Thioredoxin reductase (TrxR) is a pivotal regulator of redox homeostasis, while dysregulation of redox homeostasis is a hallmark for cancer cells. Thus, there is considerable potential to inhibit the aberrantly upregulated TrxR in cancer cells to discover selective cancer therapeutic agents. Nevertheless, the structural types of TrxR inhibitors presented currently are still relatively limited. We herein report that PACMA 31, previously reported to inhibit protein disulfide isomerase (PDI), is a potent TrxR inhibitor. PACMA 31 possesses a pharmacophore scaffold that is structurally different from the announced TrxR inhibitors and exhibits effective cytotoxicity against cervical cancer cells. Our results reveal that PACMA 31 selectively inhibits TrxR over the related glutathione reductase (GR) and in the presence of reduced glutathione (GSH). Further studies with mutant enzyme and molecular docking suggest that the propynamide fragment of PACMA 31 interacts covalently with the selenocysteine residue of TrxR. Moreover, PACMA 31 effectively and selectively curbs TrxR activity in cells and further stimulates the production of reactive oxygen species (ROS) at low micromolar concentrations, which in turn triggers the accumulation of oxidized thioredoxin (Trx) and GSSG in cells. Follow-up studies demonstrate that PACMA 31 targets TrxR in cells to induce oxidative stress-mediated cancer cell apoptosis. Our results provide a new structural type of TrxR inhibitor that may serve as a useful probe for investigating the biology of TrxR-implicated pathways, and uncover a new target of PACMA 31 that contributes to it becoming a candidate for cancer treatment.

硫氧还蛋白还原酶 (TrxR) 是氧化还原稳态的关键调节因子，而氧化还原稳态失调是癌细胞的标志。因此，抑制癌细胞中异常上调的 TrxR 以发现选择性癌症治疗剂具有相当大的潜力。然而，目前呈现的TrxR抑制剂的结构类型仍然相对有限。我们在此报告 PACMA 31 是一种有效的 TrxR 抑制剂，之前报道过该 PACMA 31 可抑制蛋白质二硫键异构酶 (PDI)。PACMA 31 拥有一个药效团支架，其结构不同于已公布的 TrxR 抑制剂，并且对宫颈癌细胞表现出有效的细胞毒性。我们的结果表明，PACMA 31 选择性抑制 TrxR 而不是相关的谷胱甘肽还原酶 (GR)，并且存在还原型谷胱甘肽 (GSH)。对突变酶和分子对接的进一步研究表明，PACMA 31 的丙炔酰胺片段与 TrxR 的硒代半胱氨酸残基共价相互作用。此外，PACMA 31 有效且选择性地抑制细胞中的 TrxR 活性，并在低微摩尔浓度下进一步刺激活性氧 (ROS) 的产生，进而引发氧化硫氧还蛋白 (Trx) 和 GSSG 在细胞中的积累。后续研究表明 PACMA 31 靶向细胞中的 TrxR 以诱导氧化应激介导的癌细胞凋亡。我们的研究结果提供了一种新结构类型的 TrxR 抑制剂，可作为研究 TrxR 相关通路生物学的有用探针，并揭示 PACMA 31 的新靶点，使其成为癌症治疗的候选药物。