ABSTRACT Our overall goal, which is fully responsive to PAR-20-271, is to develop a selective and effective inhibitor of the multi-functional DNA repair enzyme exonuclease 1 (EXO1) that can be used both as a research tool (chemical probe) and as a pre-clinical starting point toward the development of a potential cancer therapeutic drug. There is no EXO1-specific small molecule inhibitor listed in the Chemical Probe Portal or other literature. We will achieve our goal through discovery research, from implementing a primary high-throughput screen (HTS) that we have already developed, to validating hits via a well-developed “critical path” of secondary assays, to performing early hit-to-lead optimization via purchase of commercially available analogs of validated chemical scaffolds and limited focused medicinal chemistry. EXO1 represents a druggable target, as it contains functionally essential exonuclease activity for double-strand break response and repair (DSBRR) for processing of stalled replication forks, which are critical pathways by which cells counteract endogenous DNA damage and replication stress. Compared to normal cells, cancer cells carry a significantly higher burden of double-strand breaks and replication stress, which generates a therapeutic window for treating cancer. To exploit this, current therapeutic approaches primarily target proteins acting in repair pathways or in checkpoint signaling pathways controlling repair. Many cancer cells are already defective in DSBRR; thus, EXO1 inhibition will cause cancer cell-specific cell death through a synthetic lethality mechanism. Furthermore, EXO1is will display greater specificity than currently used PARP inhibitors, because PARPs participate in a wide array of other cellular processes, whereas EXO1 does not. Our group was the first to clone the human EXO1 gene and to characterize its biochemical properties. We have expressed and purified the full-length and active EXO1 enzyme at scale, developed a robust fluorescence-based enzyme inhibition assay, and performed a pilot HTS in our own core facility. Thus, in collaboration with the Prebys Center of Sanford Burnham Prebys Medical Discovery Institute, we are well positioned to 1) identify inhibitors of EXO1 exonuclease by performing HTS of a well-curated ~320,000 compound library; 2) validate hits for potency and selectivity; 3) perform “structure-activity relationship (SAR)-by-catalog” and limited focused medicinal chemistry and benchmark absorption, distribution, metabolism, and excretion (ADME)/pharmacokinetic (PK) characterization of best probes; and 4) determine the mode of action (MOA) and biological effects of validated EXO1i candidate probes. All of our Aims are responsive to and within the scope of PAR-20-271. The development of novel EXO1is will not only allow us to provide a critical tool (i. e. chemical probe) to test mechanistic insights into the replication-repair interface but will also support development of a novel chemoth...