ANTICANCER AGENTS FARNESYL TRANSFERASE INHIBITORS

1. ANTICANCER AGENTS FARNESYL TRANSFERASE INHIBITORS Chapter 21

2. Ras Protein • Notes • Signalling protein that is crucial to cell growth and division • Abnormal form is present in 30% of cancers • Prevalent in colonic and pancreatic cancers • Abnormal Ras is coded by a mutated ras gene • Small G-protein • Binds GDP in resting state and GTP in active state • Active Ras normally autocatalyses hydrolysis of GTP back to GDP • Abnormal Ras fails to hydrolyse GTP • Abnormal Ras remains permanently active • Three human Ras proteins (H-Ras, N-Ras and K-Ras)

3. Farnesyltransferase • Notes • Zinc metalloproteinase • Catalyses attachment of a farnesyl group to Ras • Hydrophobic farnesyl group anchors Ras to the inner part of the cell membrane • Farnesylation is necessary for Ras to become activated during signal transduction • Inhibition of farnesyltransferase should inhibit this process

4. FTase Further processing Farnesyltransferase Enzyme mechanism Methyl ester

5. Farnesyltransferase • Notes • Farnesyldiphosphate (FPP) binds first to the active site • FPP aids binding of Ras protein to the active site • Magnesium and iron ions are present as cofactors • Magnesium ion interacts with the pyrophosphate group • Results in a better leaving group • Iron ion interacts with the thiol group of cysteine • Results in a better nucleophile

6. C = cysteine • a = valine, isoleucine or leucine • X = methionine, glutamine or serine C-a-a-X Substrate FT Substrates Substrates share a terminal tetrapeptide moiety called the CaaX peptide

7. FT Inhibitors • Aims • Good inhibitory activity vs enzyme • Ability to cross the cell membrane to reach the enzyme • Metabolic stability • Aqueous solubility • Oral absorption • Favourable pharmacokinetic properties

8. C = cysteine • a = valine, isoleucine or leucine • X = methionine, glutamine or serine C-a-a-X C-a-Phe-X Substrate Inhibitor FT Inhibitors • Notes • Inhibitors were developed to mimic the terminal tetrapeptide moiety - CaaX peptide • Tetrapeptides having Phe next to X act as inhibitors • Serve as lead compounds

9. Val Met Cys Phe Lead compound • Disadvantages • Terminal carboxylic acid likely to be ionised - bad for absorption • Peptide bonds are susceptible to enzyme-catalysed hydrolysis • Poor stability to digestive or metabolic enzymes (e.g. aminopeptidases)

10. Peptide bond isostere Val Met Ester Ester Cys Phe Methylene- amino link Methylene- amino link Methylene- amino link Lead compound Peptidomimetic Peptidomimetic Peptidomimetic Peptidomimetic Drug design • Notes • Modifications carried out to remove peptide nature - peptidomimetics • Ester masks polar carboxylic acid or carboxylate ion - acts as prodrug • Methyleneamino link replaces N-terminal peptide bond • Methyleneamino link introduces a resistance to aminopeptidases • Peptide bond isostere introduced to mimic central peptide bond • Isostere should be capable of mimicing any binding interactions • Isostere should be stable to enzyme-catalysed hydrolysis

11. Peptide bond isostere Stable methylene-amino link Stable methylene-amino link Terminal amino group R=H FTI 276 R=iPr FTI 277 Thiol Aromatic substituent Examples of FT Inhibitors • Notes • Thiol group forms important interactions with the zinc ion cofactor • Methyleneamino link is stable to aminopeptidases • Aromatic substituent is important for inhibitory activity • Aromatic ring acts as a peptide bond isostere • Terminal amino group is ionised • Terminal amino group forms an ionic bond to the phosphate group of FPP • Terminal carboxylate group is important to binding

12. Stable methylene-amino link Peptide bond isostere Sulfone R=H L739750 R=iPr L744832 Terminal amino group Thiol Aromatic substituent Examples of FT Inhibitors • Notes • Thiol group forms important interactions with the zinc ion cofactor • Methyleneamino link is stable to aminopeptidases • Aromatic substituent is important for inhibitory activity • Methyleneoxy group acts as the peptide bond isostere • Terminal amino group is ionised • Terminal amino group forms an ionic bond to the phosphate group of FPP • Terminal carboxylate group is important to binding • Sulfone increases activity over a methylthio group

13. Peptide bond isostere Masking group Masking group Masking group Masking group AZD-3409 AZD-3409 AZD-3409 Pyrrolidine Aromatic substituent Examples of FT Inhibitors • Notes • Thiol and carboxylic acid groups are both masked in the prodrug • Lowers the toxicity risk of the thiol group • Protects the thiol from possible metabolism • Pyrrolidine ring introduces conformational rigidity • Potent inhibitor (Ki < 1 nM) • Also inhibits geranylgeranyltransferase - catalyses prenylation with geranylgeranyldiphosphate • Agents inhibiting both enzymes are potentially advantageous

14. Structure I IC50 1.4 nM Structure I IC50 1.4 nM Imidazole ring Examples of FT Inhibitors • Notes • Non-peptide inhibitor • Imidazole ring acts as the zinc ligand • Decreases the risk of toxicity due to a free thiol group

15. Lonafarnib IC50 1.9 nM Examples of FT Inhibitors • Notes • Non-peptide inhibitor • Developed from lead compound discovered by screening compound libraries • 10,000 times more active than the lead compound • No ligand for the zinc cofactor is present!

16. Steric shield Steric shield Imidazole ring Lonafarnib IC50 1.9 nM Sch 226374 IC50 0.36 nM Sch 226374 IC50 0.36 nM Sch 226374 IC50 0.36 nM • Non-peptide inhibitor • Developed from lonafarnib by structure-based drug design • Imidazole ring introduced as zinc ligand • Aromatic ring introduced as a steric shield vs metabolism Examples of FT Inhibitors

17. Imidazole Quinolone I; IC50 180 nM I; IC50 180 nM • Lead compound • Identified from screening compound libraries • Imidazole ring present - zinc ligand • Both aromatic rings are important to activity Development of Tipifarnib

18. Imidazole Quinolone I; IC50 180 nM II; IC50 35 nM • Strategy - variation of substituents • Activity increases with introduction of meta-chloro substituent Development of Tipifarnib

19. Imidazole Quinolone I; IC50 180 nM III; IC50 15 nM • Strategy - variation of substituents • Activity increases with addition of N-methyl substituent Development of Tipifarnib II; IC50 35 nM

20. Imidazole Quinolone I; IC50 180 nM • Strategy - variation of ring substitution • Activity increases IV; IC50 2.5 nM Development of Tipifarnib II; IC50 35 nM III; IC50 15 nM

21. Imidazole Quinolone I; IC50 180 nM • Extension strategy • Extra functional group • Extra binding interactions • Activity increases III; IC50 15 nM Tipifarnib; IC50 0.6 nM Development of Tipifarnib II; IC50 35 nM IV; IC50 2.5 nM

22. Other Factors • Notes • FT-Inhibitors show potential as anticancer agents • Anticancer activity may not necessarily be due solely to FT-inhibition • FTIs inhibit farnesylation of H-Ras, N-Ras and K-Ras • But N-Ras and K-Ras can by prenylated by GGTase • GGTase provides alternative mechanism of attaching Ras to cell membranes • FTI’s still have anticancer activity in cells expressing excess K-Ras • Inhibition of FT may affect other cellular processes other than Ras