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Chemical Approaches to the
Disruption of Telomerase Function
Chemical Approaches to the
Disruption of Telomerase Function
Joseph Stringer
Blackwell Group
1.25.07
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
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Traditional Cancer
Treatments
Radiotherapy – damaging DNA by ionization
not selective/highly toxic
Surgery – removal of malignant tumor
difficult to remove/invasive
Chemotherapy – use of drugs
side effects
Telomerase inhibitors – selective/minimal side effects
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Biology Basics
Human body
Systems
Organs
Tissue
Cells
Nucleus
Chromosomes
DNA
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DNA Basics
5'
3'
3'
5'
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End Replication
Problem
5'
3'
replication
5'
3'
5'
3'
3'
5'
replication
5'
3'
5'
3'
replication
5'
3'
3'
5'
Critically short DNA
Cell death
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Human Telomere
- Long telomeres have many protective proteins
- Critically short telomeres have few protective proteins
- Critically short telomeres are vulnerable
www.cancer.org
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Human Telomere
……………………TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG
……………………AATCCCAATCCCAATCCC
(5,000-20,000 bases)
Double-stranded
Rest of DNA
(100-400 bases)
Single-stranded
Telomere region
-Telomere DNA does NOT code for any
genetic information
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Cell Division
The telomerase enzyme maintains
telomere length in cancer cells,
preventing cell death
Normal cell – cell death
Cancer cell – “immortal”
Shay, J.W., et al. Nature Reviews Drug Discovery 2006, online.
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Telomerase Enzyme
Active in ~85% of cancer cells
Absent/undetectable in
normal, healthy cells
Telomerase NOT active
Telomerase active
Normal cell – cell death
Cancer cell – “immortal”
Shay, J.W., et al. Nature Reviews Drug Discovery 2006, online.
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Telomerase Timeline
Telomere ligand
Inhibits telomerase
(Hurley)
Telomerase does
NOT cause cancer
(Wright/Shay)
Telomerase discovered
(Blackburn/Greider)
Telomerase activity in cancer
cells, but not healthy cells
(Kim)
Crystal structure
of human telomere
(Neidle)
Wright, W., Shay, J., Nature Reviews Drug Discovery 2006, online.
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Targeting Telomerase
Activity
Inhibit telomerase
assembly proteins
Telomerase enzyme
-RNAi
-RT inhibitors (HIV)
-Artificial peptides
RNA template
-Peptide nucleic acid (PNA)
-Antagonist oligonucleotides
Telomere
-Stabilizing ligands
Gellert, G.C., et al. Drug Discovery Today 2005, 2, 159-164.
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Guanine - Quadruplex
……TTAGGGTTAGGGTTAGGGTTAGGGTTAGGG
……AATCCCAAT
Highly stable G-quadruplex (G4)
can inhibit telomerase activity
Zahler, A.M., et al. Nature 1991, 350, 718-719.
Gabelica, V., et al. J. Am. Chem. Soc. 2006, 128, 2641-2648
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G4 Inhibitors Proposed Mechanism
Telomerase
3'
5'
…………TTAGGGTTAGGGTTAGGGTTAGGG
…………AATCCCAATCCC
(TTAGGG)n
Telomere elongation…
cell lives
+ G4 Ligand
…………TTAGGGTTAGGGTTA
…………AATCCCAATCCC
Inhibition of
elongation …
cell dies
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G4 Selectivity
vs.
duplex DNA
G4 DNA
- Structural diversity provides basis for selective
recognition between duplex DNA vs. G4 DNA
- π stacking potential on guanine faces
Neidle, S., Read, M.A., Biopolymers 2001, 56, 195-208.
Baker, E.S., et al. J. Am. Chem. Soc. 2006, 128, 2641-2648.
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G4 Ligand Design
Cryptolepine
Proflavine
Ethidium bromide
Common DNA intercalators
DNA
Intercalated DNA
- DNA intercalators are toxic
- Characterized by large, flat aromatic core,
possibly protonated in center
- Need to design ligands selective for G4 DNA
Chan, A., et al. J. Med. Chem. 2005, 48, 7315-7321.
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Classes of G4 Ligands
Polycycles
Macrocycles
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Acridine Derivative
- Synthesized in 2001 based on parent acridine
intercalator
- EC50 115 nM
- 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.
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BRACO19 Synthesis
Read, M., et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4844-4849.
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BRACO19
G4 DNA
Read, M., et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4844-4849.
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Quinoline Derivatives
EC50 ~ 6.3µM
ΔTm G4
ΔTm dsDNA
quinoline der.
13.0°C
0.0°C
BRACO19
27.5°C
-
Guyen, B., et al. Org. Biomol. Chem. 2004, 2, 981-988.
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Quinoline Synthesis
Guyen, B., et al. Org. Biomol. Chem. 2004, 2, 981-988.
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G4 Crystal Structure
Side view
=
Axial view
Interaction with (-) charged
phosphate backbone
π stacking
partial (+) charge
Parkinson, G.N., Lee, M.P.H., Neidle, S., Nature 2002, 417, 876-880.
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“Clicked” Triazoles
- π stacking with guanine
faces
ΔTm G4
ΔTm
dsDNA
triazole
18.7°C
0.0°C
BRACO19
27.5°C
-
Moorhouse, A.D., et al. J. Am. Chem. Soc. 2006, 128, 15972-15973.
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“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.
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Classes of G4 Ligands
Polycycles
Macrocycles
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Telomestatin
- First isolated in 2001
from Streptomyces
anulatus
- EC50 5 nM
- 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.
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Cyclic Oxazoles
- Minimal duplex
DNA stabilization
- 8 steps, ~14%
overall yield
R
stereochem.
ΔTm G4
ΔTm dsDNA
(CH2)4NH2
R,R,R
6.4°C
0.0°C
telomestatin
-
27.4°C
0.0°C
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.
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Heterocycle-Peptides
- Peptides introduce versatility
- Additional interaction with G4
grooves/phosphate groups
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.
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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.
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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.
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Metal Complexes
ΔTm G4
ΔTm dsDNA
Ni(II) complex
32.8°C
0.0°C
telomestatin
27.4°C
0.0°C
- Generation of
aromatic motif
- >50:1 G4 DNA vs.
duplex DNA
-
Reed, J.E., et al. J. Am. Chem. Soc. 2006, 128, 5992-5993.
EC50 120 nM
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Metal Complexes
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G4 Ligand Issues
1. G4 structures can be polymorphic in vivo
Gabelica, V., et al. J. Am. Chem. Soc. 2007, 129, 895-904.
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G4 Ligand Issues
2. Do G-quadruplex structures exist elsewhere in
DNA ?
YES
- Difficult to predict based on DNA sequence
- A few have been found in promoter regions of
oncogenes – dual mechanism?
Siddiqui-Jain, A., et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11593-11598.
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Future Directions of
G4 Ligands
Need deeper understanding of G4-ligand
interactions
Metal complexes ?
Possible use as a gene suppressor ?
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Telomerase Inhibitors:
Therapeutic Future
“For every complex problem there is a
solution that is simple, neat, and wrong”
- H.L. Mencken
-
Need more in vivo testing
Used in combination with traditional therapy
What about other ~15% of cancer cells ?
Cure for cancer ?
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Acknowledgements
Prof. Helen E. Blackwell
Team Blackwell
*Ben Gorske
Blake Carlson
*Grant Geske
Aleeza Roth
*Jenny O’Neill
Dr. Matt Bowman
*Qi Lin
*Sarah Fowler
*Daniel Fritz
*Brent Bastian
*Margie Mattmann
*Christie McInnis
*Reto Frei
*Beth Mascato
*Rick McDonald
Prof. John Berry
Wa Neng Thao
Margaret Wong
*Lingyin Li
...I get by with a little help
from my friends
* Practice talk attendees
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Supplemental Slides
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Telomere Capping
- Dynamic equilibrium between G4 and non G4 state
- Telomere must be in linear form for telomerase
activity
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FRET Analysis (ref 26)
FRET analysis
Fluorescence Resonance
Energy Transfer
- Correlates temperature
change with a
stabilized/unstabilized DNA
structure
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TRAP Assay (ref 26)
Telomere Repeat Amplification
Protocol
- Used for quantitative and
qualitative telomerase
inhibition
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• www.txccc.org
• www.childrenscancernetwork.org
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DNA Replication
- Requires initial RNA primer
- Replication only proceeds
in 5→3 direction
Primer removal
DNA base pair
addition
http://www.senescence.info/telomeres.html
- After removal of terminal
RNA primer, gap is left
- DNA cannot add to the 5'
end (wrong direction)
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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
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G4 Ligand Issues
1. Selectivity G-quadruplex vs. duplex DNA
vs.
Minimize toxicity
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G4 Ligand Char.
Partial positive charge in center
- π stacking ability of central core
- Positively charged substituents to interact with negatively charged
phosphate backbone
Neidle, S., Lee, M.P.H., Parkinson, G. N. Nature. 2002, 417, 876-880.
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Telomere StructureGuanine (G)-Tetrad
- Higher order structure of
single stranded, Guanine
rich DNA
- Guanines are co-planar
- Occurs in telomeres when
left “uncapped” by
protective proteins
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G4 Inhibitors Proposed Mechanism
- Stabilization of G-quadruplex leads to telomerase inhibition
Mergny, J-L., et al. Nature Medicine. 1998, 4, 1366-1367.
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Human Telomere
- Protects against gene deletion
- Usually capped by protective proteins
- When telomeres become critically short, they become
uncapped and are vulnerable
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Telomestatin Binding
- External binding
>70 fold selectivity
G4 vs. duplex DNA
Hurley, L.H., et al. J. Am. Chem. Soc. 2002, 124, 4844-4849.
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