DOI: 10.25549/usctheses-c40-382548
title: Development of specific chemical tools to dissect protein functions

description: Eukaryotic protein kinases are a large family of enzymes that catalyze the phosphoryl group transfer from ATP to substrate proteins to regulate numerous essential biological processes. Many protein kinases have been identified as attractive targets for cancer therapy. However, it is rather challenging to develop small-molecule agents that specifically target a single kinase because of the large number of kinases and the high conservation within the kinase family. To meet this challenge, our laboratory has developed a chemical-genetic approach which exploits the power and efficiency of covalent chemistry. The approach involves introducing a cysteine residue to the active site to sensitize kinases of interest to selective, covalent blockade. This dissertation details my efforts towards the design, synthesis, and biological characterization of novel electrophilic small molecules for covalently and specifically targeting diverse proteins. ❧ Chapter 1 focuses on the creation of isoform-selective RAF inhibitors. RAFs, as the top player of kinase-cascade in mitogen-activated protein kinase (MAPK) signaling, play critical roles in cell proliferation, differentiation and survival. The three isoforms of RAFs in human are highly homologous in sequence and have overlapping but non-redundant functions. To dissect individual RAF’s function in MAPK signaling, we introduced a cysteine residue to the active site of BRAF to sensitize the kinase to electrophilic quinazolines. We optimized these inhibitors based on the known SAR from clinical anticancer agents, and further demonstrated these electrophilic inhibitors covalently and specifically target cysteine-engineered RAFs but not the wild-type ones in cells. ❧ Chapter 2 describes the deconvolution of RAF inhibitor polypharmocology using the isoform-selective RAF inhibitors created with our chemical-genetic approach. Although two FDA-approved RAF inhibitors, vemurafenib and dabrafenib, produce remarkable clinical efficacy initially, patients develop resistance to such therapies rapidly. Most of the resistance mechanisms elucidated recently converge on the formation of RAF dimers that appear to be resistant to vemurafenib and dabrafenib. Close structural analogs of vemurafenib have been recently reported to overcome the resistance mediated by dimeric RAFs, but the mechanism for these so-called paradox-breakers is poorly understood. Employing the chemical-genetic approach, we succeeded in generating two isoform-selective RAF inhibitors that mimics vemurafenib or the paradox breaker. These unprecedented tools allowed us to systematically dissect the effects of inhibitor binding on the activity and dimerization of various RAF dimers in cells for the first time. Our findings provide novel insights into the complex actions of RAF inhibitors and can help guide the design of improved RAF inhibitors in the future. ❧ Chapter 3 centers on the identification and characterization of novel covalent modulators for the vacuolar ATPase. The vacuolar H+ ATPase (V-ATPase) is a complex multisubunit machine that regulates important cellular processes through controlling acidity of intracellular compartments in eukaryotes. We have discovered electrophilic quinazolines that covalently modify a soluble catalytic subunit of V-ATPase with high potency and exquisite proteomic selectivity as revealed by fluorescence imaging and chemical proteomic activity-based profiling. The site of covalent modification was mapped to a cysteine residue located in a region of V-ATPase subunit A that is thought to regulate the dissociation of V-ATPase. With well-defined mechanism of action and high proteomic specificity, the described quinazolines offer a powerful set of chemical probes to investigate the physiological and pathological roles of V-ATPase.