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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
2
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
3
Biology Basics
Human body
Systems
Organs
Tissue
Cells
Nucleus
Chromosomes
DNA
4
DNA Basics
5'
3'
3'
5'
5
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
6
Human Telomere
- Long telomeres have many protective proteins
- Critically short telomeres have few protective proteins
- Critically short telomeres are vulnerable
www.cancer.org
7
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
8
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.
9
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.
10
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.
11
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.
12
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
13
G4 Inhibitors Proposed Mechanism
Telomerase
3'
5'
…………TTAGGGTTAGGGTTAGGGTTAGGG
…………AATCCCAATCCC
(TTAGGG)n
Telomere elongation…
cell lives
+ G4 Ligand
…………TTAGGGTTAGGGTTA
…………AATCCCAATCCC
Inhibition of
elongation …
cell dies
14
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.
15
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.
16
Classes of G4 Ligands
Polycycles
Macrocycles
17
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.
18
BRACO19 Synthesis
Read, M., et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4844-4849.
19
BRACO19
G4 DNA
Read, M., et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4844-4849.
20
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.
21
Quinoline Synthesis
Guyen, B., et al. Org. Biomol. Chem. 2004, 2, 981-988.
22
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.
23
“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.
24
“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.
25
Classes of G4 Ligands
Polycycles
Macrocycles
26
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.
27
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.
28
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.
29
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.
30
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.
31
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
32
Metal Complexes
33
G4 Ligand Issues
1. G4 structures can be polymorphic in vivo
Gabelica, V., et al. J. Am. Chem. Soc. 2007, 129, 895-904.
34
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.
35
Future Directions of
G4 Ligands
Need deeper understanding of G4-ligand
interactions
Metal complexes ?
Possible use as a gene suppressor ?
36
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 ?
37
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
38
Supplemental Slides
39
Telomere Capping
- Dynamic equilibrium between G4 and non G4 state
- Telomere must be in linear form for telomerase
activity
40
FRET Analysis (ref 26)
FRET analysis
Fluorescence Resonance
Energy Transfer
- Correlates temperature
change with a
stabilized/unstabilized DNA
structure
41
TRAP Assay (ref 26)
Telomere Repeat Amplification
Protocol
- Used for quantitative and
qualitative telomerase
inhibition
42
• www.txccc.org
• www.childrenscancernetwork.org
43
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)
44
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
45
G4 Ligand Issues
1. Selectivity G-quadruplex vs. duplex DNA
vs.
Minimize toxicity
46
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.
47
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
48
G4 Inhibitors Proposed Mechanism
- Stabilization of G-quadruplex leads to telomerase inhibition
Mergny, J-L., et al. Nature Medicine. 1998, 4, 1366-1367.
49
Human Telomere
- Protects against gene deletion
- Usually capped by protective proteins
- When telomeres become critically short, they become
uncapped and are vulnerable
50
Telomestatin Binding
- External binding
>70 fold selectivity
G4 vs. duplex DNA
Hurley, L.H., et al. J. Am. Chem. Soc. 2002, 124, 4844-4849.
51