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'
3' 5'
replication

5' 3'
3' 5'
replication

3' 5'
5' 3'

replication

5' 3'
Cell death
3' 5'
Critically short DNA 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) (100-400 bases)
Double-stranded Single-stranded
Rest of DNA
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 Telomerase does
(Hurley) 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 Telomerase enzyme
assembly proteins -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'
(TTAGGG)n
5'
…………TTAGGGTTAGGGTTAGGGTTAGGG
Telomere elongation…
…………AATCCCAATCCC cell lives

+ G4 Ligand

…………TTAGGGTTAGGGTTA Inhibition of
…………AATCCCAATCCC 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

-
- 45:1 selectivity for G4 DNA vs. duplex DNA
EC50 115 nM
- 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 - Generation of aromatic

motif
Ni(II) complex 32.8°C 0.0°C - >50:1 G4 DNA vs.
duplex DNA
telomestatin 27.4°C 0.0°C
- EC50 120 nM

Reed, J.E., et al. J. Am. Chem. Soc. 2006, 128, 5992-5993.

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 *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 ...I get by with a little help
*Christie McInnis from my friends
*Reto Frei
* Practice talk attendees

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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
- After removal of terminal
RNA primer, gap is left
- DNA cannot add to the 5'
end (wrong direction)

http://www.senescence.info/telomeres.html
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 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

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

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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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