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酸肌醇（PI）4小分子抑制剂 激酶IIIb（PI 4KIIIb），具有有效的抗SARS-CoV-2和过量细胞因子释放的双重活性 与COVID-19疾病有关。SARS-CoV的进入已被证明依赖于PI 4KIIIb，并且强 通过siRNA敲低PI 4KIIIb后，可以抑制进入，SARS-CoV-2被认为 通过类似的机制进入细胞。这可能反映了富集磷酸化的 PI的同种型，如PI-4，在进入时病毒融合所需的脂质细胞器中。我们已经开发 PI 4KIIIb的有效和特异性小分子抑制剂，并优化它们以获得高口服生物利用度。我们 先导抑制剂STF-1019对肠道病毒（EV）具有纳摩尔效力， PI 4KIIIb，并且是在EV-71的动物模型中已经证明体内功效的唯一分子，并且 无毒性。我们现在已经表明，STF-1019对SARS-CoV-2的EC 50为210 nM，CC 50为 >100 μ M，反映了~500的治疗指数（TI）。最后，可能是由于PI 4KIIIb在高尔基体介导的 我们最近还表明STF-1019可以有效地抑制LPS诱导的IL-6分泌， 来自人PBMC。然而，STF-1019的代谢稳定性是次优的，需要与代谢调节剂共同施用。 CYP 3A 4代谢的抑制剂（即利托那韦），以实现最佳的持续组织暴露。我们假设 1）STF-1019的SAR和主要代谢物表明我们的先导PI 4KIIIb抑制剂可以进一步 优化以增加其活性和代谢稳定性，以实现最佳暴露曲线; 2）修饰 进一步增加PI 4 KIIIb抑制可以为可能增加代谢的修饰提供缓冲 稳定性以牺牲功效为代价; 3）优化的抑制剂将在体外和体内抑制SARS-CoV-2; 4）优化的抑制剂将在体外和体内抑制SARS-CoV-2。 优化的抑制剂将对抗性的发展具有高屏障; 5）由于其正交性， 作用机制，我们的PI 4KIIIb抑制剂可以与其他药物联合使用，以最大限度地提高疗效; 6)STF-1019对IL-6的抑制反映了调节其他细胞因子释放的能力，并且这种非细胞因子的抑制作用可能与STF-1019对IL-6的抑制有关。 抗病毒活性可以在解决与严重急性胰腺炎相关的细胞因子风暴方面具有很大的额外益处。 COVID-19感染; 7）确定关键药代动力学、体外ADME-Tox参数和初始 我们优化的电极导线的临床前体内毒性评估可以促进其转化开发， 这是未来IND包装的基础。我们建议通过以下方式检验这些假设：1）鉴定STF-1019 具有最大体内谷值：EC 90比率的类似物（和备用化合物）; 2）测定 针对SARS-CoV-2的优化的PI 4KIIIb抑制剂及其对细胞因子产生的影响; 3）确定 耐药性的相对屏障，以及与其他药物协同作用的潜力;以及4）提名PI 4KIIIb 抑制剂IND候选物，通过使优化的先导物经受初始体外ADME-tox和IND-使能 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.