PMCID: PMC4047876 ; Inhibition of distinct ubiquitin E3 ligases might represent a powerful therapeutic tool. ITCH is a HECT domain-containing E3 ligase that promotes the ubiquitylation and degradation of several proteins, including p73, p63, c-Jun, JunB, Notch and c-FLIP, thus affecting cell fate. Accordingly, ITCH depletion potentiates the effect of chemotherapeutic drugs, revealing ITCH as a potential pharmacological target in cancer therapy. Using high throughput screening of ITCH auto-ubiquitylation, we identified several putative ITCH inhibitors, one of which is clomipramine--a clinically useful antidepressant drug. Previously, we have shown that clomipramine inhibits autophagy by blocking autophagolysosomal fluxes and thus could potentiate chemotherapy in vitro. Here, we found that clomipramine specifically blocks ITCH auto-ubiquitylation, as well as p73 ubiquitylation. By screening structural homologs of clomipramine, we identified several ITCH inhibitors and putative molecular moieties that are essential for ITCH inhibition. Treating a panel of breast, prostate and bladder cancer cell lines with clomipramine, or its homologs, we found that they reduce cancer cell growth, and synergize with gemcitabine or mitomycin in killing cancer cells by blocking autophagy. We also discuss a potential mechanism of inhibition. Together, our study (i) demonstrates the feasibility of using high throughput screening to identify E3 ligase inhibitors and (ii) provides insight into how clomipramine and its structural homologs might interfere with ITCH and other HECT E3 ligase catalytic activity in (iii) potentiating chemotherapy by regulating autophagic fluxes. These results may have direct clinical applications. ; This work has been supported by the Medical Research Council and MRCT; grants from AIRC (2008-2010_33-08) (#5471) (2011-IG11955), AIRC 5xmille (#9979), Telethon Grant GGPO9133, Ministry of Education and Science of the Russian Federation (11.G34.31.0069), to GM. This work was also supported by the Agencia Nacional de Promoción Científica y Tecnológica, Argentina (PICT-2011-2778 to CNC and PICT-2011-1231 and PICT-2011-2783 to MR) and FOCEM-Mercosur (COF 03/11). C.N.C. thanks Molsoft LLC for providing an academic license for the ICM program. Research in the laboratory of AC is supported by grants from the Dr Miriam and Sheldon G. Adelson Medical Research Foundation (AMRF), the Israel Science Foundation (ISF), the I-CORE Program of the Planning and Budgeting Committee and the ISF (Grant 1775/12), the EU TreatPolyQ Network and the Deutsch-Israelische Projektkooperation (DIP). AC is an Israel Cancer Research Fund (ICRF) USA Professor. ; Peer-reviewed ; Publisher Version