Discovery of Novel Peptidic Inhibitors of the Non-Catalytic Function of Focal Adhesion Kinase (FAK)

PROJECT SUMMARY ABSTRACT Focal Adhesion KinaseFAKis a major cancer drug target that is overexpressed in multiple tumor typesFAK is a critical regulator of tumor survivalinvasionproliferationmetastasisand immune evasionCurrent FAK inhibitors that target the ATP binding pocket of the kinase domain do no effectively inhibit FAK in cancer because FAK also functions as a scaffolding proteinThe Focal Adhesion TargetingFATdomain of FAK is an interesting alternative drug target due to its requirement for FAK localizationactivityand downstream effectsDisruption and mutation of the FAT domain causes significant effects on tumor cell apoptosisproliferationinvasionand metastasisSpecificallythe FAT domain interacts with the alpha helical LDand LDmotifs of Paxillin to promote its biological effectsThe structure of the FAT Paxillin complex has been solved by x ray crystallography however has been challenging to target with small moleculesIn this projectwe will use hydrocarbon stapled alpha helical peptides that have the advantage of enhanced proteolytic stabilitycell permeabilityand potent inhibition of the entire protein interaction interfaceWe have preliminary data of Stapled Peptideshowing low micromolar inhibition of FAK Paxillin binding and NMR SPR data validating the binding site of the peptideIn the first specific aimwe will perform structure activity relationshipsSARon stapled alpha helical peptides for enhanced binding and competitive inhibitionWe will perform SAR on stapled peptides by changing hydrocarbon stapling strategymodifying Nand C terminal amino acidsand adding alternative amino acidsIn additionwe will utilize molecular modeling to optimize peptide protein contactssynthesize stapled peptides of homologous peptide sequencesand characterize biophysical biochemical properties of stapled peptidesIn the second specific aimwe will characterize and optimize lead peptides for cellular effectsWe will perform robust assays to measure cell permeability of stapled peptidescharacterize peptides for protease resistance and effects on membrane lysisand test peptides in cellular efficacy assays to assess the effects of stapled peptides on cancer cellsIn the third specific aimwe will test lead peptides with in vitro DMPK assays and preliminary in vivo efficacy modelsWe will characterize peptides using plasma bindingmetabolic stabilityand CYP inhibition assaysWe will also test lead peptides in mouse xenograft models alone and in combination with chemotherapyIn allthese specific aims will be used to discover peptide inhibitors of FAK non catalytic function that can be the basis for future clinical development PROJECT NARRATIVE This research will identify innovative peptide based drugs that target alternative regions of the cancer protein focal adhesion kinaseFAKIt will enhance technologies to target protein protein interactions in cancer cells and improve upon current therapies that target FAKFurthermorethis application will help establish peptide chemistry that can be used to create cell permeable peptide drugs