ANTICANCER AGENTS PROTEIN KINASE INHIBITORS

1. ANTICANCER AGENTS PROTEIN KINASE INHIBITORS Chapter 21

2. Protein Kinases • Enzymes that catalyse phosphorylation reactions on protein substrates • 500-2000 estimated protein kinases in a cell • Protein kinases are present in the cytoplasm • Protein kinase receptors - dual role as receptor and enzyme • Overexpression can result in cancer • Tyrosine kinases, serine-threonine kinases and histidine kinases • ATP used as enzyme cofactor - phosphorylating agent

3. ATP ADP Protein Kinases Tyrosine kinases

4. ATP ADP ATP ADP Protein Kinases Serine-threonine kinases

5. Protein Kinases • Active Site • Contains the binding site for the protein substrate • Contains the binding site for the ATP cofactor • Clinically useful inhibitors target the ATP binding site • ATP binding site is similar but not identical for all protein kinases • Allows selectivity of inhibitor action

6. H N O C 2 Gln-767 H Hydrophobic pocket N H C 3 H C 3 O H HBD H C N 3 Leu-768 O Cleft HBA Met-769 N S H O Ribose pocket 1. Protein Kinases ATP binding site

7. Gln-767 Hydrophobic pocket O H HBD N Leu-768 O Cleft HBA Met-769 N S H H N O C 2 H N O H C 3 H C 3 Ribose pocket C H 3 Protein Kinases ATP binding site • Purine base is buried deep into the binding site • Purine forms two hydrogen bonding interactions to the binding site • Ribose sugar binds to a ‘ribose binding pocket’ • Triphosphate chain lies along a cleft towards the enzyme surface • Triphosphate interacts with two metal ions and amino acids • Specificity surface is an area of unoccupied binding site • An empty hydrophobic pocket lies opposite the ribose binding pocket • The gatekeeper residue is an amino acid situated at the entrance to the hydrophobic pocket • The size of the gatekeeper residue is important in drug design • The nature of amino acids in the binding pockets is important to drug design

8. Protein Kinase Inhibitors • Notes • Type I inhibitors act on the active conformation of the enzyme • Type I inhibitors bind to the ATP binding site and block access to ATP • Type II inhibitors act on the inactive conformation of the enzyme • Type II inhibitors bind to the enzyme and stabilise the inactive conformation • Type II inhibitors are likely to be more selective Type I inhibitors Gefitinib, erlotinib, SU11248 and seliciclib Type II inhibitors Imatinib, lapatinib, sorafenib and vatalanib

9. Gefitinib (Iressa) • Notes • Developed by Astra Zeneca • Inhibits the kinase active site of the epidermal growth factor receptor • The EGF-receptor is a tyrosine kinase receptor • Gefitinib is a 4-anilinoquinazoline structure

10. Gefitinib (Iressa) Lead compound Secondary amine Small lipophilic group Electron-donating substituents • Notes • The secondary amine, electron-donating substituents and small lipophilic group are all important for activity • Useful in vitro activity • Lower in vivo activity due to rapid metabolism • Metabolised by cytochrome P450 enzymes

11. Cytochrome P450 enzymes + Oxidation Gefitinib (Iressa) Metabolism of the lead compound • Notes • Methyl group and para-position of aromatic ring are susceptible positions • Blocking metabolism should improve the half life of the drug

12. Gefitinib (Iressa) Drug design • Notes • Fluoro-substituent blocks para-hydroxylation of the aromatic ring • Fluorine is similar in size to hydrogen and has no steric effect • Methyl group is replaced by a chlorosubstituent • Chlorine and methyl group have similar sizes and lipophilicities • Chlorine acts as a bio-isotere for the methyl group • Chlorine is resistant to oxidation • Compound is less active in vitro, but more active in vivo

13. Spacer Ionisable Gefitinib Gefitinib (Iressa) Drug design Morpholine • Notes • Morpholine ring increases water solubility • Morpholine nitrogen allows generation of water soluble amine salts • Spacer allows morpholine to protrude out of the active site • Remains solvated when the drug is bound • Avoids a desolvation penalty

14. HBA HBA HBA HBA Gefitinib (Iressa) • Binding interactions • Identified by a molecular modelling experiment • Gefitinib is docked with a model binding site • Binds to the ATP binding site • Aniline ring occupies the normally vacant hydrophobic pocket opposite the ribose binding pocket • Quinazoline binds to the same region as the purine ring of ATP

15. 3. Gefitinib (Iressa) Synthesis of gefitinib and analogues

16. Aniline ring Aniline ring Quinazoline ring Quinazoline ring Lapatinib Erlotinib (Tarceva) IC50 2 nM 4. Lapatinib and Etlotinib • Notes • 4-Anilinoquinazoline structures - compare gefitinib • EGF-receptor kinase inhibitors

17. Pyrrole Pyrimidine HBA HBD 5. PKI 166 • Notes • Pyrrolopyrimidine structure • EGF-receptor kinase inhibitor • Different binding mode from ATP or anilinoquinazolines

18. Gefitinib Gefitinib PKI 166 PKI 166 HBA HBA HBA HBD HBD ATP ATP HBA 5. PKI 166 • Comparison of binding interactions • ATP and EGF-receptor kinase inhibitors all contain a pyrimidine ring • Different binding modes are possible

19. 6. Imatinib (Glivec or Gleevec) • Notes • First protein kinase inhibitor to reach the market • Selective inhibitor for a hybrid tyrosine kinase (Bcr-Abl) • Bcr-Abl is active in certain tumour cells

20. Anilino substituent Pyrimidine • Phenylaminopyrimidine structure • Identified by random screening of compound libraries • Originally identified as a PKC inhibitor • PKC is a serine-threonine kinase 6. Imatinib (Glivec or Gleevec) Lead compound

21. Amide Increased inhibition of PKC Inhibits tyrosine kinases as well 6. Imatinib (Glivec or Gleevec) Drug design Pyridine

22. Conformational blocker Imatinib • Increased activity vs tyrosine kinases • No activity against serine-threonine kinases Spacer • Piperazine increases activity, selectivity and water solubility • Spacer inserted to avoid aniline structure 6. Imatinib (Glivec or Gleevec) Drug design Piperazine

23. 6. Imatinib (Glivec or Gleevec) • Binding interactions • Identified from a crystal structure of an inhibitor-Abl kinase complex • Amide serves as an ‘anchoring group’ and orientates the molecule • Amide binds to Glu and Asp • Glu and Asp are important to the catalytic mechanism

24. 6. Imatinib (Glivec or Gleevec) Binding interactions • Other interactions determine target selectivity • A hydrogen bond to the gatekeeper Thr is essential to activity • N-Alkylation eliminates activity

25. Ionic bond Glu 6. Imatinib (Glivec or Gleevec) Binding interactions • Molecular modelling studies suggest that the piperazinyl group interacts with a glutamate residue • Imatinib inhibits protein kinases containing this glutamate residue (Abl, c-Kit and PDGF-R) Piperazinyl group

26. Conformational blocker 6. Imatinib (Glivec or Gleevec) Binding interactions • Conformational blocker aids selectivity • Binds to a hydrophobic pocket that is not accessible if a larger gatekeeper residue was present

27. Mutation to Isoleucine 6. Imatinib (Glivec or Gleevec) • Drug resistance • Mutation of the gatekeeper residue to isoleucine introduces resistance (T315I mutation) • Isoleucine unable to form an important hydrogen bond to the amine

28. 6. Imatinib (Glivec or Gleevec) Synthesis of imatinib and analogues

29. 7. Second Generation Bcr-Abl inhibitors

30. 7. Second Generation Bcr-Abl inhibitors • Notes • Inhibits two protein kinase targets (Abl and Src) • Currently in clinical trials • Less likely to fall prey to drug resistance

31. Notes • Allosteric inhibitor of Bcr-Abl • Does not bind to ATP binding site • Stabilises inactive form of the enzyme • Binds to an autoregulatory cleft • Potential agent for treating leukaemia • Notes • Binds to the protein substrate site • Currently under study 7. Second Generation Bcr-Abl inhibitors

32. 8. Inhibitors of cyclin-dependent kinases • Cyclin-dependent kinases • CDKs are involved in control of the cell cycle and are overexpressed in many cancer cells • Serine-threonine kinases • Activated by cyclins • Inhibited by cyclin-dependent kinase inhibitors • Synthetic inhibitors bind to the ATP binding site

33. HBD HBA Benzopyran Piperidine Phenyl ring 8. Inhibitors of cyclin-dependent kinases • Benzopyran binds to the adenine binding region • Piperidine binds to the region occupied by the first phosphate of ATP • Phenyl lies over the ribose binding pocket • Undergoing clinical trials

34. HBD HBA Benzopyran Piperidine Phenyl ring R-Roscovitine (seliciclib) 7-Hydroxystaurosporin is undergoing clinical trials Shows selectivity for CDK2 Undergoing clinical trials 8. Inhibitors of cyclin-dependent kinases

35. 9. Kinase Inhibitors of FGF-R and VEGF-R • FGF-R and VEGF-R • FGF-R = fibroblast growth factor receptor • VEGF-R = vascular endothelial growth factor receptor • Associated with angiogenesis • Inhibitors bind to the ATP binding site • Currently undergoing clinical trials

36. Anilino substituent Pyrrole Oxindole Phthalazine HBA HBD Pyridine • SU 5416 in clinical trials for treatment of colorectal cancer • Oxindole binds to same region as adenine of ATP Phase III clinical trials in 2006 9. Kinase Inhibitors of FGF-R and VEGF-R

37. 10. Multi-tyrosine receptor kinase inhibitors • Notes • Designed to be selective against a range of tyrosine receptor kinases implicated in tumours • Drug resistance unlikely to occur for all kinase targets • Equivalent of combination therapy (poly-pharmacology) • Sometimes called ‘dirty drugs’ • Promising agents against tumours that are driven by several abnormalities

38. 10. Multi-tyrosine receptor kinase inhibitors • Notes • Sorafenib approved as a VEGF-R kinase inhibitor • Sunitinib approved in 2006 - inhibits VEGF-R, PDGF-R and KIT receptor kinases • Vatalanib undergoing clinical trials

39. Lead compound; IC50 17 mM 10. Multi-tyrosine receptor kinase inhibitors • Design of sorafenib • Lead compound found by high throughput screening • 200 000 compounds tested • Tested against recombinant Raf-1 kinase Urea

40. Lead compound IC50 17 mM II; IC50 1.7 mM III; Poor activity 10. Multi-tyrosine receptor kinase inhibitors Design of sorafenib - variation of substituents • Notes • Methyl substituent is optimum for activity • 10-fold increase in activity • Phenoxy group is bad for activity

41. VI; Poor activity 10. Multi-tyrosine receptor kinase inhibitors Design of sorafenib - variation of rings Isoxazole Lead compound IC50 17 mM • Notes • Variation of rings also carried out systematically • Isoxazole ring is not good for activity • Conventional medicinal chemistry strategies fail to achieve further improvement

42. Isoxazole Phenoxy group IV; IC50 1.1 mM IV; IC50 1.1 mM 10. Multi-tyrosine receptor kinase inhibitors Design of sorafenib Lead compound IC50 17 mM • Notes • Parallel synthesis - 1000 analogues synthesised with all possible combinations of rings and substituents • Structure IV has slightly increased activity - contradicts results from conventional studies • Isoxazole ring and phenoxy substituent are good for activity when combined in the same structure - synergistic effect • Structure IV taken as new lead compound

43. Isoxazole Phenoxy group IV; IC50 1.1 mM • Ring variation • 5-fold increase in activity • Increase in aqueous solubility and cLogP V; IC50 0.23 mM 10. Multi-tyrosine receptor kinase inhibitors Design of sorafenib Lead compound IC50 17 mM Pyridine

44. Isoxazole Phenoxy group IV; IC50 1.1 mM Ring variation Substituent variation Substituent variation Sorafenib IC50 12 nM Sorafenib IC50 12 nM 1000-fold increase in activity 10. Multi-tyrosine receptor kinase inhibitors Design of sorafenib Lead compound IC50 17 mM Pyridine V; IC50 0.23 mM

45. HBD HBD HBA HBA 10. Multi-tyrosine receptor kinase inhibitors Sorafenib - binding interactions • Notes • Urea functional group acts as a binding anchor (compare imatinib) • Hydrogen bonds are formed to catalytic Asp and Glu • Binding orientates the molecule • Positions each half into two selectivity regions

46. 11. Inhibitors of heat shock protein 90 • Notes • HSP 90 is a kinase protein and acts as a molecular chaperone • Important to survival of cells - inhibition likely to lead to cell death • HSP 90 interacts selectively with many of the proteins implicated in tumours • Targeting HSP 90 may be effective against tumour cells resistant against other drugs • Resistant cells contain mutated proteins - rely more on HSP 90 during the folding process • Resistant cells likely to be more vulnerable to inhibitors of HSP 90

47. Natural product • Potent inhibitor • Urethane group is crucial to activity • Binds to region occupied by adenine • Poor solubility • Reactive quinone moiety Quinone Urethane 11. Inhibitors of heat shock protein 90 Notes Inhibitors bind to the ATP binding site Lead compound - geldanamycin

48. Alvespimycin Tanespimycin IPI 504 11. Inhibitors of heat shock protein 90 Geldanamycin analogues