Combined approaches to Drug Discovery

In lecture, we will be focusing at different points on natural products, receptor-based design, and pharmacophore-based design of drugs

In reality, drug development may combine these elements into a synthetic approach

This lecture will illustrate how a combination of approaches led to the development of new anti-cancer therapeutics based around a natural product, the molecule taxol

Taxol

Overview of Talk:

I. Cancer & the Microtubule Cytoskeleton

II. Cellular Target: the Protein Tubulin

III. Structure of Taxol & Mechanism of Action

IV. History & Development of Taxol

V. Resistance & the Future of Taxane-based Therapies

The Search for Anti-Cancer Drugs

Cancer is caused by normal cells that acquire mutations causing them to proliferate and eventually metastasize, spreading throughout the body and causing inevitable death

Small molecules that are selectively toxic to dividing cells have potential as anti-cancer drugs, by killing tumor cells but not most cells of the body

The Microtubule Cytoskeleton

The microtubule cytoskeleton is a highly regulated system affecting:- transport of materials within the cell- progression through cell division (mitosis)

The cytoskeleton is dynamically restructured:

Molecules that block polymerization or stabilize microtubules can stop mitosis, by preventing cytoskeletal reorganization

Tubules are therefore a logical target for anticancer drugs - Stop microtubule disassembly = stop cell division

The Protein Tubulin

Microtubules are composed of the protein tubulin

(1) Tubulin forms dimers, which consist of an and a subunit

(2) Dimers stack together into protofilaments, which are linear strings

(3) Protofilaments bind laterally to form hollow, cylindrical microtubules

Tubule

Downing, 2000

Structure of Tubulin

Tubulin protein exists as two 450 a.a. monomers, and

- Each binds a high-energy GTP molecule

An - dimer then forms (GTP-tubulin)

Dimers polymerize to form long protofilaments

Polymerization causes hydrolysis of the -GTP, which destabilizes the microtubule

- GDP-tubulin wants to relax into a new conformation, which dissociates from the microtubule

GTPtubulin

GDPtubulin

collapse

All that holds the microtubule together is a fast-growing cap of recently added GTP-tubulin

Loss of GTP- tubulin cap...

Karp, 1999

Structure of TaxolTaxol (“Paclitaxel”) comprises:

(A) a diterpene core (taxane skeleton) (B) 3 phenyl ring-bearing side chains (C) 2 acetoxy moeities

Structure of TaxolTaxol comprises:

(A) a diterpene core (B) 3 side chains bearing aromatic rings (C) 2 acetoxy moeities

Structure of TaxolTaxol comprises:

(A) a diterpene core (B) 3 phenyl ring-bearing side chains (C) 2 acetoxy moeities

1) addition of -phenylalanine2) oxidation of side chain C-2’3) addition of benzoyl group to side chain

Taxol: Mechanism of ActionTaxol was discovered to have an unprecedented mechanism of action: it stabilizes microtubules, preventing them from de-polymerizing

- Microtubule scaffold normally positions chromosomes, then collapses as the replicated chromosomes are pulled apart during cytokinesis

- Effect of Taxol is to trap mitotic cells within a cage of microtubules, preventing disassembly of the scaffold

Selectively kills dividing cancer cells

Crystal Structure of Tubulin Dimer

Taxol bound to -tubulin

GDP

3.7-Å resolutioncrystal structure

2 -sheets surrounded by 12 helices

Nogales et al., 1998Caplow et al., 1994

Kd = 15 nM

Taxol bound to -tubulin B9-B10 loop region in -tubulin

Taxol occupies a hydrophobic cleft in -tubulin, which is filled by an 8-residue extender connecting B9-B10 in -tubulin

Snyder et al., 2001

Empty binding pocket is very hydrophobic

Taxol fits neatly into the available space

Snyder et al., 2001

Binding of Taxol to -Tubulin

Histidine 229 residue of -tubulin is interposed between the C-2 and C-3’ phenyl rings of bound taxol, preventing hydrophobic collapse of the taxol rings against each other

Snyder et al., 2001

- explains why linking these rings with a tether yields inactive drugs: the rings interact hydrophobically with the protein, not with each other

H-bond between C-2’OH & backbone carbonyl of Arg-369

meta but not para substitutions on this ring are still active, due to space in the hydrophobic pocket

Mechanism of Action:

Lateral InteractionsTaxol binds very near the M loop of -tubulin, which makes lateral contacts between adjacent protofilaments

Stabilizes microtubules by strengthening lateral interactions between protofilments

- stabilizes a conformation of M loop that favors lateral contacts

- or - - counteracts destabilizing effects of GTP hydrolysis w/ compensating structural change (GTP- = Taxol-)

Nogales et al., 1999

Development of Microtubule-based Therapies

A drug like Taxol probably could not have been developed through rational drug design

(1) Microtubules are structural proteins that do not normally bind to small molecules

- no ligand, like a receptor would have

- no natural substrate, like an enzyme would have

Development of Microtubule-based Therapies

A drug like Taxol probably could not have been developed through rational drug design

(2) Microtubules are so unstable, they cannot be crystalized for structural studies - except by treating them with Taxol!

- the binding site of Taxol was thus defined by electron crystallographic studies of tubulin, because it was there in the crystals already

The Search for Anti-Cancer Drugs

The National Cancer Institute has long searched for natural products with anti-cancer potential

Extracts of plants, animals, and microbes are screened against a panel of cultured tumor cells

- extracts showing novel patterns of activity are then investigated, often by university researchers

Ethnobotany is the study of how cultures use plants for medicinal or other purposes

-

Taxol and EthnobotanyYew trees have long been recognized as toxic, or used medicinally

- Julius Ceasar noted a rival king killed himself with a yew potion- Name “Taxus” is from Greek word toxon, or poison

- Pliny the Elder noted that people died after drinking wine stored in casks made of yew wood

- Poisonous nature of Yew is noted in Hamlet & Macbeth

- Brewed by native Americans to treat fever, arthritis

- Sacred tree to Celtic druids

Pacific Yew treeTaxus brevifolia

Isolation of Taxol1951: As part of a National Cancer Institute (NCI) initiative to isolate new anti-cancer drugs, 35,000 plants were screened for

anti-tumor bioactivity

- Screening means testing extracts against cell lines derived from a diverse array of human tumors (breast, stomach, lung, etc)

1964: Extract of Pacfic Yew bark showed anti-tumor activity, but limited supply of bark (endangered tree) delayed isolation of

the active component

1971: Taxol identified as the anti-tumor molecule in Yew bark, butnot explored as a drug for a further 12 years

Cabri & DiFabio, 2000

Development of Taxol as an Anti-cancer Drug

1983: NCI-sponsored Phase 1 clinical trials delayed by allergic reactions to solvent in which Taxol was dissolved

-- also, supply of Taxol remained a persistent problem

1989: Results showed 30% response among patients with advancedovarian cancer (an otherwise untreatable disease)

treatmentcontrol

http://www.taxol.com

Development of Taxol

1991: Special partnership formed between NCI and the company Bristol-Myers-Squibb through a Commercial Research & Development Agreement (CRADA)

- BMS assumed full risk & responsibility for developing Taxol as a drug, in exchange for full access to NCI’s medicinal data

1992: Taxol approved as second-line treatment for ovarian cancer if previous chemotherapies have failed

- Taxol was not originally patented; BMS has relied on proprietary rights to NCI clinical and scientific data to maintain its exclusive market position

Cabri & DiFabio, 2000

Current use of Taxol

Currently approved as a treatment for:

(1) first-line treatment of ovarian cancer, in combination with the drug Cisplatin (1998)

(2) first-line treatment of non-small cell lung cancer (1999)

(3) breast cancer, in combination with other chemotherapy (1999)

(4) second-line treatment of AIDS-related Kaposi’s sarcoma (1997)

Current use of Taxol

- Taxanes used in ~25% of US cancer treatements

- Account for > $2 billion in worldwide sales (2005)

Taxol Supply: Trees vs. Patients?Taxus brevifolia is part of old-growth forests in the Northwest

- Requested to be put on Endangered Species list in 1990 by environmental groups (denied)

Took the bark of 6 trees, each 100 years old, to produce enough Taxol for one treatment!

- 13,000 kg of bark per 1 kg of pure Taxol

Ethical issue: how to balance future supply against immediate needs?

Taxol Supply: Semi-synthesisSemi-synthetic Taxol approved for treatment in 1995, following discovery that the related European Yew produced 10 DAB, a biosynthetic intermediate in its needles (a renewable resource)

Taxol10 DAB

Taxol Supply: Total synthesis4 total syntheses reported:

1) Holton (Florida State), 1994

2) Nicolaou (Scripps), 1994

- initial assembly of diterpene skeleton, followed by derivatization

3) Danishefsky (Columbia), 1995

- attachment of functionalized segments

4) Mukiyama, 1999

Taxol Supply: Biotechnology1) Plant cell culture: Phyton Catalytic Inc., Ithaca NY holds US rights to taxol production by plant cell fermentation (75,000 L)

2) A fungus (1994) and a bacterium Erwinia sp. (1995) isolated from Taxus tree produce taxol in culture (but low yield)

- lateral gene transfer from host plant to pathogen?? - culture of pathogens could be an alternative to harvesting trees

3) Taxadiene synthase and other genes important in taxol biosynthesis isolated by Crouteau (Washinton State)

- could lead to large-scale fermentation by genetic engineering(move genes into E. coli, yeast)

Roadblocks in the Development of Taxol

1. Supply of the natural product

2. Complexity of synthesis

3. Poor solubility in water (testing, delivery, reactions to solvent)

4. Weak correlation of in vitro activity in bioassays with in vivo activity against tumor xenografts

Taxol Resistance by Human Tumors4 mutations commonly confer Taxol resistance in human ovarian cancer cell lines:

1) Phenyalanine-270 Valine

2) Alanine-364 Threonine

3) Threonine-274 Isoleucine

4) Arginine-282 Glutamine

Taxol Resistance by Human Tumors4 mutations commonly confer Taxol resistance in human ovarian cancer cell lines:

1) Phenyalanine-270 Valine

- loss of Phe here eliminates strong hydrophobic packing with methyl group of Taxol’s C-4 acetate

Taxol Resistance by Human Tumors4 mutations commonly confer Taxol resistance in human ovarian cancer cell lines:

3) Threonine-274 Isoleucine

- loss of Thr here eliminates H-bonding between Taxol’s O-21 and side chain -OH of Thr274

Future of Microtubule-based Therapies

Rational alteration of the Taxane skeleton has resulted in improved drugs such as Taxotere (“docetaxel”)

ReferencesCabri, W. & Di Fabio, R. (2000) From bench to market: The evolution of chemical syntheses. Oxford University Press, N.Y., 266 pp.

Caplow, M., Shanks, J. & Ruhlen, R. (1994) How Taxol modulates microtubule disassembly. J. Biol. Chem. 269, 23399-23402.

Downing, K.H. (2000) Structural basis for the interaction of tubulin with proteins and drugs that affect microtubule dynamics. Annu. Rev. Cell Devel. Biol. 16, 89-111.

Giannakakou, P., Gussio, R., Nogales, E., Downing, K.H., Zaharevitz, D., Bollbuck, B., Poy, G., Sackett, D., Nicolaou, K.C. & Fojo, T. (2000) A common pharmacophore for epothilone and taxanes: Molecular basis for drug resistance conferred by tubulin mutations in human cancer cells. Proc. Natl. Acad. Sci. USA 97, 2904-2909.

Nogales, E., Wolf, S. & Downing, K.H. (1998) Structure of the tubulin dimer by electron crystallography. Nature 391, 199-203.

Nogales, E., Whittaker, M. Milligan, R., & Downing, K.H. (1999) High-resolution model of the microtubule. Cell 96, 79-88.

Snyder, J.P., Nettles, J.H., Cornett, B., Downing, K.H. & Nogales, E. (2001) The binding conformation of Taxol in -tubulin: A model based on electron crystalographic density. Proc. Natl. Acad. Sci. USA 98, 5312-5316.

Data from clinical trials obtained from http://www.taxol.com, a website managed by Bristol-Myers Squibb corporation.

- references should follow format of PNAS, but include full title

ReferencesCabri, W. & Di Fabio, R. (2000) From bench to market: The evolution of chemical syntheses. Oxford University Press, N.Y., 266 pp.

Caplow, M., Shanks, J. & Ruhlen, R. (1994) How Taxol modulates microtubule disassembly. J. Biol. Chem. 269, 23399-23402.

Downing, K.H. (2000) Structural basis for the interaction of tubulin with proteins and drugs that affect microtubule dynamics. Annu. Rev. Cell Devel. Biol. 16, 89-111.

Giannakakou, P., Gussio, R., Nogales, E., Downing, K.H., Zaharevitz, D., Bollbuck, B., Poy, G., Sackett, D., Nicolaou, K.C. & Fojo, T. (2000) A common pharmacophore for epothilone and taxanes: Molecular basis for drug resistance conferred by tubulin mutations in human cancer cells. Proc. Natl. Acad. Sci. USA 97, 2904-2909.

Nogales, E., Wolf, S. & Downing, K.H. (1998) Structure of the tubulin dimer by electron crystallography. Nature 391, 199-203.

Nogales, E., Whittaker, M. Milligan, R., & Downing, K.H. (1999) High-resolution model of the microtubule. Cell 96, 79-88.

Snyder, J.P., Nettles, J.H., Cornett, B., Downing, K.H. & Nogales, E. (2001) The binding conformation of Taxol in -tubulin: A model based on electron crystalographic density. Proc. Natl. Acad. Sci. USA 98, 5312-5316.

Data from clinical trials obtained from http://www.taxol.com, a website managed by Bristol-Myers Squibb corporation.

- primary literature citations (experimental studies)

ReferencesCabri, W. & Di Fabio, R. (2000) From bench to market: The evolution of chemical syntheses. Oxford University Press, N.Y., 266 pp.

Caplow, M., Shanks, J. & Ruhlen, R. (1994) How Taxol modulates microtubule disassembly. J. Biol. Chem. 269, 23399-23402.

Downing, K.H. (2000) Structural basis for the interaction of tubulin with proteins and drugs that affect microtubule dynamics. Annu. Rev. Cell Devel. Biol. 16, 89-111.

Giannakakou, P., Gussio, R., Nogales, E., Downing, K.H., Zaharevitz, D., Bollbuck, B., Poy, G., Sackett, D., Nicolaou, K.C. & Fojo, T. (2000) A common pharmacophore for epothilone and taxanes: Molecular basis for drug resistance conferred by tubulin mutations in human cancer cells. Proc. Natl. Acad. Sci. USA 97, 2904-2909.

Nogales, E., Wolf, S. & Downing, K.H. (1998) Structure of the tubulin dimer by electron crystallography. Nature 391, 199-203.

Nogales, E., Whittaker, M. Milligan, R., & Downing, K.H. (1999) High-resolution model of the microtubule. Cell 96, 79-88.

Snyder, J.P., Nettles, J.H., Cornett, B., Downing, K.H. & Nogales, E. (2001) The binding conformation of Taxol in -tubulin: A model based on electron crystalographic density. Proc. Natl. Acad. Sci. USA 98, 5312-5316.

Data from clinical trials obtained from http://www.taxol.com, a website managed by Bristol-Myers Squibb corporation.

- secondary literature citations (book, review article)