Gossypol Inhibits Neddylation Of Cullin RING E3 Ubiquitin Ligases:mechanistic And Functional Studies

Cullin-RING E3 Ligase(CRL)is the largest family of ubiquitin ligases in cells,responsible for the ubiquitination of 20%of the intracellular substrates degraded by the ubiquitin-proteasome system.Neddylation is a kind of post-translation modification similar to protein ubiquitination.The most important Neddylation substrate in cells is the scaffold protein Cullin in the CRL ubiquitin ligase.When Cullin molecules are modified by Neddylation,the activity of CRL ubiquitin ligase can be activated,thus promoting the ubiquitination modification and degradation of the substrates of CRL ubiquitin ligase.Accumulating experimental data have validated CRL5,consisting of SAG and Cull in-5 as an attractive anticancer target.To identify small molecule inhibitor(s)of CUL5 Neddylation,we performed AlphaScreen-based high-throughput screening of a library of 7500 FDA approval compounds and identified Gossypol.Gossypol is a kind of yellow polyphenols double naphthalene aldehyde compound,is lidan malvaceae plants,trees,cotton and upland cotton mature seeds,a polyphenol extract from the bark of the root class material,because of its antitumor activity is widely attention,and has entered a phase II clinical trial in the treatment of lymphocytic leukemia,prostate cancer,non-small cell lung cancer.However,whether Gossypol has additional mechanism of anti-cancer activity is unknown.In this study,we report that Gossypol can effectively inhibit Cullin Neddylation.By using a variety of assays,including in vitro and in vivo Neddylation,biochemical,cellular thermal transfer and molecular docking,we showed that Gossypol targets E3s rather than Nedd8,E1 or E2s,and blocks attachment of Nedd8 to Cullin family members,particularly cullin-1 and cullin-5,thus inactivating their enzymatic activity,leading to accumulation of substrates,such as MCL1 and NOXA.Biologically,combination of Gossypol with McL-1 inhibitor S63845 synergistically inhibited the growth of PC3 prostate cancer cells.Our study showed a novel activity of Gossypol as an effective Neddylation E3 inhibitor,which extended our current understanding of its mechanism of anti-tumor function.