chemical approaches to the disruption of telomerase function chemical approaches to the disruption...

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