PROJECT SUMMARY: Over the last decade, essential metabolic dependencies of many cancers have been revealed. These dependencies have highlighted that certain nodes of metabolism are attractive drug targets to drive cancer cell death. For the most part, oncogenic metabolic proteins have not been drugged, and many lack obvious binding pockets for pharmacological manipulation. Among the most prominent examples of un-drugged metabolic cancer targets are creatine kinases (CKs). CKs are essential for growth and metastasis of numerous cancers, especially aggressive acute myeloid leukemias (AML). The essentiality of CKs for aggressive AMLs is distinct, as somatic tissues do not rely on CKs for viability. However, despite being a highly actionable drug target in AML, no potent inhibitors exist against CKs. We recently developed a mass spectrometric (MS) platform that allows for rapid screening of small molecules for covalent engagement with protein cysteines across the proteome. With preliminary data, we have combined this platform with a small molecule screening library to systematically identify drug leads targeting cysteines on un-drugged proteins. In doing so, we have identified a lead scaffold that is potently inhibitory against the CK family of enzymes, by selectively targeting a key active site cysteine residue. Moreover, at high nanomolar concentrations, this CK inhibitor is selectively cytotoxic to AML cancers that depend on CKs. The goal of this project is to develop this new scaffold class of CK inhibitor for clinical application in AML cancers. We will test the hypothesis that rational development of this scaffold will improve potency and AML toxicity by targeting key interactions at the CK active site pocket. Moreover, we will determine whether this new CK inhibitor class is an effective therapeutic in a mouse model of AML in vivo. In Aim 1 we will combine molecular modeling approaches and our preliminary SAR data to rationally develop a series of molecules to systematically probe inhibitory potency against the CK active site pocket. Activity and selectivity of these molecules will be determined using our MS platform, isolated CK kinetics, and cellular AML models. In parallel, using patient-derived cellular models of AML, for which CK is essential, we will determine clinically relevant disease-modifying outputs, including viability, colony forming, and on-target toxicity. In Aim 2 we will determine therapeutic efficacy of our CK inhibitor chemotype in mouse models EVI1-positive AML that rely essentially on CKs. Taken together, this project will advance a first-in-class inhibitor of CKs, which is an un-drugged metabolic dependency of AML cancers. Successful completion of these Aims would position this new chemotype for treatment of AML, and provide a new chemical probe for understanding the role CKs play in AML and cellular metabolism.