Structure-based design of potent and selective inhibitors of the HECT ligase NEDD4

Among the human E3 ubiquitin ligases, NEDD4 (Neural precursor cell expressed developmentally down-regulated 4) plays a critical role in development and cancer, making it a compelling therapeutic target. However, no specific NEDD4 inhibitors have advanced in drug development. In this study, we reveal the inhibitory mechanism of Norclomipramine, a tricyclic antidepressant, which inhibits NEDD4-mediated ubiquitin chain elongation by binding to a hydrophobic pocket in the Ub exosite of the N-lobe. Building on this mechanism, we conducted a focused medicinal chemistry campaign, resulting in the development of covalent inhibitors that specifically target the non-catalytic cysteine C627. These compounds exhibit selective binding to NEDD4 over other family members, effectively inhibiting NEDD4-mediated polyubiquitination while leaving monoubiquitinated substrates unaffected. Among these, compound 32 emerged as a potent lead (IC50 = 0.12 µM) with favorable pharmacokinetic properties, including oral bioavailability, paving the way for future in vivo efficacy studies. NEDD4 is a key E3 ubiquitin ligase involved in development and cancer, however, specific inhibitors against NEDD4 remain underdeveloped. Here, the authors identify the inhibitory mechanism of norclomipramine and develop covalent inhibitors targeting NEDD4’s non-catalytic cysteine C627, with one compound showing potent inhibition and promising pharmacokinetics, advancing therapeutic strategies against NEDD4-related diseases.