Project Summary Ubiquitin specific protease 8 (USP8) is a cysteine protease that can deubiquinate receptor tyrosine kinases. USP8 regulates endocytosis and through this activity modulates the degradation of diverse substrates by the autophagy- lysosome system. In oncology, USP8 has been shown to deubiquinate and stabilize mutant epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC). Preliminary studies show that USP8 inhibition promotes ubiquitin-mediated degradation of mutant EGFR in NSCLC cell lines and primary patient samples both sensitive and resistant to EGFR kinase inhibition. These results suggest that UPS8 inhibition could provide a therapeutic strategy for overcoming gefitinib-resistance in NSCLC. However, the USP8 inhibitor used in these preliminary studies is an early generation inhibitor that lacks the requisite potency and selectivity of a high-quality probe compound. The goal of this research program is to develop the first reversible USP8 inhibitors with the requisite potency, selectivity, and cell permeability to interrogate the potential of USP8 as a therapeutic target in NSCLC. The first aim is to perform medicinal chemistry optimization of lead scaffolds identified from a high throughput screen. I will design analogs of each compound to broadly survey regions of the molecule that are required for activity and positions that can be modified to modulate properties such as solubility and stability. For these studies, one position in the molecule will be modified at a time then productive changes will be combined. In the second aim, these inhibitors will be characterized with respect to their cellular target engagement, anti-proliferative effects, and effects on the stability of EGFR in mutant and wild type NSCLC cell lines. In the third aim, I will use the newly developed USP8 inhibitors as probes in phenotypic, proteomic, and transcriptomic assays across a broad set of cancer cell lines to uncover new USP8 biology in an unbiased manner. This proposal therefore outlines the development of the first USP8 inhibitors with the required potency and selectivity to be used as cellular probes to study USP8 biology, and contributes to evidence for USP8 inhibition as a therapeutic strategy for NSCLC.