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Combined approaches to Drug Discovery

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

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Combined approaches to Drug Discovery

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  1. 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

  2. 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

  3. 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

  4. 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

  5. The Protein Tubulin Microtubules are composed of the protein tubulin (1) Tubulin formsdimers, which consist of an a and a b subunit (2) Dimers stack together into protofilaments, which are linear strings (3) Protofilaments bind laterally to form hollow, cylindrical microtubules Tubule Downing, 2000

  6. Structure of Tubulin Tubulin protein exists as two 450 a.a. monomers, a and b - Each binds a high-energy GTP molecule An a - b dimer then forms (GTP-tubulin) Dimers polymerize to form long protofilaments Polymerization causes hydrolysis of the b-GTP, which destabilizes the microtubule - GDP-tubulin wants to relax into a new conformation, which dissociates from the microtubule

  7. All that holds the microtubule together is a fast-growing cap of recently added GTP-tubulin GTP tubulin GDP tubulin Loss of GTP- tubulin cap... collapse Karp, 1999

  8. Structure of Taxol Taxol (“Paclitaxel”) comprises: (A) a diterpene core (taxane skeleton) (B) 3 phenyl ring-bearing side chains (C) 2 acetoxy moeities

  9. Structure of Taxol Taxol comprises: (A) a diterpene core (B) 3 side chains bearing aromatic rings (C) 2 acetoxy moeities

  10. Structure of Taxol Taxol comprises: (A) a diterpene core (B) 3 phenyl ring-bearing side chains (C) 2 acetoxy moeities

  11. 1) addition of b-phenylalanine 2) oxidation of side chain C-2’ 3) addition of benzoyl group to side chain

  12. Taxol: Mechanism of Action Taxol 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

  13. Crystal Structure of Tubulin Dimer 3.7-Å resolution crystal structure 2 b-sheets surrounded by 12 a helices Taxol bound to b-tubulin Kd = 15 nM Nogales et al., 1998 Caplow et al., 1994 GDP

  14. Taxol bound to b-tubulin B9-B10 loop region in a-tubulin Taxol occupies a hydrophobic cleft in b-tubulin, which is filled by an 8-residue extender connecting B9-B10 in a-tubulin Snyder et al., 2001

  15. Empty binding pocket is very hydrophobic Taxol fits neatly into the available space Snyder et al., 2001

  16. Binding of Taxol to b-Tubulin - explains why linking these rings with a tether yields inactive drugs: the rings interact hydrophobically with the protein, not with each other Snyder et al., 2001 Histidine 229 residue of b-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

  17. 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

  18. Mechanism of Action: Lateral Interactions Taxol binds very near the M loop of b-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-b = Taxol-b) Nogales et al., 1999

  19. 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

  20. 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

  21. 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 -

  22. Taxol and Ethnobotany Yew 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 tree Taxus brevifolia

  23. Isolation of Taxol 1951: 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, but not explored as a drug for a further 12 years Cabri & DiFabio, 2000

  24. 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 advanced ovarian cancer (an otherwise untreatable disease) treatment control http://www.taxol.com

  25. 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

  26. Current use of Taxol Currentlyapproved 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)

  27. Current use of Taxol - Taxanes used in ~25% of US cancer treatements - Account for > $2 billion in worldwide sales (2005)

  28. 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?

  29. Taxol Supply: Semi-synthesis Semi-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) 10 DAB Taxol

  30. Taxol Supply: Total synthesis 4 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

  31. Taxol Supply: Biotechnology 1) 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)

  32. 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

  33. Taxol Resistance by Human Tumors 4 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

  34. Taxol Resistance by Human Tumors 4 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

  35. Taxol Resistance by Human Tumors 4 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

  36. Future of Microtubule-based Therapies Rational alteration of the Taxane skeleton has resulted in improved drugs such as Taxotere (“docetaxel”)

  37. References Cabri, 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. USA97, 2904-2909. Nogales, E., Wolf, S. & Downing, K.H. (1998) Structure of the ab tubulin dimer by electron crystallography. Nature391, 199-203. Nogales, E., Whittaker, M. Milligan, R., & Downing, K.H. (1999) High-resolution model of the microtubule. Cell96, 79-88. Snyder, J.P., Nettles, J.H., Cornett, B., Downing, K.H. & Nogales, E. (2001) The binding conformation of Taxol in b-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

  38. References Cabri, 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. USA97, 2904-2909. Nogales, E., Wolf, S. & Downing, K.H. (1998) Structure of the ab tubulin dimer by electron crystallography. Nature391, 199-203. Nogales, E., Whittaker, M. Milligan, R., & Downing, K.H. (1999) High-resolution model of the microtubule. Cell96, 79-88. Snyder, J.P., Nettles, J.H., Cornett, B., Downing, K.H. & Nogales, E. (2001) The binding conformation of Taxol in b-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)

  39. References Cabri, 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. USA97, 2904-2909. Nogales, E., Wolf, S. & Downing, K.H. (1998) Structure of the ab tubulin dimer by electron crystallography. Nature391, 199-203. Nogales, E., Whittaker, M. Milligan, R., & Downing, K.H. (1999) High-resolution model of the microtubule. Cell96, 79-88. Snyder, J.P., Nettles, J.H., Cornett, B., Downing, K.H. & Nogales, E. (2001) The binding conformation of Taxol in b-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)

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