication Problem
3'
3'
5'
5'
5'
3'
5'
3'
replication
replication
replication
Cell death
Critically short DNA
3'5'
5'
3'
5'3'
5'
3'
7
Human Telomere
- Long telomeres have many protective proteins- Critically short telomeres have few protective
proteins- Critically short telomeres are vulnerable
www.cancer.org
8
Human Telomere
……………………TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG
……………………AATCCCAATCCCAATCCC (5,000-20,000 bases) (100-400 bases)
Double-stranded Single-stranded
Rest of DNA
Telomere region
-Telomere DNA does NOT code for any genetic information
9
Cell Division
Cancer cell – “immortal”
Shay, J.W., et al. Nature Reviews Drug Discovery 2006, online.
The telomerase enzyme maintainstelomere length in cancer cells,preventing cell death
Normal cell – cell death
10
Telomerase Enzyme
Shay, J.W., et al. Nature Reviews Drug Discovery 2006, online.
Active in ~85% of cancer cells
Absent/undetectable in normal, healthy cells
Telomerase active
Telomerase NOT active
Normal cell – cell death Cancer cell – “immortal”
11
Telomerase Timeline
Telomerase discovered(Blackburn/Greider)
Telomerase activity in cancercells, but not healthy cells(Kim)
Telomere ligandInhibits telomerase(Hurley)
Telomerase doesNOT cause cancer(Wright/Shay)
Crystal structureof human telomere(Neidle)
Wright, W., Shay, J., Nature Reviews Drug Discovery 2006, online.
12
Targeting Telomerase Activity
Inhibit telomeraseassembly proteins
Inhibit telomeraseassembly proteins Telomerase enzyme
-RNAi-RT inhibitors (HIV)-Artificial peptides
Telomerase enzyme-RNAi-RT inhibitors (HIV)-Artificial peptides
RNA template-Peptide nucleic acid (PNA)-Antagonist oligonucleotides
RNA template-Peptide nucleic acid (PNA)-Antagonist oligonucleotides
Telomere-Stabilizing ligands
Telomere-Stabilizing ligands
Gellert, G.C., et al. Drug Discovery Today 2005, 2, 159-164.
13
Guanine - Quadruplex
Zahler, A.M., et al. Nature 1991, 350, 718-719.Gabelica, V., et al. J. Am. Chem. Soc. 2006, 128, 2641-2648
Highly stable G-quadruplex (G4)can inhibit telomerase activity
……TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG……AATCCCAAT
14
G4 Inhibitors -Proposed Mechanism
5'
3'
+ G4 Ligand
…………TTAGGGTTAGGGTTA…………AATCCCAATCCC
…………TTAGGGTTAGGGTTAGGGTTAGGG…………AATCCCAATCCC
(TTAGGG)n
Inhibition ofelongation …cell dies
Telomere elongation… cell lives
Telomerase
15
G4 Selectivity
- Structural diversity provides basis for selective recognition between duplex DNA vs. G4 DNA
- π stacking potential on guanine faces
vs.
duplex DNA G4 DNA
Neidle, S., Read, M.A., Biopolymers 2001, 56, 195-208.Baker, E.S., et al. J. Am. Chem. Soc. 2006, 128, 2641-2648.
16
G4 Ligand Design
- DNA intercalators are toxic- Characterized by large, flat aromatic core,
possibly protonated in center- Need to design ligands selective for G4
DNA
Common DNA intercalators
Chan, A., et al. J. Med. Chem. 2005, 48, 7315-7321.
Cryptolepine Proflavine Ethidium bromide
DNA Intercalated DNA
17
Classes of G4 Ligands
Polycycles Macrocycles
18
Acridine Derivative
- Synthesized in 2001 based on parent acridine intercalator- - 45:1 selectivity for G4 DNA vs. duplex DNA- Phase I/II clinical trial (Antisoma)
Read, M., et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4844-4849.
EC50 115 nM
19
BRACO19 Synthesis
Read, M., et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4844-4849.
20
BRACO19
Read, M., et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4844-4849.
G4 DNA
21
Quinoline Derivatives
Guyen, B., et al. Org. Biomol. Chem. 2004, 2, 981-988.
ΔTm G4 ΔTm dsDNA
quinoline der.
13.0°C 0.0°C
BRACO19 27.5°C -
EC50 ~ 6.3µM
22
Quinoline Synthesis
Guyen, B., et al. Org. Biomol. Chem. 2004, 2, 981-988.
23
G4 Crystal Structure
Parkinson, G.N., Lee, M.P.H., Neidle, S., Nature 2002, 417, 876-880.
Axial view
Side view
=
π stackingpartial (+) charge
Interaction with (-) chargedphosphate backbone
24
“Clicked” Triazoles
- π stacking with guanine faces
Moorhouse, A.D., et al. J. Am. Chem. Soc. 2006, 128, 15972-15973.
ΔTm G4
ΔTm dsDNA
triazole 18.7°C 0.0°C
BRACO19 27.5°C -
25
“Clicked” Triazoles
- “Click chemistry”, highly flexible- Selective for G4 DNA vs. duplex DNA- Generation of π stacking motif
Moorhouse, A.D., et al. J. Am. Chem. Soc. 2006, 128, 15972-15973.
26
Classes of G4 Ligands
Polycycles Macrocycles
27
Telomestatin
- First isolated in 2001 from Streptomyces anulatus
- - Total synthesis
finished in 2006, 21 steps, <1% overall yield
- First natural product shown to bind selectively to G4 DNA
Shin-ya, K., et al. J. Am. Chem. Soc. 2001, 123, 1262-1263.Doi, T., et al. Org. Lett. 2006, 8, 4165-4167.
EC50 5 nM
28
Cyclic Oxazoles
- Minimal duplex DNA stabilization
- 8 steps, ~14% overall yield
Minhas, G.S., et al. Bioorg. Med. Chem. Lett. 2006, 16, 3891-3895.Jantos, K., et al. J. Am. Chem. Soc. 2006, 128, 13662-13663.
R stereochem.
ΔTm G4 ΔTm dsDNA
(CH2)4NH2 R,R,R 6.4°C 0.0°C
telomestatin
- 27.4°C 0.0°C
29
Heterocycle-Peptides
Schouten, J.A., et al. J. Am. Chem. Soc. 2003, 125, 5594-5595.Green, J.J., et al. J. Am. Chem. Soc. 2006, 128, 9809-9812.
- Peptides introduce versatility
- Additional interaction with G4 grooves/phosphate groups
30
Heterocycle-Peptides
>50:1 selectivity G4 DNA vs. duplex DNA
Schouten, J.A., et al. J. Am. Chem. Soc. 2003, 125, 5594-5595.Green, J.J., et al. J. Am. Chem. Soc. 2006, 128, 9809-9812.
31
Metal Complexes
- Ni(II) forces planarity, resulting in π stacking
- Piperidine interaction with phosphate backbone
Reed, J.E., et al. J. Am. Chem. Soc. 2006, 128, 5992-5993.
32
Metal Complexes
- Generation of aromatic motif
- >50:1 G4 DNA vs. duplex DNA
-
Reed, J.E., et al. J. Am. Chem. Soc. 2006, 128, 5992-5993.
ΔTm G4 ΔTm dsDNA
Ni(II) complex
32.8°C 0.0°C
telomestatin
27.4°C 0.0°C EC50 120 nM
33
Metal Complexes
34
G4 Ligand Issues
1. G4 structures can be polymorphic in vivo
Gabelica, V., et al. J. Am. Chem. Soc. 2007, 129, 895-904.
35
G4 Ligand Issues
2. Do G-quadruplex structures exist elsewhere in DNA ?
- Difficult to predict based on DNA sequence- A few have been found in promoter regions
of oncogenes – dual mechanism?
YES
Siddiqui-Jain, A., et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11593-11598.
36
Future Directions of G4 Ligands
Need deeper understanding of G4-ligandinteractions
Possible use as a gene suppressor ?
Metal complexes ?
37
Telomerase Inhibitors: Therapeutic Future
- Need more in vivo testing- Used in combination with traditional
therapy- What about other ~15% of cancer cells ?- Cure for cancer ?
“For every complex problem there is asolution that is simple, neat, and wrong”
- H.L. Mencken
38
AcknowledgementsProf. Helen E. Blackwell
Team Blackwell*Ben Gorske Blake Carlson *Beth Mascato*Grant Geske Aleeza Roth *Rick
McDonald*Jenny O’Neill Dr. Matt Bowman Prof. John
Berry*Qi Lin Wa Neng Thao*Sarah Fowler Margaret Wong*Daniel Fritz *Lingyin Li*Brent Bastian*Margie Mattmann*Christie McInnis*Reto Frei* Practice talk attendees
...I get by with a little helpfrom my friends
39
Supplemental Slides
40
Telomere Capping
- Dynamic equilibrium between G4 and non G4 state
- Telomere must be in linear form for telomerase activity
41
FRET Analysis (ref 26)
FRET analysisFluorescence Resonance
Energy Transfer
- Correlates temperature change with a stabilized/unstabilized DNA structure
42
TRAP Assay (ref 26)
Telomere Repeat Amplification Protocol
- Used for quantitative and qualitative telomerase inhibition
43
• www.txccc.org• www.childrenscancernetwork.org
44
DNA Replication
- Requires initial RNA primer
- Replication only proceeds in 5→3 direction
- After removal of terminal RNA primer, gap is left
- DNA cannot add to the 5' end (wrong direction)
http://www.senescence.info/telomeres.html
Primer removal
DNA base pairaddition
45
Telomere Capping
- Cell can replicate with a capped state, until telomere gets short, then it will uncap and telomerase will add length
- When a telomere is very short, it cannot be capped efficiently, and the single stranded G-rich DNA can form G-quadruplexes, thus making a target for G4 ligands
- Cancer cells with short telomeres must “expose” their loose end (become linear) to add on and keep living
46
G4 Ligand Issues
1. Selectivity G-quadruplex vs. duplex DNA
Minimize toxicity
vs.
47
G4 Ligand Char.
- π stacking ability of central core
- Positively charged substituents to interact with negatively charged phosphate backbone
Partial positive charge in center
Neidle, S., Lee, M.P.H., Parkinson, G. N. Nature. 2002, 417, 876-880.
48
Telomere Structure-Guanine (G)-Tetrad
- Higher order structure of single stranded, Guanine rich DNA
- Guanines are co-planar
- Occurs in telomeres when left “uncapped” by protective proteins
49
G4 Inhibitors -Proposed Mechanism
- Stabilization of G-quadruplex leads to telomerase inhibition
Mergny, J-L., et al. Nature Medicine. 1998, 4, 1366-1367.
50
Human Telomere
- Protects against gene deletion
- Usually capped by protective proteins
- When telomeres become critically short, they become uncapped and are vulnerable
51
Telomestatin Binding
Hurley, L.H., et al. J. Am. Chem. Soc. 2002, 124, 4844-4849.
- External binding
>70 fold selectivity G4 vs. duplex DNA