High throughput screening for inhibitors of the HECT ubiquitin E3 ligase ITCH identifies antidepressant drugs as regulators of autophagy.
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.
1] Medical Research Council, Toxicology Unit, Hodgkin Building, Leicester University, Leicester LE1 9HN, UK [2] Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET- Partner Institute of the Max Planck Society, Polo Científico Tecnológico, Buenos Aires, Argentina.
M
M
Rossi
Medical Research Council, Toxicology Unit, Hodgkin Building, Leicester University, Leicester LE1 9HN, UK.
B
B
Rotblat
Medical Research Council-Technology, London WC2B 4AN, UK.
K
K
Ansell
Medical Research Council, Toxicology Unit, Hodgkin Building, Leicester University, Leicester LE1 9HN, UK.
I
I
Amelio
Second University of Naples, Department of Biochemistry, Biophysics and General Pathology, Naples, Italy.
M
M
Caraglia
Second University of Naples, Department of Biochemistry, Biophysics and General Pathology, Naples, Italy.
G
G
Misso
Biochemistry Laboratory, IDI-IRCCS, and Department of Experimental Medicine and Surgery, University of Rome 'Tor Vergata', Rome, Italy.
F
F
Bernassola
Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA)-CONICET- Partner Institute of the Max Planck Society, Polo Científico Tecnológico, Buenos Aires, Argentina.
C N
CN
Cavasotto
Medical Research Council, Toxicology Unit, Hodgkin Building, Leicester University, Leicester LE1 9HN, UK.
R A
RA
Knight
Cancer and Vascular Biology Research Center, The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa 9649, Israel.
A
A
Ciechanover
1] Medical Research Council, Toxicology Unit, Hodgkin Building, Leicester University, Leicester LE1 9HN, UK [2] Biochemistry Laboratory, IDI-IRCCS, and Department of Experimental Medicine and Surgery, University of Rome 'Tor Vergata', Rome, Italy.
G
G
Melino
D000928:Antidepressive Agents / Q000494:pharmacology*; D001343:Autophagy / Q000187:drug effects*; D001665:Binding Sites; D045744:Cell Line, Tumor; D002997:Clomipramine / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D015203:Reproducibility of Results; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
D000928:Antidepressive Agents; D004791:Enzyme Inhibitors; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D002997:Clomipramine