Welsh Kate, Milutinovic Snezana, Ardecky Robert J, Gonzalez-Lopez Marcos, Ganji Santhi Reddy, Teriete Peter, Finlay Darren, Riedl Stefan, Matsuzawa Shu-Ichi, Pinilla Clemencia, Houghten Richard, Vuori Kristiina, Reed John C, Cosford Nicholas D P
Sanford Burnham Prebys Medical Discovery Institute, 10901 N. Torrey Pines Rd, La Jolla, CA, 92037, United States of America.
Torrey Pines Institute for Molecular Studies, 3550 General Atomics Ct, San Diego, CA, 92121, United States of America & 11350 SW Village Parkway, Port St. Lucie, FL, 34987, United States of America.
PLoS One. 2016 Sep 12;11(9):e0161952. doi: 10.1371/journal.pone.0161952. eCollection 2016.
Members of the Inhibitor of APoptosis (IAP) protein family suppress apoptosis within tumor cells, particularly in the context of immune cell-mediated killing by the tumor necrosis factor (TNF) superfamily cytokines. Most IAPs are opposed endogenously by the second mitochondrial activator of caspases (SMAC), which binds to selected baculovirus IAP repeat (BIR) domains of IAPs to displace interacting proteins. The development of SMAC mimetics as novel anticancer drugs has gained impetus, with several agents now in human clinical trials. To further understand the cellular mechanisms of SMAC mimetics, we focused on IAP family members cIAP1 and cIAP2, which are recruited to TNF receptor complexes where they support cell survival through NF-κB activation while suppressing apoptosis by preventing caspase activation. We established fluorescence polarization (FP) assays for the BIR2 and BIR3 domains of human cIAP1 and cIAP2 using fluorochrome-conjugated SMAC peptides as ligands. A library of SMAC mimetics was profiled using the FP assays to provide a unique structure activity relationship (SAR) analysis compared to previous assessments of binding to XIAP. Potent compounds displayed mean inhibitory binding constants (Ki) of 9 to 27 nM against the BIR3 domains of cIAP1 and cIAP2, respectively. Selected compounds were then characterized using cytotoxicity assays in which a cytokine-resistant human tumor cell line was sensitized to either TNF or lymphotoxin-α (LT-α). Cytotoxicity correlated closely with cIAP1 and cIAP2 BIR3 binding activity with the most potent compounds able to reduce cell viability by 50%. Further testing demonstrated that active compounds also inhibit RIP1 binding to BIR3 of cIAP1 and cIAP2 in vitro and reduce steady-state cIAP1 protein levels in cells. Altogether, these data inform the SAR for our SMAC mimetics with respect to cIAP1 and cIAP2, suggesting that these IAP family members play an important role in tumor cell resistance to cytotoxicity mediated by TNF and LT-α.
凋亡抑制蛋白（IAP）家族成员可抑制肿瘤细胞内的凋亡，尤其是在肿瘤坏死因子（TNF）超家族细胞因子介导的免疫细胞杀伤情况下。大多数IAP在内源上受到半胱天冬酶的第二个线粒体激活剂（SMAC）的对抗，SMAC与IAP的选定杆状病毒IAP重复（BIR）结构域结合，以取代相互作用的蛋白质。SMAC模拟物作为新型抗癌药物的开发已获得动力，目前有几种药物正在进行人体临床试验。为了进一步了解SMAC模拟物的细胞机制，我们重点研究了IAP家族成员cIAP1和cIAP2，它们被招募到TNF受体复合物中，在那里它们通过激活NF-κB来支持细胞存活，同时通过阻止半胱天冬酶激活来抑制凋亡。我们使用荧光染料偶联的SMAC肽作为配体，为人cIAP1和cIAP2的BIR2和BIR3结构域建立了荧光偏振（FP）测定法。使用FP测定法对一个SMAC模拟物文库进行了分析，以提供与先前对XIAP结合评估相比独特的构效关系（SAR）分析。强效化合物对cIAP1和cIAP2的BIR3结构域的平均抑制结合常数（Ki）分别为9至27 nM。然后使用细胞毒性测定法对选定的化合物进行表征，其中一种细胞因子抗性人类肿瘤细胞系对TNF或淋巴毒素-α（LT-α）敏感。细胞毒性与cIAP1和cIAP2 BIR3结合活性密切相关，最有效的化合物能够将细胞活力降低50%。进一步的测试表明，活性化合物还在体外抑制RIP1与cIAP1和cIAP2的BIR3结合，并降低细胞中的稳态cIAP1蛋白水平。总之，这些数据为我们的SMAC模拟物关于cIAP1和cIAP2的SAR提供了信息，表明这些IAP家族成员在肿瘤细胞对TNF和LT-α介导的细胞毒性的抗性中起重要作用。
