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Phenotypic high-throughput screening platform identifies novel chemotypes for necroptosis inhibition.
Cell Death Discovery ( IF 7 ) Pub Date : 2020-02-11 , DOI: 10.1038/s41420-020-0240-0
Hugo Brito 1 , Vanda Marques 1 , Marta B Afonso 1 , Dean G Brown 2 , Ulf Börjesson 3 , Nidhal Selmi 3 , David M Smith 4 , Ieuan O Roberts 5 , Martina Fitzek 6 , Natália Aniceto 1 , Rita C Guedes 1 , Rui Moreira 1 , Cecília M P Rodrigues 1
Affiliation  

Regulated necrosis or necroptosis, mediated by receptor-interacting kinase 1 (RIPK1), RIPK3 and pseudokinase mixed lineage kinase domain-like protein (MLKL), contributes to the pathogenesis of inflammatory, infectious and degenerative diseases. Recently identified necroptosis inhibitors display moderate specificity, suboptimal pharmacokinetics, off-target effects and toxicity, preventing these molecules from reaching the clinic. Here, we developed a cell-based high-throughput screening (HTS) cascade for the identification of small-molecule inhibitors of necroptosis. From the initial library of over 250,000 compounds, the primary screening phase identified 356 compounds that strongly inhibited TNF-α-induced necroptosis, but not apoptosis, in human and murine cell systems, with EC50 < 6.7 μM. From these, 251 compounds were tested for RIPK1 and/or RIPK3 kinase inhibitory activity; some were active and several have novel mechanisms of action. Based on specific chemical descriptors, 110 compounds proceeded into the secondary screening cascade, which then identified seven compounds with maximum ability to reduce MLKL activation, IC50 >100 μM, EC50 2.5-11.5 μM under long-term necroptosis execution in murine fibroblast L929 cells, and full protection from ATP depletion and membrane leakage in human and murine cells. As a proof of concept, compound SN-6109, with binding mode to RIPK1 similar to that of necrostatin-1, confirmed RIPK1 inhibitory activity and appropriate pharmacokinetic properties. SN-6109 was further tested in mice, showing efficacy against TNF-α-induced systemic inflammatory response syndrome. In conclusion, a phenotypic-driven HTS cascade promptly identified robust necroptosis inhibitors with in vivo activity, currently undergoing further medicinal chemistry optimization. Notably, the novel hits highlight the opportunity to identify new molecular mechanisms of action in necroptosis.

中文翻译:

表型高通量筛选平台鉴定了抑制坏死性凋亡的新化学型。

由受体相互作用激酶 1 (RIPK1)、RIPK3 和假激酶混合谱系激酶结构域样蛋白 (MLK​​L) 介导的调节性坏死或坏死性凋亡与炎症、感染性和退行性疾病的发病机制有关。最近发现的坏死性凋亡抑制剂表现出中等特异性、次优药代动力学、脱靶效应和毒性,阻碍了这些分子进入临床。在这里,我们开发了一种基于细胞的高通量筛选(HTS)级联,用于鉴定坏死性凋亡的小分子抑制剂。从超过 250,000 种化合物的初始库中,初步筛选阶段鉴定出 356 种化合物,它们在人和小鼠细胞系统中强烈抑制 TNF-α 诱导的坏死性凋亡,但不抑制细胞凋亡,EC50 < 6.7 μM。其中,测试了 251 种化合物的 RIPK1 和/或 RIPK3 激酶抑制活性;有些是活跃的,有些具有新颖的作用机制。根据特定的化学描述符,110种化合物进入二级筛选级联,然后鉴定出7种具有最大降低MLKL激活能力的化合物,在小鼠成纤维细胞L929细胞中长期坏死性凋亡执​​行下IC50>100μM,EC50 2.5-11.5μM,并全面保护人类和鼠类细胞免受 ATP 耗尽和膜渗漏。作为概念证明,化合物 SN-6109 与 RIPK1 的结合模式类似于 necrostatin-1,证实了 RIPK1 抑制活性和适当的药代动力学特性。SN-6109 在小鼠身上进行了进一步测试,显示出对 TNF-α 诱导的全身炎症反应综合征的功效。总之,表型驱动的 HTS 级联迅速鉴定出具有体内活性的强效坏死性凋亡抑制剂,目前正在进行进一步的药物化学优化。值得注意的是,这些新颖的热门作品凸显了识别坏死性凋亡新分子作用机制的机会。
更新日期:2020-02-11
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