Chemical Biology Strategies for the Control of Protein Function and the Interrogation of Cyclin/CDK Interactions

Abstract

Protein-protein interactions (PPIs) are ubiquitous in the cell, and it is difficult to overstate their importance in biology. They form the backbone for a majority of signaling pathways, they are required for post-translational modifications, and for transcription and translation. Hence, aberrant PPIs have been implicated in several disease states and they have become important drug targets. A lot of work has gone into understanding the features of PPIs and what drives them with the goals of predicting them a priori as well as using them as tools in biochemistry. In this dissertation, we utilize and interrogate specific protein-protein interactions for different purposes in each chapter. In Chapter 2, we detail our efforts toward developing potent and selective bivalent inhibitors for protein phosphatase 1 (PP1). The bivalent inhibitor strategy involves utilizing the Jun-Fos coiled coil interaction to aid in discovering bivalent inhibitors, a strategy successfully used for protein kinases. Even though we did not discover bivalent inhibitors for PP1, we demonstrate that acid-amide analogs of norcantharidin are unstable and not effective phosphatase inhibitors. Chapter 3 follows our studies toward engineering an orthogonal Bcl-xL/Bad interface for studying biochemistry. We make several rational mutations on Bcl-xL and use a Bad phage-displayed library with the goal of selecting high affinity and potentially selective Bad peptides. In the process, we obtain a Bad peptide blueprint for Bcl-xL and Bcl-xL R139A via phage display. Finally, in Chapter 4, we profile cyclin/CDK interactions in vitro and in cell lysate using the split-luciferase assay. We confirm most of the interactions known to date in this assay, and we discover many new interactions and highlight their potential significance in signaling pathways and in diseases.