Abstract:
The catalytically inactive caspase-8-homologous protein, c-FLIP, is a potent antiapoptotic protein highly expressed in various types of cancers. c-FLIP competes with caspase-8 for binding to the adaptor protein FADD (Fas-Associated Death Domain) following death receptors\' (DRs) activation via the ligands of the TNF-R family. As a consequence, the extrinsic apoptotic signaling pathway involving DRs is inhibited. The inhibition of c-FLIP activity in tumor cells might enhance DR-mediated apoptosis and overcome immune and anticancer drug resistance. Based on an in silico approach, the aim of this work was to identify new small inhibitory molecules able to bind selectively to c-FLIP and block its anti-apoptotic activity. Using a homology 3D model of c-FLIP, an in silico screening of 1880 compounds from the NCI database (National Cancer Institute) was performed. Nine molecules were selected for in vitro assays, based on their binding affinity to c-FLIP and their high selectivity compared to caspase-8. These molecules selectively bind to the Death Effector Domain 2 (DED2) of c-FLIP. We have tested in vitro the inhibitory effect of these nine molecules using the human lung cancer cell line H1703, overexpressing c-FLIP. Our results showed that six of these newly identified compounds efficiently prevent FADD/c-FLIP interactions in a molecular pull-down assay, as well as in a DISC immunoprecipitation assay. The overexpression of c-FLIP in H1703 prevents TRAIL-mediated apoptosis; however, a combination of TRAIL with these selected molecules significantly restored TRAIL-induced cell death by rescuing caspase cleavage and activation. Altogether, our findings indicate that new inhibitory chemical molecules efficiently prevent c-FLIP recruitment into the DISC complex, thus restoring the caspase-8-dependent apoptotic cascade. These results pave the way to design new c-FLIP inhibitory molecules that may serve as anticancer agents in tumors overexpressing c-FLIP.
摘要:
催化失活的caspase-8同源蛋白,c-FLIP,是一种有效的抗凋亡蛋白,在各种类型的癌症中高度表达。在通过TNF-R家族的配体激活死亡受体(DR)后,c-FLIP与caspase-8竞争结合衔接蛋白FADD(Fas-相关死亡结构域)。因此,涉及DR的外源性凋亡信号通路被抑制.肿瘤细胞中c-FLIP活性的抑制可能会增强DR介导的凋亡并克服免疫和抗癌药物的耐药性。基于计算机模拟方法,这项工作的目的是鉴定能够选择性结合c-FLIP并阻断其抗凋亡活性的新的小抑制分子.使用c-FLIP的同源3D模型,对来自NCI数据库(国家癌症研究所)的1880种化合物进行了计算机筛选。选择了九种分子进行体外测定,基于它们对c-FLIP的结合亲和力和与caspase-8相比的高选择性。这些分子选择性结合c-FLIP的死亡效应物结构域2(DED2)。我们已经使用过表达c-FLIP的人肺癌细胞系H1703在体外测试了这9种分子的抑制作用。我们的结果表明,这些新鉴定的化合物中的六个在分子下拉法中有效地防止了FADD/c-FLIP相互作用,以及在DISC免疫沉淀测定中。c-FLIP在H1703中的过表达阻止TRAIL介导的细胞凋亡;然而,TRAIL与这些选择的分子的组合通过挽救半胱天冬酶裂解和活化显著恢复TRAIL诱导的细胞死亡。总之,我们的发现表明,新的抑制性化学分子有效地防止c-FLIP募集到DISC复合物中,从而恢复caspase-8依赖性凋亡级联反应。这些结果为设计新的c-FLIP抑制分子铺平了道路,这些分子可以在过表达c-FLIP的肿瘤中用作抗癌剂。