1. Applying pharmacophore-based approach to improving Taxol Started with a natural product that was highly active - can other molecules with the same mechanism of action be isolated? - can we determine the pharmacophore for tubulin binding? - can we improve the drug with this knowledge?

2. Alternative Microtubule-based Therapies Following the discovery of Taxol, efforts to develop analogous drugs have employed tubulin polymerization assays - “Assay” means a lab test in which crude extracts, fractions from chromatographic separations, or pure compounds are applied to a system (proteins, cells, or whole animals) - An active compound (also called a “hit”) is one that has a desired effect (it inhibits a protein, kills bacteria, cures a mouse of cancer) Now that we recognize stabilizing microtubules is an effective way to treat cancer, no need to screen for generically toxic molecules; we can be much more directed in our approach

3. Alternative Microtubule-based Therapies In a tubulin polymerization assay, chemicals are tested to see if they can inhibit depolymerization of microtubules - assays are performed in vitro, meaning in a test tube, not in a living cell Add tubulin, GTP, and the chemical to be tested - Wait 30 min - Measure turbidity (cloudiness of solution); as tubulin polymerizes, microtubules come out of solution, making it turbid - Look under electron microscope; measure length of resulting microtubules

4. Alternative Microtubule-based Therapies Using in vitro assays, 3 classes of natural molecules have been discovered which also stabilize microtubules: (1) Epothilones A and B (from soil bacteria) - water-soluble (easier delivery) - bacteria can be mass-cultured (2) Eleutherobin (from a tropical soft coral) (3) Discodermalide (from a tropical sponge) All 3 types competitively exclude Taxol, indicating they bind to the same site on tubulin and share one mechanism of action Understanding this mechanism should make possible the design of more potent synthetic drugs for cancer therapy

5. Microtubule Stabilizing Molecules 3 are diterpene-based, but no obvious structural similarities (-)-Taxol (-)-Eleutherobin (+)-Discodermalide (-)-Epothilone B (R=Me)

6. Alternative Microtubule-based Therapies Taxol and Epothilone Taxol and Discodermolide Although flat structures look very different, 3D models show how other drugs can fill the same space as Taxol in the tubulin binding pocket Giannakakou et al., PNAS 2000

7. Tubulin-binding pharmacophore Taxol Epothilone B Spatially conserved atoms shown in red 6.95 Å 6.93 Å Structural overlap Pharmacophore

8. Tubulin-binding pharmacophore Taxol Epothilone B Spatially conserved atoms shown in red Structural overlap Epothilones are especially prone to resistance mutations costing an H-bond to C-7 OH

9. Tubulin-binding pharmacophore Taxol Epothilone B Spatially conserved atoms shown in red Structural overlap Epothilones are especially prone to resistance mutations costing an H-bond to C-7 OH Taxanes are more resistant, due to nearby alternate H-bond acceptors at C-10 and C-9

10. Tubulin-binding pharmacophore Taxol Epothilone B Structural overlap C-12 methyl group of Epothilone B makes it 14 times more potent than Epothilone A (missing this -CH 3 ) thiazole ring packs against Phe-270 side chain

11. Can the pharmacophore model explain the activity of eleutherobin?

12. C-7 methyl- packs against –CH 3 of Thr 274 - explains why eleutherobin works against Thr 274  Isoleucine mutant cells eleutherobin epothilone B

13. Receptor-Based Drug Design Knowing a protein is critical for the etiology of a disease, you can... 1. Design an assay using that protein to identify drug candidates 2. Starting w/ the structure of a protein-ligand complex, design synthetic analogues or make modifications to create an inhibitor - Exploit unique features of the protein of interest to improve activity and confer specificity 3. Based on crystal or solution structures of the protein of interest, use molecular modeling to design a novel small molecule that will fit a potential binding site (de novo rational drug design)

14. Receptor-Based Examples 1. Targeting a single protein essential for disease progression Gleevec, a new anti-cancer drug 2. Taking advantage of unique features of a protein target

15. Treating chronic myeloid leukemia Chronic myeloid leukemia (CML) is a form of cancer in which white blood cells proliferate out of control This form of cancer is characterized by the presence of the Philadelphia Chromosome, a translocation (or swapping) of the ends of chromosomes 9 and 22 - 1st chromosomal defect linked to cancer (1960) - One chromosome is shortened, another becomes longer

16. NormalTranslocated Philadelphia Chromosome The shorter (missing DNA) Philadelphia chromosome The translocation chromosome, w/ extra DNA

18. c-ABL c-BCR BCR/ABL BCR/ABL is a fusion protein, combining parts of 2 different genes c-ABL can bind DNA, actin, other proteins

19. 1987: shown BCR/ABL fusion protein has tyrosine kinase activity - Many oncogenes, mutated genes that predispose a cell to become cancerous, are now known to be tyrosine kinases A kinase is an enzyme that phosphorylates another protein, by attaching a phosphate group onto an -OH - There are 2 kinds: (1) serine/threonine kinases (2) tyrosine kinases

20. Many tyrosine kinases participate in signaling cascades that trigger cell division, in response to signals like growth factors - Are normally tightly regulated by the cell to control proliferation - Mutation can change kinases into oncogenes (= cancer-causing genes) that are turned on constitutively (= constantly), instead of just when cell gets the signal to start dividing Sebolt-Leopold & English, Nature 2006

21. - phosphorylated substrate protein assumes a new conformation, thus changing from inactive to active form Kinase Activity

22. Bcr/Abl fusion protein is switched permanently “on”, sending the internal signals for cell division without the appropriate external signals (like growth factors) Bcr/Abl is the unique cause of CML, making it a perfect target for rational drug design Drugs that target this kind of modified protein represent a new form of cancer treatment - Such cytostatic drugs target cancer selectively, rather than indiscriminately killing all dividing cells, unlike most earlier chemotherapies (which are cytotoxic) - Drug discovery can begin by designing, atom by atom, new medicines specific for particular types of cancer

23. A bunch of synthetic molecules were made by the pharmaceutical company Ciba-Geigy (now part of Novartis), to test for tyrosine kinase inhibition Sent to a university researcher (Druker) who had developed a specific bioassay to test for activity of the BCR/ABL fusion protein A drug called STI-571 (Gleevec®) was found to inhibit Bcr/Abl effectively enough to work as a drug, and specific enough to have minimal side effects on normal cells Targets only a subset of all cellular kinases: (1) BCR/ABL (2) receptors for platelet-derived growth factor (PDGF) and stem cell factor (SCF) (3) the proto-oncogene c-Kit

24. Gleevec

25. Clinical success of Gleevec FDA approved in 2001

26. Surprisingly, Gleevec binds to the inactive conformation of ABL Jams activation loop, which cannot switch to “open” conformation Locks protein into a permanently inactive conformation

27. Mechanism of Action Upon signal, ABL auto-phosphorylates Tyrosine 393, a residue on the “activation loop” - The activation loop is a conserved regulatory element in kinases, controlling their catalytic activity by switching between different activity states Once phosphorylated, the activation loop swings out into its “open” conformation - In the open conformation, 3 residues (Asp 381-Phe 382-Gly 383) are positioned to catalyze substrate phosphorylation - Also, catalytic site is no longer blocked by the collapsed loop

28. Normally: (1) signal to cell (2) ABL auto-phosphorylates (3) activation loop swings out: - exposes catalytic site - aligns 3 essential residues on the loop w/ the catalytic core (4) substrate protein binds, gets phosphorylated on tyrosine

29. Mechanism of Action The open conformation is similar in all kinases, but the inactive (“closed”) conformation is different in each protein Gleevec wedges into the ATP-binding pocket and sticks into the hydrophobic core of the kinase, locking Abl into its inactive conformation - Gleevec cannot bind to the active form of Abl - Gleevec cannot bind to the inactive form of other kinases Illustrates a whole new approach to drug design: exploit the unique features of the inactive shapes of specific kinases - Prevent them from activating + driving cell proliferation

30. Cancer cell + Gleevec: (1) BCR/ABL fusion is always “on” (2) Gleevec binds, locks activation loop into inactive conformation (3) Catalytic site is thus blocked (4) Substrate proteins cannot be phosphorylated (5) No out-of-control cell division for BCR/ABL blood cells (6) No effect on normal cells

31. Tyr253 Leu370 Val256 Phe382 Asp381 Lys271 Ile313 Thr315 Met318 Glu286 Met290 Complex H-bond network holds Gleevec in ATP-binding site of ABL Brown residues form a hydrophobic cage; promote binding through van der Waals interactions

32. Tyr253 Leu370 Val256 Phe382 Asp381 Lys271 Ile313 Thr315 Met318 Glu286 Met290 21 amino acid residues contact the drug - 6 H-bonds total - mostly van der Waals Threonine 315 is a critical residue - responsible for ABL-Gleevec specificity - related kinases lack this residue, so Gleevec can’t bind to them

33. Gleevec Resistance Patients pass through distinct stages: chronic phaseblast crisis 5-6 years3-6 months (BCR/ABL)(additional genetic defects) Gleevec treatment results: remissioneventual resistance Late-stage cancers continue to proliferate even with Gleevec Found that all contain 1 of 2 types of mutations: (1) extra copies of BCR/ABL oncogene (gene amplification) (2) mutations that decrease drug binding, OR keep ABL locked into its active conformation (to which Gleevec doesn’t bind)

34. BCR/ABL gene was sequenced for 6 patients who relapsed during Gleevec treatment, and all had the same mutation: Threonine 315 Isoleucine 315 - In what 2 ways does this change prevent Gleevec from binding? A single amino acid change confers drug resistance, resulting in rapid death of the patient