期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2014
卷号：111
期号：47
页码：16736-16741
DOI：10.1073/pnas.1412152111
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceThe ubiquitin system controls a wide range of processes in cells and provides attractive drug targets for the treatment of cancer and other diseases. However, it has proved difficult to obtain inhibitors of the ligases that conjugate ubiquitin to substrates, of which there are hundreds. One class, the HECT (homologous to E6AP C terminus) domain ligases, receives ubiquitin from an E2 enzyme and transfers it to substrate. We have selected bicyclic peptides that block the E2 binding site of individual HECT ligases, as well as a small molecule, heclin (HECT ligase inhibitor), that broadly inhibits these ligases in cells. These inhibitors demonstrate that HECT domains are druggable targets and provide tools to study ubiquitination. The same approach could be used to select further HECT inhibitors. The human genome encodes several hundred E3 ubiquitin ligases containing RING domains, and around 28 containing HECT domains. These enzymes catalyze the transfer of ubiquitin from E2 enzyme thioesters to a huge range of substrates and play crucial roles in many cellular functions. This makes them attractive potential therapeutic targets. However, they have proven difficult to inhibit: very few good inhibitors exist for RING domain ligases, and none have been described for HECT ligases. Here we show that bicyclic peptides isolated by phage display [Heinis C, Rutherford T, Freund S, Winter G (2009) Nat Chem Biol. 5(7):502-507] can target the E2 binding sites on the HECT domains of Smurf2, Nedd4, Mule/Huwe1, and WWP1, and thus act as specific inhibitors of these enzymes in vitro. By screening for displacement of one of these peptides from Smurf2, we were able to identify a small molecule, heclin (HECT ligase inhibitor), which inhibits several HECT ligases in tissue culture cells. In vitro, heclin does not block E2 binding but causes a conformational change that results in oxidation of the active site Cys. This demonstrates that HECT domains are potentially druggable and provides molecules that may be of experimental use. Heclin kills HEK293 cells growing in culture, consistent with an essential role for HECT ligase activity in mammalian cells.
