PROJECT SUMMARY Melanoma is an extremely aggressive cancer with high mortality. Its phenotypic plasticity and heterogeneity enable it to adapt to diverse physiological settings and defeat treatment approaches. Targeted therapies and check point inhibitors became available in the past decade; however, these drugs work in only a subset of patients and drug resistance eventually emerges, even in initial responders. Therefore, new targets and clinical approaches for melanoma are an unmet medical need. We identified glutaryl-CoA dehydrogenase (GCDH) as one such target. GCDH expression correlates with aggressive cancers and low survival in melanoma patients. Our data demonstrates that GCDH knockdown results in apoptotic cell death in melanoma cells. Melanomas seem uniquely sensitive to toxic glutarate metabolites resulting from GCDH deficiencies, since suppression of the Dehydrogenase E1 and Transketolase Domain Containing 1 (DHTKD1) enzyme, catalyzing the preceding reaction in the catabolic pathway and converting 2-oxoadipate to glutaryl-CoA, rescues melanoma cells from apoptosis. Pancreatic cancer cells, but not other cancers nor normal cells, share this overreliance on GCDH and undergo apoptosis upon GCDH knockdown. We hypothesize that small-molecules interferring with GCDH will result in the obliteration of melanoma cells through apoptosis. We propose to identify chemical probes of GCDH to further validate the enzyme as a molecular target for melanoma. In a pilot screen, we established and validated all of the assays proposed herein. We will perform large-scale HTS, hit confirmation and optimization, and validate the identified hits in a panel of diverse cell lines for melanoma and other cancers. Compounds identified will provide desirable pharmacological tools to study the pathophysiology of GCDH in melanoma and other cancers, and the molecular mechanisms of lysine metabolism liability in melanoma, as well as provide potential starting points for future therapeutic treatments. This 4-year project will pursue the following Specific Aims, consistent with the expectations in PAR-20-271: AIM 1 Generate GCDH protein, optimize conditions and perform screening to identify compounds targeting GCDH. Optimize tertiary assays for hit validation. Primary assays will target binding activity and inhibition, and tertiary assays will monitor cellular target engagement and protein level. AIM 2 Perform hit selection, confirmation and profiling using a panel of secondary assays. Functional hit profiles will be established using inhibition assays for a representative of acyl-CoA dehydrogenases and mechanism of action studies by using protein thermal shift with substrate analogs and enzyme kinetic studies to establish competition profiles with the substrates. AIM 3 Perform hit validation and scaffold prioritization using biochemical and cellular assays. GCDH target engagement will be confirmed in an in-cell protein stability assay. Effects on cellular protein levels w...