Oral small molecule inhibitors of NSP4-mediated membrane-associated RNA replication of SARS-CoV-2 and other RNA viruses

NSP4 介导的 SARS-CoV-2 和其他 RNA 病毒膜相关 RNA 复制的口服小分子抑制剂

• 批准号: 10514275
• 负责人: JEFFREY S GLENN
• 金额: $ 926.66万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-16 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10514275
• 关键词: 2019-nCoV; Antiviral Agents; Antiviral resistance; Back; Binding Proteins; Biochemical; Biological Assay; Biological Availability; Caco-2 Cells; Cell Survival; Cells; Characteristics; Chikungunya virus; Clinic; Collaborations; Collection; Combined Modality Therapy; Cryoelectron Microscopy; Data; Dengue Virus; Development; Dose; Drug Kinetics; Genome; Hamsters; Hepatitis C virus; Impairment; In Vitro; Individual; Interferons; Internet; Intracellular Membranes; Lead; Libraries; Lipids; Lung; Maximum Tolerated Dose; Mediating; Membrane; Middle East Respiratory Syndrome Coronavirus; Modeling; Modification; Mus; Mutation; N-terminal; Nonstructural Protein; Oral; Outpatients; Peptides; Permeability; Pharmaceutical Chemistry; Predisposition; Process; Protease Inhibitor; Proteins; RNA Viruses; RNA replication; Resistance development; Role; SARS coronavirus; SARS-CoV-2 genome; SARS-CoV-2 infection; SARS-CoV-2 protease; Testing; Vesicle; Viral; Virion; Virulent; Virus; analog; anti-viral efficacy; base; clinical development; electron tomography; experimental study; humanized mouse; in vivo; inhibitor; mouse model; next generation; novel; nucleoside analog; pandemic disease; prevent; remdesivir; screening; small molecule inhibitor; synergism; virology

ABSTRACT: Our overall objective is to advance to the clinic oral small molecule inhibitors of NSP4-mediated membrane-associated replication of SARS-CoV-2 and other RNA viruses of pandemic concern. Positive-strand RNA viruses replicate their genomes in association with intracellular membranes or novel membrane structures induced by specific viral non-structural (NS) proteins. SARS-CoV-2 also induces intracellular membrane structures to support its replication and its NSP4 protein has recently been implicated in this process. Inspection of NSP4 revealed an N-terminal amphipathic helix (AH). Addition of the latter to lipid vesicles in vitro specifically induced their aggregation, suggesting this segment may mediate part of NSP4’s membrane altering activity. Excitingly, STF-3577, an optimized analog of an inhibitor we previously identified against a similar function mediated by hepatitis C virus’ NS4B, prevents NSP4 AH-mediated lipid vesicle aggregation in a dose-dependent fashion with an IC50 of 480nM. Cryo electron microscopy and tomography of SARS-CoV-2 infected cells treated with STF-3577 revealed an impairment in the characteristic viral induced intracellular membrane rearrangements and associated nascent virions, along with a corresponding accumulation of possible precursor small individual membrane vesicles. Importantly, addition of STF-3577 to SARS-CoV-2 infected cells inhibited genome replication with an EC50 of 803nM with no effect on cell viability at the highest concentration tested (20 uM). No natural mutations have been observed in the NSP4 AH targeted by STF-3577. STF-3577 has high oral bioavailability, is well tolerated in 7-day repeat dosing, just two doses decreased virus lung titers >3 log in SARS- CoV-2-infected mice, and it has strong in vitro synergy with SARS-CoV-2 protease inhibitors. We hypothesize that: 1) STF-3577 represents an attractive lead molecule for entering IND-enabling studies; 2) a focused medicinal chemistry strategy can identify next generation/back up more potent analogs of STF-3577; 3) the inhibition of lipid vesicle aggregation assay represents an ideal biochemical assay to help guide the medicinal chemistry optimization of potency effort; 4) similar assays with candidate NSP4 peptides from other viruses can be used to guide the development of inhibitors targeting additional RNA viruses of pandemic concern; 5) STF- 3577 and its optimized analogs represent ideal combination partners for other direct-acting anti-SARS-CoV-2 agents (e.g., protease inhibitors); and 6) there may be a high barrier to the development of resistance to STF- 3577. We will test these hypotheses by: 1) optimizing STF-3577’s anti-SARS-CoV-2 potency and pharmacokinetics; 2) determining the in vivo activity of the optimized NSP4 inhibitors against SARS-CoV-2 in mice and hamsters; 3) expanding the virology data package; 4) nominating a NSP4 inhibitor IND candidate; and 5) exploring targeting NSP4 function in other RNA viruses of pandemic potential. Successful accomplishment of the above will yield an exciting new class of antivirals to treat outpatient infections of SARS-CoV-2, as both a mono- or synergistic combination therapy, and other RNA viruses of pandemic potential.

摘要：我们的总体目标是将NSP 4介导的口服小分子抑制剂推向临床。 SARS-CoV-2和其他大流行关注的RNA病毒的膜相关复制。正链 RNA病毒复制它们的基因组与细胞内膜或新的膜结构有关 由特定的病毒非结构（NS）蛋白诱导。SARS-CoV-2还诱导细胞内膜 支持其复制的结构和其NSP 4蛋白最近涉及该过程。检查 NSP 4的N-末端为两亲性螺旋（AH）。将后者加入体外脂质囊泡中， 诱导它们的聚集，表明该片段可能介导NSP 4的膜改变活性的一部分。 令人兴奋的是，STF-3577，一种我们以前发现的针对类似功能的抑制剂的优化类似物， 由丙型肝炎病毒的NS 4 B介导，以剂量依赖性方式阻止NSP 4 AH介导的脂质囊泡聚集。 IC 50为480 nM。经处理的SARS-CoV-2感染细胞的冷冻电子显微镜和断层扫描 与STF-3577的联合作用显示了特征性病毒诱导的细胞内膜重排的损伤 和相关的新生病毒粒子，沿着可能的前体小个体的相应积累 膜囊泡重要的是，向SARS-CoV-2感染的细胞中加入STF-3577抑制了基因组 在最高测试浓度（20 μ M）下，EC 50为803 nM，对细胞活力没有影响。没有 在STF-3577靶向的NSP 4 AH中观察到天然突变。STF-3577具有较高的口服 生物利用度，在7天重复给药中耐受良好，在SARS中仅两个剂量降低病毒肺滴度> 3log。 CoV-2感染小鼠，并与SARS-CoV-2蛋白酶抑制剂有较强的体外协同作用。我们假设 1）STF-3577是一种有吸引力的先导分子，可用于IND研究; 2） 药物化学策略可以鉴定下一代/备份更有效的STF-3577类似物; 3） 脂质囊泡聚集抑制试验代表了一种理想的生化试验，有助于指导药物 效力努力的化学优化; 4）用来自其它病毒的候选NSP 4肽的类似测定可 用于指导开发针对其他大流行性RNA病毒的抑制剂; 5）STF- 3577及其优化的类似物是其他直接作用的抗SARS-CoV-2的理想组合伙伴 代理（例如，蛋白酶抑制剂）;和6）可能存在对STF抗性发展的高屏障- 3577.我们将通过以下方法来验证这些假设：1）优化STF-3577的抗SARS-CoV-2效力， 药代动力学; 2）确定优化的NSP 4抑制剂在体内对SARS-CoV-2的活性， 小鼠和仓鼠; 3）扩展病毒学数据包; 4）提名NSP 4抑制剂IND候选物;和 5)探索靶向具有大流行潜力的其他RNA病毒中的NSP 4功能。成功完成 以上的研究将产生一种令人兴奋的新型抗病毒药物来治疗SARS-CoV-2的门诊感染， 单一或协同组合疗法，以及其他具有大流行潜力的RNA病毒。