FARNESYL TRANSFERASE INHIBITORS ANTICANCER AGENTS FARNESYL TRANSFERASE INHIBITORS Chapter 21

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)

Farnesyl transferase 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 farnesyl transferase should inhibit this process

Farnesyl transferase Enzyme mechanism FTase Further processing Methyl ester

Farnesyl transferase Notes Farnesyl diphosphate (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

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

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

FT Inhibitors C-a-a-X Substrate C-a-Phe-X Inhibitor C = cysteine a = valine, isoleucine or leucine X = methionine, glutamine or serine 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

Lead compound Cys Val Phe Met 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)

Drug design Peptidomimetic Lead compound Peptidomimetic Peptidomimetic Peptide bond isostere Ester Methylene- amino link Peptidomimetic Lead compound Cys Val Phe Met Ester Methylene- amino link Peptidomimetic Peptidomimetic Methylene- amino link Peptidomimetic 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

Examples of FT Inhibitors Peptide bond isostere Stable methylene-amino link Stable methylene-amino link Terminal amino group Thiol Aromatic substituent R=H FTI 276 R=iPr FTI 277 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

Stable methylene-amino link Examples of FT Inhibitors Peptide bond isostere Stable methylene-amino link Sulfone Aromatic substituent Terminal amino group Thiol R=H L739750 R=iPr L744832 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

Examples of FT Inhibitors Peptide bond isostere Masking group AZD-3409 Masking group AZD-3409 AZD-3409 Pyrrolidine Aromatic substituent 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 geranylgeranyl diphosphate Agents inhibiting both enzymes are potentially advantageous

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

Examples of FT Inhibitors Lonafarnib IC50 1.9 nM 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!

Examples of FT Inhibitors Lonafarnib IC50 1.9 nM Imidazole ring Steric shield Sch 226374 IC50 0.36 nM Steric shield 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

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

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

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

FT-Inhibitors show potential as anticancer agents 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