ANTICANCER AGENTS FARNESYL TRANSFERASE INHIBITORS Chapter 21

ANTICANCER AGENTSANTICANCER AGENTSFARNESYL TRANSFERASE INHIBITORSFARNESYL TRANSFERASE INHIBITORS

Chapter 21Chapter 21

Ras ProteinRas 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 transferaseFarnesyl 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

PPO

farnesyl diphosphate

Ras

HN

NH

HN

NH

O

O

O

OH

OHS

S

MetVal

Cys FTaseFTaseRas

HN

NH

HN

NH

O

O

O

OH

OS

S

FurtherFurtherprocessingprocessing

Ras

HN

OMe

O

S

Methyl esterMethyl ester

Farnesyl transferaseFarnesyl transferaseEnzyme mechanismEnzyme mechanism

PPO

farnesyl diphosphate

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 peptideInhibitors 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

H2NNH

HN

NH

OH

O

O

HS

SMe

O

O

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

H2NNH

HN

NH

OH

O

O

HS

SMe

O

O

CysCys

ValVal

PhePhe

MetMet

Drug designDrug design

NotesNotes•Modifications carried out to remove peptide nature - peptidomimeticsModifications carried out to remove peptide nature - peptidomimetics•Ester masks polar carboxylic acid or carboxylate ion - acts as prodrugEster masks polar carboxylic acid or carboxylate ion - acts as prodrug•Methyleneamino link replaces Methyleneamino link replaces NN-terminal peptide bond -terminal peptide bond •Methyleneamino link introduces a resistance to aminopeptidasesMethyleneamino link introduces a resistance to aminopeptidases•Peptide bond isostere introduced to mimic central peptide bondPeptide bond isostere introduced to mimic central peptide bond•Isostere should be capable of mimicing any binding interactionsIsostere should be capable of mimicing any binding interactions•Isostere should be stable to enzyme-catalysed hydrolysisIsostere should be stable to enzyme-catalysed hydrolysis

H2NNH

XY

NH

OR

O

O

HS

SMe

PeptidomimeticPeptidomimetic

Methylene-Methylene-amino linkamino link

H2NNH

XY

NH

OR

O

O

HS

SMe


EsterEster


H2NNH

XY

NH

OR

O

O

HS

SMe


Peptide bond Peptide bond isostereisostere

EsterEster


H2NNH

XY

NH

OR

O

O

HS

SMe


Examples of FT InhibitorsExamples of FT Inhibitors

H2NNH

HS

NH

O

OR

O

SMe

R=H FTI 276R=iPr FTI 277

TerminalTerminalamino groupamino group

ThiolThiol AromaticAromaticsubstituentsubstituent

NotesNotes•Thiol group forms important interactions with the zinc ion cofactorThiol group forms important interactions with the zinc ion cofactor•Methyleneamino link is stable to aminopeptidasesMethyleneamino link is stable to aminopeptidases•Aromatic substituent is important for inhibitory activityAromatic substituent is important for inhibitory activity•Aromatic ring acts as a peptide bond isostereAromatic 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 FPPTerminal amino group forms an ionic bond to the phosphate group of FPP•Terminal carboxylate group is important to bindingTerminal carboxylate group is important to binding

Stable Stable methylene-methylene-amino linkamino link

H2NNH

HS

NH

O

OR

O

SMe

Peptide bondPeptide bondisostereisostere


H2NNH

HS

NH

O

OR

O

SMe


H2NNH

ONH

OR

O

O

HS

SO2Me

SulfoneSulfone

AromaticAromaticsubstituentsubstituent

TerminalTerminalamino groupamino group

ThiolThiol

R=H L739750R=iPr L744832

NotesNotes•Thiol group forms important interactions with the zinc ion cofactorThiol group forms important interactions with the zinc ion cofactor•Methyleneamino link is stable to aminopeptidasesMethyleneamino link is stable to aminopeptidases•Aromatic substituent is important for inhibitory activityAromatic substituent is important for inhibitory activity•Methyleneoxy group acts as the peptide bond isostereMethyleneoxy 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 FPPTerminal amino group forms an ionic bond to the phosphate group of FPP•Terminal carboxylate group is important to bindingTerminal carboxylate group is important to binding•Sulfone increases activity over a methylthio groupSulfone increases activity over a methylthio group



H2NNH

ONH

OR

O

O

HS

SO2Me

AZD-3409AZD-3409HN

NH

O

O

O

F

NH

S

O

N

SMe

PyrrolidinePyrrolidine

Aromatic Aromatic substituentsubstituent


NotesNotes•Thiol and carboxylic acid groups are both masked in the prodrugThiol and carboxylic acid groups are both masked in the prodrug•Lowers the toxicity risk of the thiol groupLowers the toxicity risk of the thiol group•Protects the thiol from possible metabolismProtects the thiol from possible metabolism•Pyrrolidine ring introduces conformational rigidityPyrrolidine ring introduces conformational rigidity•Potent inhibitor (KPotent inhibitor (Kii < 1 nM) < 1 nM)•Also inhibits geranylgeranyltransferase - catalyses prenylation with Also inhibits geranylgeranyltransferase - catalyses prenylation with geranylgeranyl diphosphategeranylgeranyl diphosphate•Agents inhibiting both enzymes are potentially advantageousAgents inhibiting both enzymes are potentially advantageous

Masking Masking groupgroup


AZD-3409AZD-3409HN

NH

O

O

O

F

NH

S

O

N

SMe




AZD-3409AZD-3409HN

NH

O

O

O

F

NH

S

O

N

SMe

O

SN

NN

NH

ONC

S

Structure IStructure IICIC5050 1.4 nM 1.4 nM


NotesNotes•Non-peptide inhibitorNon-peptide inhibitor•Imidazole ring acts as the zinc ligandImidazole ring acts as the zinc ligand•Decreases the risk of toxicity due to a free thiol groupDecreases the risk of toxicity due to a free thiol group

Imidazole ringImidazole ring

O

SN

NN

NH

ONC

S

Structure IStructure IICIC5050 1.4 nM 1.4 nM

N

Br

N

Br

Cl

O

N NH2

O

Lonafarnib Lonafarnib ICIC5050 1.9 nM 1.9 nM


NotesNotes•Non-peptide inhibitor Non-peptide inhibitor •Developed from lead compound discovered by screening compound librariesDeveloped from lead compound discovered by screening compound libraries•10,000 times more active than the lead compound10,000 times more active than the lead compound•No ligand for the zinc cofactor is present! No ligand for the zinc cofactor is present!


•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

NN

N

Cl

O O

N

O

NN

Me

Sch 226374 Sch 226374 ICIC5050 0.36 nM 0.36 nM

N

Br

N

Br

Cl

O

N NH2

O

Lonafarnib Lonafarnib ICIC5050 1.9 nM 1.9 nM

Steric Steric shieldshield

NN

N

Cl

O O

N

O

NN

Me

Sch 226374 Sch 226374 ICIC5050 0.36 nM 0.36 nM

Imidazole Imidazole ringring

Steric Steric shieldshield

NN

N

Cl

O O

N

O

NN

Me

Sch 226374 Sch 226374 ICIC5050 0.36 nM 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

N

HN

Cl

N

O

I; ICI; IC5050 180 nM 180 nM

ImidazoleImidazole

QuinoloneQuinolone

N

HN

Cl

N

O

I; ICI; IC5050 180 nM 180 nM

•Strategy - variation of substituents •Activity increases with introduction of meta-chloro substituent

N

HN

Cl

N

O

Cl

II; ICII; IC5050 35 nM 35 nM


ImidazoleImidazole

QuinoloneQuinolone

N

HN

Cl

N

O

I; ICI; IC5050 180 nM 180 nM

•Strategy - variation of substituents •Activity increases with addition of N-methyl substituent

N

HN

Cl

N

O

Cl


N

MeN

Cl

N

O

Cl

III; ICIII; IC5050 15 nM 15 nM


ImidazoleImidazole

QuinoloneQuinolone

N

HN

Cl

N

O

I; ICI; IC5050 180 nM 180 nM

N

HN

Cl

N

O

Cl


N

MeN

Cl

N

O

Cl


MeN

ClN

N

O

Cl

Me

IV; ICIV; IC5050 2.5 nM 2.5 nM

•Strategy - variation of ring substitution •Activity increases


ImidazoleImidazole

QuinoloneQuinolone

N

HN

Cl

N

O

I; ICI; IC5050 180 nM 180 nM

N

MeN

Cl

N

O

Cl


IV; ICIV; IC5050 2.5 nM 2.5 nM

MeN

Cl

ClN

N

Me

H2N

O

Tipifarnib; ICTipifarnib; IC5050 0.6 nM 0.6 nM

•Extension strategy •Extra functional group•Extra binding interactions•Activity increases


ImidazoleImidazole

QuinoloneQuinolone

N

HN

Cl

N

O

I; ICI; IC5050 180 nM 180 nM

N

HN

Cl

N

O

Cl


Other FactorsOther Factors

NotesNotes

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

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

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

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

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

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

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ANTICANCER AGENTS FARNESYL TRANSFERASE INHIBITORS Chapter 21