The overall goal of this proposal is to dissect the molecular mechanisms of metabolic regulation of ATP-citrate lyase (ACLY) and to characterize ACLY inhibitors for cancer therapy. ACLY is the predominant source of nucleocytosolic acetyl-CoA, an essential building block for the production of fatty acids, cholesterol, isoprenoids and protein acetylation. Elevated ACLY activity is found in metabolic disorders, cardiovascular diseases and many cancers, prompting the development of several ACLY inhibitors. While many ACLY inhibitors have been developed, only bempedoic acid, which forms an active bempedoyl-CoA adduct in hepatocytes, has been approved by the FDA for therapeutic use. Risk of hepatocellular carcinoma is elevated in individuals with metabolic disorders, many of whom may be candidates for treatment with bempedoic acid; yet metabolic regulation of ACLY activity and its functional role in hepatocellular carcinoma remain poorly understood. Elevated levels of ACLY acetylation at K540, K546 and K554 and phosphorylation at S455 and S481, and retention of exon 14 encoding a region with S481, have also been correlated with cancer, thus also suggesting roles for ACLY posttranslational and posttranscriptional modification in cancer metabolism. ACLY is an ~500 kD multidomain homotetrameric enzyme that uses citrate, CoA and ATP cosubstrates to produce oxaloacetate (OAA) and acetyl-CoA. Until recently, the lack of structural information on intact human ACLY has hampered understanding of its molecular mechanism of catalysis and the structure-based development of inhibitors. The Wellen lab recently reported on various disease-associated phenotypes associated with dysregulated ACLY function; and the Marmorstein lab reported on the cryo-EM structures of ACLY in different reaction states, along with associated biochemical and biophysical studies, to elucidate the molecular basis for acetyl-CoA production by ACLY. The latter findings lead to several unresolved questions underlying the metabolic regulation of ACLY and set the stage for the structure-based development of more potent and selective ACLY inhibitors for therapeutic applications. These recent studies now position the Wellen and Marmorstein labs to work together to resolve important gaps in knowledge in metabolic regulation and inhibition of ACLY, through the following specific aims: (1) Evaluate the role of metabolic binders in ACLY activity, (2) Determine the molecular mechanism of how posttranslational modifications and exon 14 retention impact ACLY regulation, and (3) Evaluate the molecular mode of action of ACLY inhibitors. Together, these studies will reveal the molecular mechanisms for how ACLY activity and regulation is mediated by the binding of metabolites, and posttranscriptional and posttranslational modification and will lead to the rational development of ACLY drugs to treat cancer.