Chemical and Genetic Tools for Probing the Cooperativity of Molecular Chaperones

The cytosol of the mammalian cell is a highly crowded environment, where all of the molecular processes necessary for sustaining life take place. In order to maintain a healthy balance of protein synthesis, folding, trafficking, and degradation in this environment, the cell relies heavily on a class of proteins known as molecular chaperones. These chaperones, such as the 70 and 90 kDa heat shock proteins (Hsp70 and Hsp90), are the most abundant proteins in the cell, and make up a highly interconnected, cooperative network of hundreds of individual members. Recent efforts have been made to explore chaperones as drug targets, due to their implications in various diseases, including cancer and neurodegeneration. However, these proteins have proven to be extremely difficult drug targets, in large part due to the complexity of their functions in a cell. It has become increasingly clear that before we can develop successful therapeutics for this system, there is a need for new tools to dissect the underlying biology of the chaperone network. This thesis describes efforts from our group toward building a set of tools to study the cooperative nature of molecular chaperones. This cooperation exists on various levels, from the interdomain allostery of the Hsp70 protein itself, to this chaperone’s interactions with its co-chaperones, and ultimately its role in the greater network of protein homeostasis (proteostasis). We carried out a high throughput ATPase screen to identify a new chemical scaffold for inhibiting the interaction of Hsp70 with its nucleotide exchange factor class of co-chaperones. This inhibitor series was further optimized through synthetic diversification, and secondary biochemical assays. Additionally, we aimed to increase the power of established chemical probes by combining them with a chaperone-centric functional genomic platform, in the form of an shRNA screen. We screened a focused chaperone shRNA library in combination with chaperone inhibitors in various cell lines to identify targetable weaknesses in the proteostasis network. Ultimately, the work herein presents a starting point for the synthesis of new allosteric inhibitors of Hsp70, as well as novel targets for the development of advanced prostate cancer therapeutics