Friday, April 26, 2024
4/26/2024
PROJECT SUMMARY ABSTRACT Focal Adhesion Kinase (FAK) is a major cancer drug target that is overexpressed in multiple tumor types. FAK is a critical regulator of tumor survival, invasion, proliferation, metastasis, and immune evasion. Current 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 protein. The Focal Adhesion Targeting (FAT) domain of FAK is an interesting alternative drug target due to its requirement for FAK localization, activity, and downstream effects. Disruption and mutation of the FAT domain causes significant effects on tumor cell apoptosis, proliferation, invasion, and metastasis. Specifically, the FAT domain interacts with the alpha helical LD2 and LD4 motifs of Paxillin to promote its biological effects. The structure of the FAT-Paxillin complex has been solved by x-ray crystallography however has been challenging to target with small molecules. In this project, we will use hydrocarbon stapled alpha helical peptides that have the advantage of enhanced proteolytic stability, cell permeability, and potent inhibition of the entire protein interaction interface. We have preliminary data of Stapled Peptide 3 showing low micromolar inhibition of FAK-Paxillin binding and NMR/SPR data validating the binding site of the peptide. In the first specific aim, we will perform structure-activity relationships (SAR) on stapled alpha helical peptides for enhanced binding and competitive inhibition. We will perform SAR on stapled peptides by changing hydrocarbon stapling strategy, modifying N- and C-terminal amino acids, and adding alternative amino acids. In addition, we will utilize molecular modeling to optimize peptide-protein contacts, synthesize stapled peptides of homologous peptide sequences, and characterize biophysical/biochemical properties of stapled peptides. In the second specific aim, we will characterize and optimize lead peptides for cellular effects. We will perform robust assays to measure cell permeability of stapled peptides, characterize peptides for protease resistance and effects on membrane lysis, and test peptides in cellular efficacy assays to assess the effects of stapled peptides on cancer cells. In the third specific aim, we will test lead peptides with in vitro DMPK assays and preliminary in vivo efficacy models. We will characterize peptides using plasma binding, metabolic stability, and CYP inhibition/assays. We will also test lead peptides in mouse xenograft models alone and in combination with chemotherapy. In all, these specific aims will be used to discover peptide inhibitors of FAK non-catalytic function that can be the basis for future clinical development.
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