Chemical Approaches to theDisruption of Telomerase Function

Chemical Approaches to theDisruption of Telomerase Function

Joseph StringerBlackwell Group 1.25.07

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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 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

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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'

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'

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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) (100-400 bases)

Double-stranded Single-stranded

Rest of DNA

Telomere region

-Telomere DNA does NOT code for any genetic information

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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

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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”

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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.

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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.

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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

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G4 Inhibitors -Proposed Mechanism

5'

3'

+ G4 Ligand

…………TTAGGGTTAGGGTTA…………AATCCCAATCCC

…………TTAGGGTTAGGGTTAGGGTTAGGG…………AATCCCAATCCC

(TTAGGG)n

Inhibition ofelongation …cell dies

Telomere elongation… cell lives

Telomerase

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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.

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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

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Classes of G4 Ligands

Polycycles Macrocycles

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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

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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

Read, M., et al. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4844-4849.

G4 DNA

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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

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Quinoline Synthesis

Guyen, B., et al. Org. Biomol. Chem. 2004, 2, 981-988.

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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

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“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 -

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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

- - 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

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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

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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

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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

- 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

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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 ?

- 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.

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Future Directions of G4 Ligands

Need deeper understanding of G4-ligandinteractions

Possible use as a gene suppressor ?

Metal complexes ?

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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

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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

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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 analysisFluorescence 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

- 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

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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

Minimize toxicity

vs.

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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.

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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

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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

Hurley, L.H., et al. J. Am. Chem. Soc. 2002, 124, 4844-4849.

- External binding

>70 fold selectivity G4 vs. duplex DNA