Optimizing a small molecule inhibitor of SARS-CoV-2 replication and associated cytokine storm

我们的目标是开发一种新型的磷脂酰肌醇(PI)4小分子抑制剂 同时具有抗SARS-CoV-2和过度释放细胞因子双重活性的KI4KIIb 与新冠肺炎病有关。SARS-CoV的进入已被证明依赖于PI4KIIIb，并且很强 通过siRNA敲除PI4KIIIb后，进入被抑制，SARS-CoV-2被认为是 通过类似的机制进入单元格。这可能反映了对磷酸化的浓缩的要求 PI的异构体，如PI-4，在病毒进入时融合所需的脂质细胞器中。我们已经开发出 高效和特定的PI4KIIIb小分子抑制剂，并对其进行了优化，以实现高口服生物利用度。我们的 铅抑制剂STF-1019对肠道病毒(EV)具有纳摩尔效力，EV也依赖于 PI4KIIIb，是唯一在EV-71动物模型中证明了体内疗效的分子，以及 没有毒性。我们现在已经证明了STF-1019‘S对SARS-CoV-2的EC50为210 nm，CC50为 &gt；100微米，反映治疗指数(TI)~500。最后，可能是由于PI4KIIIb在高尔基体介导的 分泌方面，我们最近还发现STF-1019可以有效地抑制内毒素诱导的IL-6的分泌 来自人的PBMC。然而，S的代谢稳定性是次优的，需要与 通过CYP3A4抑制其代谢的利托那韦，以获得最佳的持续组织暴露。我们假设 结果表明：1)STF-1019‘S SAR和主要代谢产物表明，我们的先导PI4KIIIb抑制剂可以进一步 优化以提高其活性和新陈代谢稳定性，以实现最佳暴露曲线；2)修改 进一步增加PI4KIIIb抑制可以为可能增加代谢的修饰提供缓冲 稳定性以牺牲疗效为代价；3)优化后的抑制剂在体外和体内都能抑制SARS-CoV-2；4) 优化后的缓蚀剂将具有较高的阻力发展障碍；5)由于其正交性 作用机制，我们的PI4KIIIb抑制剂可以与其他药物联合使用，以最大限度地发挥疗效； 6)STF1019‘S对IL-6的抑制反映了一种调节其他细胞因子释放的能力，而这种非 抗病毒活性在应对与重症相关的细胞因子风暴方面可能有很大的额外好处 新冠肺炎感染；7)关键药代动力学、体外ADME-TOX参数和初始浓度的测定 我们优化的铅的临床前体内毒性评估可以促进其翻译开发，并且 形成未来IND一揽子计划的基础。我们建议通过以下方式验证这些假设：1)识别STF-1019 具有最大体内谷值的类似物(和备份化合物)：EC90；2)测定其体内活性 抗SARS-CoV-2 PI4KIIIb抑制剂的优化及其对细胞因子产生的影响；3)测定 抗药性的相对障碍，以及与其他药物协同作用的可能性；以及4)提名PI4KIIIb 通过使优化的铅在体外进行初始ADME-TOX和IND使能来抑制IND候选 临床前动物安全性研究。

Our goal is to develop towards an IND a novel class of small molecule inhibitors of phosphoinositide (PI) 4 kinase IIIb (PI4KIIIb) with potent dual activity against both SARS-CoV-2 and the excess cytokine release associated with COVID-19 disease. Entry of SARS-CoV has been shown to depend on PI4KIIIb, and strong inhibition of entry was achieved following knockdown of PI4KIIIb via siRNA, and SARS-CoV-2 is believed to enter cells via a similar mechanism. This likely reflects a requirement for enrichment of phosphorylated isoforms of PI, such as PI-4, in the lipid organelle required for viral fusion upon entry. We have developed potent and specific small molecule inhibitors of PI4KIIIb, and optimized them for high oral bioavailability. Our lead inhibitor, STF-1019 has nanomolar efficacy against enteroviruses (EV) which are also dependent on PI4KIIIb, and is the only molecule to have demonstrated in vivo efficacy in the animal model of EV-71, and without toxicity. We have now shown that STF-1019’s EC50 against SARS-CoV-2 is 210 nM, with a CC50 of >100 microM, reflecting a therapeutic index (TI) of ~500. Finally, likely due to PI4KIIIb’s role in Golgi-mediated secretion, we have also recently shown that STF-1019 can potently inhibit the LPS-induced secretion of IL-6 from human PBMC. STF-1019’s metabolic stability, however, is suboptimal, requiring co-administration with an inhibitor (i.e. ritonavir) of its metabolism by CYP3A4 for optimal sustained tissue exposure. We hypothesize that: 1) STF-1019’s SAR and major metabolites indicates that our lead PI4KIIIb inhibitor can be further optimized to increase its activity and metabolic stability to achieve an optimal exposure profile; 2) modifications that further increase PI4KIIIb inhibition can provide a buffer for modifications that may increase metabolic stability at the expense of efficacy; 3) the optimized inhibitor will inhibit SARS-CoV-2 in vitro, and in vivo; 4) the optimized inhibitor will have a high barrier to the development of resistance; 5) because of its orthogonal mechanism of action, our PI4KIIIb inhibitor can be used in combination with other agents to maximize efficacy; 6) STF-1019’s inhibition of IL-6 reflects an ability to modulate the release of other cytokines, and this non- antiviral activity can be of great additional benefit in addressing the cytokine storm associated with severe COVID-19 infection; 7) determination of key pharmacokinetic, in vitro ADME-Tox parameters and initial preclinical in vivo toxicity assessment of our optimized lead can advance its translational development, and form the basis of a future IND package. We propose the test these hypotheses by: 1) Identifying the STF-1019 analog (and back-up compound) with greatest in vivo trough:EC90 ratios; 2) determining the in vivo activity of the optimized PI4KIIIb inhibitors against SARS-CoV-2 and their effect on cytokine production; 3) determining the relative barrier to resistance, and potential for synergy with other agents; and 4) nominating a PI4KIIIb inhibitor IND candidate by subjecting the optimized lead to initial in vitro ADME-tox and IND-enabling preclinical animal safety studies.