Komarov A P, Komarova E A, Green K, Novototskaya L R, Baker P S, Eroshkin A, Osterman A L, Chenchick A A, Frangou C, Gudkov A V
Cellecta, Inc., Mountain View, CA, USA.
Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY, USA.
Cell Death Dis. 2016 Mar 17;7(3):e2145. doi: 10.1038/cddis.2016.45.
shRNA-mediated gene-silencing technology paired with cell-based functional readouts reveals potential targets directly, providing an opportunity to identify drugs against the target without knowing the precise role of the target in the pathophysiological processes of interest. By screening a lentiviral shRNA library targeting for major components of human signaling pathways and known drug targets, we identified and validated both canonical as well as 52 novel mediators of FAS and TNF ligand-induced apoptosis. Presence of potential therapeutic targets among these mediators was confirmed by demonstration of in vivo activity of siRNAs against four identified target candidates that protected mice from acute liver failure (ALF), a life-threatening disease with known involvement of death receptor (DR)-mediated apoptosis. Network-based modeling was used to predict small-molecule inhibitors for several candidate apoptosis mediators, including somatostatin receptor 5 (SSTR5) and a regulatory subunit of PP2A phosphatase, PPP2R5A. Remarkably, pharmacological inhibition of either SSTR5 or PPP2R5A reduced apoptosis induced by either FASL or TNF in cultured cells and dramatically improved survival in several mouse models of ALF. These results demonstrate the utility of loss-of-function genetic screens and network-based drug-repositioning methods for expedited identification of targeted drug candidates and revealed pharmacological agents potentially suitable for treatment of DR-mediated pathologies.
shRNA介导的基因沉默技术与基于细胞的功能读数相结合，可直接揭示潜在靶点，为在不了解靶点在相关病理生理过程中的确切作用的情况下，识别针对该靶点的药物提供了机会。通过筛选针对人类信号通路主要成分和已知药物靶点的慢病毒shRNA文库，我们鉴定并验证了FAS和TNF配体诱导凋亡的经典介质以及52种新型介质。通过证明针对四种已鉴定的候选靶点的siRNA在体内的活性，证实了这些介质中存在潜在的治疗靶点，这些靶点可保护小鼠免受急性肝衰竭（ALF）的侵害，急性肝衰竭是一种已知与死亡受体（DR）介导的凋亡有关的危及生命的疾病。基于网络的建模用于预测几种候选凋亡介质的小分子抑制剂，包括生长抑素受体5（SSTR5）和PP2A磷酸酶的调节亚基PPP2R5A。值得注意的是，对SSTR５或PPP2R5A的药理学抑制可减少培养细胞中由FASL或TNF诱导的凋亡，并显著提高几种急性肝衰竭小鼠模型的存活率。这些结果证明了功能丧失基因筛选和基于网络的药物重新定位方法在快速鉴定靶向药物候选物方面的实用性，并揭示了可能适用于治疗DR介导的病理疾病的药物制剂。
