二硫氰酸盐（DTC）是一类简单的有机化合物，广泛应用于工业和医药领域。它们是能与多种金属离子（包括铜离子）形成络合物的有效金属螯合剂。最近，已经确定了双（二乙基硫氰酸盐）-铜络合物（CuET）是抗酒精药物盖帝明（disulfiram，DSF）的代谢产物，与DSF被报道的抗癌活性有关。在细胞内，CuET导致NPL4蛋白质（p97分离酶的必要辅因子）聚集，p97与泛素-蛋白酶体系统密不可分。CuET引起的p97/NPL4功能失调会导致蛋白质毒性应激，进而产生热震应激和蛋白质未折叠响应，并引发癌细胞死亡。然而，我们不清楚NPL4抑制是CuET特有的，还是其他二硫氰酸盐-铜络合物也具有相似的作用。因此，本研究测试了20种DTCs-铜络合物，检测其与NPL4蛋白的靶向和聚集能力。令人惊讶的是，我们发现在结构不同的DTCs-铜络合物中，对NPL4的一定抑制能力相对较为常见，其中13种化合物在细胞NPL4聚集实验中得分较高。这些化合物还表现出先前报道的CuET的典型细胞表型，包括NPL4/p97蛋白的固定化、多泛素化蛋白、未折叠蛋白和热震应激的积累。此外，这些活性络合物对癌细胞也具有毒性（纳摩尔范围内最有效），我们发现NPL4聚集和细胞毒性之间存在强烈的正相关性，进一步证实了NPL4作为相关靶点。这些结果显示了DTCs-铜络合物广泛靶向NPL4，导致癌细胞致命蛋白质毒性应激的效力，并对药物开发具有重要意义。版权所有 © 2023 Elsevier Masson SAS。保留所有权利。

Dithiocarbamates (DTCs) are simple organic compounds with many applications in industry and medicine. They are potent metal chelators forming complexes with various metal ions, including copper. Recently, bis(diethyldithiocarbamate)-copper complex (CuET) has been identified as a metabolic product of the anti-alcoholic drug Antabuse (disulfiram, DSF), standing behind DSF's reported anticancer activity. Mechanistically, CuET in cells causes aggregation of NPL4 protein, an essential cofactor of the p97 segregase, an integral part of the ubiquitin-proteasome system. The malfunction of p97/NPL4 caused by CuET leads to proteotoxic stress accompanied by heat shock and unfolded protein responses and cancer cell death. However, it is not known whether the NPL4 inhibition is unique for CuET or whether it is shared with other dithiocarbamate-copper complexes. Thus, we tested 20 DTCs-copper complexes in this work for their ability to target and aggregate NPL4 protein. Surprisingly, we have found that certain potency against NPL4 is relatively common for structurally different DTCs-copper complexes, as thirteen compounds scored in the cellular NPL4 aggregation assay. These compounds also shared typical cellular phenotypes reported previously for CuET, including the NPL4/p97 proteins immobilization, accumulation of polyubiquitinated proteins, the unfolded protein, and the heat shock responses. Moreover, the active complexes were also toxic to cancer cells (the most potent in the nanomolar range), and we have found a strong positive correlation between NPL4 aggregation and cytotoxicity, confirming NPL4 as a relevant target. These results show the widespread potency of DTCs-copper complexes to target NPL4 with subsequent induction of lethal proteotoxic stress in cancer cells with implications for drug development.Copyright © 2023 Elsevier Masson SAS. All rights reserved.