Design, Development, and Evaluation of Tools to Study Cellular ADP-ribose Polymer Metabolism

Abstract

The metabolism of ADP-ribose polymers (PAR) is involved in several cellular processes with a primary focus on maintaining genomic integrity. PAR metabolism following genotoxic stress is transient due to a close coordination between poly(ADP-ribose) polymerases (PARPs) which synthesize PAR and poly(ADP-ribose) glycohydrolase (PARG) which degrades PAR. PARP-1 inhibitors have emerged as promising anticancer therapeutics by increasing chemotherapy sensitivity and selectively target tumors harboring DNA repair defects. Several pharmaceutical companies have PARP-1 inhibitors in clinical trials for treatment of cancer. PARP-1 inhibitors are generally well tolerated, although they typically have poor selectivity among PARPs, and potentially other NAD binding enzymes. The promise of PARP-1 inhibitors as cancer therapeutics has led this dissertation research towards developing alternative tools and approaches to target PAR metabolism.One approach described is an evaluation of high-throughput PARP-1 screening assays as potential tools to discover new classes of PARP-1 inhibitors. These assays were compared to a widely used radiolabeling PARP-1 assay. They were found to offer several advantages that include simplicity, sensitivity, reproducibility, accuracy and eliminating the need for radioactive materials.The primary focus of this dissertation research was to develop PARG inhibitors as an alternative way of targeting PAR metabolism. Lack of viable genetically engineered animals, effective siRNA, and useful pharmacological 20 inhibitors has prevented PARG from being evaluated as a therapeutic target. This dissertation describes the first systematic approach, using Target related Affinity Profiling (TRAP) technology, for the discovery of PARG inhibitors. Identification of several hits led to the first detailed structural activity relationship (SAR) studies defining a pharmacophore for PARG inhibition. Interestingly, these molecules show varying degrees of PARP-1 inhibition, providing the first direct evidence for homology in the active sites of PARP-1 and PARG. Evaluation of a lead inhibitor has provided the first evidence for PARG inhibition in intact cells. Further optimization resulted in a cell permeable inhibitor with reduced toxicity and poor selectivity, providing evidence for a new class of inhibitors that disrupt PAR metabolism by inhibiting both enzymes. The use of dual PARG/PARP-1 inhibitors represents a new approach for therapeutic development of anticancer agents. Finally, directions aimed to overcome remaining challenges are discussed.