Rossi M, Rotblat B, Ansell K, Amelio I, Caraglia M, Misso G, Bernassola F, Cavasotto C N, Knight R A, Ciechanover A, Melino G
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.
Medical Research Council, Toxicology Unit, Hodgkin Building, Leicester University, Leicester LE1 9HN, UK.
Cell Death Dis. 2014 May 1;5(5):e1203. doi: 10.1038/cddis.2014.113.
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.
抑制不同的泛素E3连接酶可能是一种强大的治疗工具。ITCH是一种含HECT结构域的E3连接酶，可促进包括p73、p63、c-Jun、JunB、Notch和c-FLIP在内的多种蛋白质的泛素化和降解，从而影响细胞命运。因此，ITCH的缺失增强了化疗药物的效果，表明ITCH是癌症治疗中的一个潜在药理学靶点。通过对ITCH自身泛素化的高通量筛选，我们鉴定出了几种潜在的ITCH抑制剂，其中之一是氯米帕明——一种临床常用的抗抑郁药物。此前，我们已经表明氯米帕明通过阻断自噬溶酶体通量来抑制自噬，因此在体外可增强化疗效果。在此，我们发现氯米帕明特异性地阻断ITCH自身泛素化以及p73泛素化。通过筛选氯米帕明的结构同源物，我们鉴定出了几种ITCH抑制剂以及对ITCH抑制至关重要的假定分子基团。用氯米帕明或其同源物处理一组乳腺癌、前列腺癌和膀胱癌细胞系，我们发现它们可抑制癌细胞生长，并通过阻断自噬与吉西他滨或丝裂霉素协同杀死癌细胞。我们还讨论了一种潜在的抑制机制。总之，我们的研究（i）证明了使用高通量筛选来鉴定E3连接酶抑制剂的可行性，（ii）深入了解了氯米帕明及其结构同源物如何在（iii）通过调节自噬通量增强化疗作用中干扰ITCH和其他HECT E3连接酶的催化活性。这些结果可能具有直接的临床应用价值。
