Development of small molecule inhibitors of the E3 ligase NEDD4

PROJECT SUMMARY/ABSTRACT Ubiquitination is a post-translational modification responsible for maintaining protein homeostasis within the cell. The HECT E3 ligases are a family of enzymes that catalyze ubiquitination, and dysregulation of this family of proteins is responsible for the pathogenesis of many diseases. In particular, the E3 ligase NEDD4 has been implicated in neurodegenerative disease, cardiovascular disease, and cancer. Despite the implication of NEDD4 in many diseases, studies of this E3 ligase have been hindered by a limited understanding of the structural dynamics of the HECT domain, which catalyzes ubiquitination, and the lack of selective small molecule probes. This proposal aims to elucidate the conformational dynamics of the NEDD4 HECT domain and use this information to develop small molecule inhibitors of NEDD4. Aim 1 will investigate the structural dynamics of the HECT domain. An NMR spectrum of the NEDD4 HECT domain will be obtained and assigned using different labeling techniques and NMR pulse sequences. Next, this assigned spectrum will be used to study the conformational changes of the HECT domain in different steps of the ubiquitination process by incubating the HECT domain with different protein partners and obtaining corresponding spectra. Finally, the kinetics of these conformational changes and the preference for each conformational state will be determined using a series of relaxation, saturation, and exchange NMR experiments. Aim 2 will afford small molecule inhibitors of NEDD4 and evaluate their efficacy in various cancer cell lines. Structure–activity relationship studies will be performed on a molecule previously identified in an in silico screen and analogues will be evaluated in a plate-based high throughput assay. This inhibitor will then be validated and characterized to determine its binding site and mechanism of inhibition. Finally, this inhibitor will be tested in various cancer cell lines in which NEDD4 has been identified to contribute to the promotion of cell proliferative activity. This proposal will deepen the understanding of the conformational changes the NEDD4 HECT domain undergoes to ubiquitinate its substrates and afford a small molecule inhibitor that can be used as a probe to understand the potential of NEDD4 as a therapeutic target. This proposal will be performed at Brigham and Women’s Hospital, Harvard Medical School, and Dana-Farber Cancer Institute all of which have the resources necessary to support this research. These institutions provide a collaborative, interdisciplinary environment critical to the success of this project. This proposal will also equip the applicant with new skills in protein engineering, enzymology, and protein NMR, preparing him for an independent academic career in the field of chemical biology. In addition to acquiring new technical skills, the applicant will develop his oral and written communication skills through manuscript preparation, grant writing, and presentations of his findings in departmental seminars and national conferences. He will also continue to teach and mentor undergraduate and graduate students affording him the necessary mentorship skills needed in academia.