Identification and characterization of inhibitors of HECT E3 ligases by high throughput screening

ROSSI, M.; ROTBLAT, B.; ANSELL, K.; CAVASOTTO, CLAUDIO N.; CIECHANOVER, A.; MELINO, G.

Shanghai

Congreso; Seventh International Conference SUMO, Ubiquitin, UBL Proteins: Implications for Human Diseases; 2014

Mutations, altered expression, or viral hijacking of different members of the E3 family of ubiquitin (Ub) ligases have been reported to contribute to the development of a wide variety of pathologies. Therefore, selective interference of this enzyme family might represent a powerful therapeutic tool. ITCH is a HECT domain-containing E3 Ub ligase that promotes the ubiquitylation and degradation of several proteins controlling cell growth and apoptotic processes. In agreement with these findings, it has been reported that ITCH depletion potentiates the effect of chemotherapeutic drugs, revealing ITCH as a potential pharmacological target in cancer therapy. Using high throughput screening, we identified several putative ITCH inhibitors one of which is Clomipramine, a drug that is used in the clinic to treat psychiatric disorders. We found that Clomipramine specifically blocks the HECT catalytic activity of ITCH. In addition, using Clomipramine structural analogues we identified putative molecular moieties that are essential for ITCH inhibition. To obtain qualitative insight on how Clomipramine derivatives might obstruct ITCH ubiquitylation, we performed modelling studies and identified two potential binding sites of Clomipramine within the HECT domain of ITCH. Together, our study demonstrates the feasibility of using high throughput screening to identify E3 Ub ligase inhibitors and provide insight into how Clomipramine might interfere with ITCH and other HECT E3 Ub ligases catalytic activity.