Slide 1

ANTICANCER AGENTSANTICANCER AGENTS

FARNESYL TRANSFERASE INHIBITORSFARNESYL TRANSFERASE INHIBITORS

Chapter 21Chapter 21

Ras Protein Ras Protein

NotesNotes

•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 Farnesyl transferase

NotesNotes

•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

FTaseFTase

FurtherFurther

processingprocessing

Methyl esterMethyl ester

Farnesyl transferase Farnesyl transferase

Enzyme mechanismEnzyme mechanism

Farnesyl transferaseFarnesyl transferase

NotesNotes

•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 SubstratesFT Substrates

C-a-a-XC-a-a-X

SubstrateSubstrate

•C = cysteine

• a = valine, isoleucine or leucine

•X = methionine, glutamine or serine

Substrates share a terminal tetrapeptide moiety called the CaaX peptideSubstrates share a terminal tetrapeptide moiety called the CaaX peptide

FT InhibitorsFT Inhibitors

AimsAims

•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 InhibitorsFT Inhibitors

NotesNotes

•Inhibitors were developed to mimic the terminal tetrapeptide moiety - CaaX peptide•Inhibitors were developed to mimic the terminal tetrapeptide moiety - CaaX peptide

•Tetrapeptides having Phe next to X act as inhibitors

•Serve as lead compounds

C-a-a-XC-a-a-X

SubstrateSubstrate

C-a-Phe-XC-a-Phe-X

InhibitorInhibitor

•C = cysteine

• a = valine, isoleucine or leucine

•X = methionine, glutamine or serine

Lead compoundLead compound

CysCys

ValVal

PhePhe

MetMet

DisadvantagesDisadvantages

•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)

Lead compoundLead compound

CysCys

ValVal

PhePhe

MetMet

Drug designDrug design

NotesNotes

•Modifications carried out to remove peptide nature - peptidomimetics•Modifications carried out to remove peptide nature - peptidomimetics

•Ester masks polar carboxylic acid or carboxylate ion - acts as prodrug•Ester masks polar carboxylic acid or carboxylate ion - acts as prodrug

•Methyleneamino link replaces N-terminal peptide bond•Methyleneamino link replaces N-terminal peptide bond

•Methyleneamino link introduces a resistance to aminopeptidases•Methyleneamino link introduces a resistance to aminopeptidases

•Peptide bond isostere introduced to mimic central peptide bond•Peptide bond isostere introduced to mimic central peptide bond

•Isostere should be capable of mimicing any binding interactions•Isostere should be capable of mimicing any binding interactions

•Isostere should be stable to enzyme-catalysed hydrolysis•Isostere should be stable to enzyme-catalysed hydrolysis

PeptidomimeticPeptidomimetic

Methylene-Methylene-

amino linkamino link

PeptidomimeticPeptidomimetic

EsterEster

Methylene-Methylene-

amino linkamino link

PeptidomimeticPeptidomimetic

Peptide bondisosterePeptide bondisostere

EsterEster

Methylene-Methylene-

amino linkamino link

PeptidomimeticPeptidomimetic

Examples of FT InhibitorsExamples of FT Inhibitors

R=H FTI 276

R=iPr FTI 277

TerminalTerminal

amino groupamino group

ThiolThiol

AromaticAromatic

substituentsubstituent

NotesNotes

•Thiol group forms important interactions with the zinc ion cofactor•Thiol group forms important interactions with the zinc ion cofactor

•Methyleneamino link is stable to aminopeptidases•Methyleneamino link is stable to aminopeptidases

•Aromatic substituent is important for inhibitory activity•Aromatic substituent is important for inhibitory activity

•Aromatic ring acts as a peptide bond isostere•Aromatic ring acts as a peptide bond isostere

•Terminal amino group is ionised•Terminal amino group is ionised

•Terminal amino group forms an ionic bond to the phosphate group of FPP•Terminal amino group forms an ionic bond to the phosphate group of FPP

•Terminal carboxylate group is important to binding•Terminal carboxylate group is important to binding

Stablemethylene-amino linkStablemethylene-amino link

Peptide bondPeptide bond

isostereisostere

Stablemethylene-amino linkStablemethylene-amino link

Examples of FT InhibitorsExamples of FT Inhibitors

SulfoneSulfone

AromaticAromatic

substituentsubstituent

TerminalTerminal

amino groupamino group

ThiolThiol

R=H L739750

R=iPr L744832

NotesNotes

•Thiol group forms important interactions with the zinc ion cofactor•Thiol group forms important interactions with the zinc ion cofactor

•Methyleneamino link is stable to aminopeptidases•Methyleneamino link is stable to aminopeptidases

•Aromatic substituent is important for inhibitory activity•Aromatic substituent is important for inhibitory activity

•Methyleneoxy group acts as the peptide bond isostere•Methyleneoxy group acts as the peptide bond isostere

•Terminal amino group is ionised•Terminal amino group is ionised

•Terminal amino group forms an ionic bond to the phosphate group of FPP•Terminal amino group forms an ionic bond to the phosphate group of FPP

•Terminal carboxylate group is important to binding•Terminal carboxylate group is important to binding

•Sulfone increases activity over a methylthio group•Sulfone increases activity over a methylthio group

Peptide bondPeptide bond

isostereisostere

Stablemethylene-amino linkStablemethylene-amino link

AZD-3409AZD-3409

PyrrolidinePyrrolidine

AromaticsubstituentAromaticsubstituent

Examples of FT InhibitorsExamples of FT Inhibitors

NotesNotes

•Thiol and carboxylic acid groups are both masked in the prodrug•Thiol and carboxylic acid groups are both masked in the prodrug

•Lowers the toxicity risk of the thiol group•Lowers the toxicity risk of the thiol group

•Protects the thiol from possible metabolism•Protects the thiol from possible metabolism

•Pyrrolidine ring introduces conformational rigidity•Pyrrolidine ring introduces conformational rigidity

•Potent inhibitor (Ki < 1 nM)•Potent inhibitor (Ki < 1 nM)

•Also inhibits geranylgeranyltransferase - catalyses prenylation withgeranylgeranyl diphosphate•Also inhibits geranylgeranyltransferase - catalyses prenylation withgeranylgeranyl diphosphate

•Agents inhibiting both enzymes are potentially advantageous•Agents inhibiting both enzymes are potentially advantageous

MaskinggroupMaskinggroup

AZD-3409AZD-3409

Peptide bondPeptide bond

isostereisostere

MaskinggroupMaskinggroup

AZD-3409AZD-3409

Structure IStructure I

IC50 1.4 nMIC50 1.4 nM

Examples of FT InhibitorsExamples of FT Inhibitors

NotesNotes

•Non-peptide inhibitor•Non-peptide inhibitor

•Imidazole ring acts as the zinc ligand•Imidazole ring acts as the zinc ligand

•Decreases the risk of toxicity due to a free thiol group•Decreases the risk of toxicity due to a free thiol group

Imidazole ringImidazole ring

Structure IStructure I

IC50 1.4 nMIC50 1.4 nM

LonafarnibLonafarnib

IC50 1.9 nMIC50 1.9 nM

Examples of FT InhibitorsExamples of FT Inhibitors

NotesNotes

•Non-peptide inhibitor•Non-peptide inhibitor

•Developed from lead compound discovered by screening compound libraries•Developed from lead compound discovered by screening compound libraries

•10,000 times more active than the lead compound•10,000 times more active than the lead compound

•No ligand for the zinc cofactor is present!•No ligand for the zinc cofactor is present!

Examples of FT InhibitorsExamples of FT Inhibitors

•Non-peptide inhibitor

•Developed from lonafarnib by structure-based drug design

•Imidazole ring introduced as zinc ligand

•Aromatic ring introduced as a steric shieldvs metabolism

Sch 226374Sch 226374

IC50 0.36 nMIC50 0.36 nM

LonafarnibLonafarnib

IC50 1.9 nMIC50 1.9 nM

StericSteric

shieldshield

Sch 226374Sch 226374

IC50 0.36 nMIC50 0.36 nM

ImidazoleImidazole

ringring

StericSteric

shieldshield

Sch 226374Sch 226374

IC50 0.36 nMIC50 0.36 nM

Development of TipifarnibDevelopment of Tipifarnib

•Lead compound

•Identified from screening compound libraries

•Imidazole ring present - zinc ligand

•Both aromatic rings are important to activity

I; IC50 180 nMI; IC50 180 nM

ImidazoleImidazole

QuinoloneQuinolone

I; IC50 180 nMI; IC50 180 nM

•Strategy - variation of substituents

•Activity increases with introductionof meta-chloro substituent

II; IC50 35 nMII; IC50 35 nM

Development of TipifarnibDevelopment of Tipifarnib

ImidazoleImidazole

QuinoloneQuinolone

I; IC50 180 nMI; IC50 180 nM

•Strategy - variation of substituents

•Activity increases with addition ofN-methyl substituent

II; IC50 35 nMII; IC50 35 nM

III; IC50 15 nMIII; IC50 15 nM

Development of Tipifarnib Development of Tipifarnib

ImidazoleImidazole

QuinoloneQuinolone

I; IC50 180 nMI; IC50 180 nM

II; IC50 35 nMII; IC50 35 nM

III; IC50 15 nMIII; IC50 15 nM

IV; IC50 2.5 nMIV; IC50 2.5 nM

•Strategy - variation of ring substitution

•Activity increases

Development of Tipifarnib Development of Tipifarnib

ImidazoleImidazole

QuinoloneQuinolone

I; IC50 180 nMI; IC50 180 nM

III; IC50 15 nMIII; IC50 15 nM

IV; IC50 2.5 nMIV; IC50 2.5 nM

Tipifarnib; IC50 0.6 nMTipifarnib; IC50 0.6 nM

•Extension strategy

•Extra functional group

•Extra binding interactions

•Activity increases

Development of TipifarnibDevelopment of Tipifarnib

ImidazoleImidazole

QuinoloneQuinolone

I; IC50 180 nMI; IC50 180 nM

II; IC50 35 nMII; IC50 35 nM

Other FactorsOther Factors

NotesNotes

•FT-Inhibitors show potential as anticancer agents•FT-Inhibitors show potential as anticancer agents

•Anticancer activity may not necessarily be due solely to FT-inhibition•Anticancer activity may not necessarily be due solely to FT-inhibition

•FTIs inhibit farnesylation of H-Ras, N-Ras and K-Ras•FTIs inhibit farnesylation of H-Ras, N-Ras and K-Ras

•But N-Ras and K-Ras can by prenylated by GGTase•But N-Ras and K-Ras can by prenylated by GGTase

•GGTase provides alternative mechanism of attaching Ras to cell membranes•GGTase provides alternative mechanism of attaching Ras to cell membranes

•FTI’s still have anticancer activity in cells expressing excess K-Ras•FTI’s still have anticancer activity in cells expressing excess K-Ras

•Inhibition of FT may affect other cellular processes other than Ras•Inhibition of FT may affect other cellular processes other than Ras