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 NSP4 介导的口服小分子抑制剂推进临床 SARS-CoV-2 和其他大流行关注的 RNA 病毒的膜相关复制。 RNA病毒复制与细胞内膜或新膜结构相关的基因组 由特定病毒非结构 (NS) 蛋白诱导的 SARS-CoV-2 也会诱导细胞内膜。 支持其复制的结构及其 NSP4 蛋白最近与此过程有关。 NSP4 的 N 末端两亲性螺旋 (AH) 在体外特异性地添加到脂质囊泡中。 诱导它们的聚集，表明该片段可能介导部分 NSP4 的膜改变活性。 令人兴奋的是，STF-3577，我们之前针对类似功能鉴定的抑制剂的优化类似物 由丙型肝炎病毒 NS4B 介导，以剂量依赖性方式阻止 NSP4 AH 介导的脂质囊泡聚集 经处理的 SARS-CoV-2 感染细胞的冷冻电子显微镜和断层扫描的 IC50 为 480nM。 STF-3577 揭示了特征性病毒诱导的细胞内膜重排的损害 和相关的新生病毒粒子，以及可能的前体小个体的相应积累 重要的是，向 SARS-CoV-2 感染的细胞中添加 STF-3577 会抑制基因组。 在最高测试浓度 (20 uM) 下，EC50 为 803nM，对细胞活力没有影响。 在 STF-3577 靶向的 NSP4 AH 中观察到自然突变，STF-3577 具有较高的口服活性。 生物利用度，在 7 天重复给药中具有良好的耐受性，在 SARS 中仅两次剂量就降低了病毒肺滴度 >3 log CoV-2感染的小鼠，在体外与SARS-CoV-2蛋白酶抑制剂有很强的协同作用。 ： 1) STF-3577 是进入 IND 研究的一个有吸引力的先导分子；2) 一个有针对性的药物； 药物化学策略可以识别下一代/备份更有效的 STF-3577 类似物； 抑制脂质囊泡聚集测定是一种理想的生化测定，有助于指导药物治疗 4) 对来自其他病毒的候选 NSP4 肽进行类似的测定 用于指导针对大流行病的其他 RNA 病毒的抑制剂的开发 5) STF-； 3577 及其优化类似物是其他直接作用抗 SARS-CoV-2 的理想组合伙伴 剂（例如蛋白酶抑制剂）；以及 6) 对 STF 产生耐药性可能存在很高的障碍。 3577.我们将通过以下方式测试这些假设：1) 优化 STF-3577 的抗 SARS-CoV-2 效力和 2) 确定优化的 NSP4 抑制剂针对 SARS-CoV-2 的体内活性 小鼠和仓鼠；3) 扩展病毒学数据包；4) 提名 NSP4 抑制剂 IND 候选者； 5) 探索其他具有大流行潜力的RNA病毒中针对NSP4的功能。 上述药物将产生一类令人兴奋的新型抗病毒药物，用于治疗 SARS-CoV-2 的门诊感染，因为 单一或协同联合疗法，以及其他具有大流行潜力的RNA病毒。