Chemical Biology of the Control of Neddylation by DCN1

The long-term goal is to generate and use chemical and genetic probes to study the cullin RING ubiquitin ligases (CRL’s) and understand their activation controlled by the interaction of by the Defective in Cullin Neddylation 1 (DCN1) and UBE2M proteins. Because the CRL’s ultimately control ubiquitination of many diverse proteins, thus regulating their stability, intracellular localization, and function, having spatiotemporal control over DCN1 mediated CRL activity has the potential to unravel the mechanism regulating key cellular signaling networks. The health relatedness of this project lies in two facts: 1) DCN1 is an oncoprotein, amplified in squamous cell carcinomas, that drives a highly malignant phenotype, and 2) CRL driven ubiquitination is a validated target in multiple diseases, particularly cancer and immune dysfunction. Therefore, inhibitors of the DCN1-UB2M interaction that are potent, selective, and bioavailable have the potential to be developed as antitumor drugs and possibly for other diseases. The exact nature of how ubiquitination is regulated and in turn regulates protein homeostasis has proven opaque to traditional genetics due to the highly complex and redundant nature of the ubiquitination network. Inhibitors of DCN1-UB2M will allow direct interrogation of the function of sub-portions of the network and are likely to unveil fundamental principles of the regulation of protein homeostasis. The generation of complementary cellular and mouse genetic models will enable independent verification of hypotheses. Finally, the DCN1-UBE2M interaction requires N-terminal acetylation of UBC12, a common posttranslational modification controlling protein interactions. Therefore, a strategy for targeting N-terminal acetylation dependent protein interactions could be widely applicable. The research design and methods for achieving these goals involves the integrated and recursive use of structure-driven, hypothesis-based medicinal chemistry; in vitro biochemical measures of affinity and inhibitory potency; in vivo measures of compound efficacy and pharmacodynamic responses; and in vitro and in vivo measures of compound bioavailability, distribution, metabolism, excretion, and toxicity. The overall goal is to develop inhibitors that are active in vivo and elucidate how this interaction drives cellular and tissue effects. Our aims are: Aim 1: To optimize lead inhibitors of the DCN1-UBC12 interaction to provide compounds with sufficient potency, selectivity, and bioavailability to allow the study of the consequences of inhibiting this interaction in cells and animals, and to understand structural mechanisms for inhibiting N-terminal acetylation-dependent interactions. Aim 2: To dissect the effects of acute DCN-UBC12 inhibition on the NEDD8/CUL pathway in cells. Aim 3: To study the effects of inhibiting the DCN1-UBC12 interaction in DCN1-dependent animal models and establish the pharmacodynamic and toxicokinetic consequences of inhibiting this protein interaction.