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：我们的总体目标是开发临床口服NSP4介导的小分子抑制剂。 SARS-CoV-2和其他引起大流行关注的RNA病毒的膜相关复制。正链 RNA病毒与细胞内膜或新的膜结构相关地复制它们的基因组 由特定的病毒非结构蛋白(NS)诱导。SARS-CoV-2还诱导细胞内膜 支持其复制和其NSP4蛋白的结构最近被认为与这一过程有关。检查 NSP4的N端有一个两亲性螺旋(AH)。后者在体外特异性地添加到脂泡中 诱导它们聚集，提示该片段可能介导了NSP4‘S膜改变活性的一部分。 令人兴奋的是，STF-3577，一种我们之前针对类似功能确定的抑制剂的优化类似物 丙型肝炎病毒NS4B介导的剂量依赖性抑制NSP4AH介导的脂泡聚集 时尚，IC50为480 Nm。SARS-CoV-2感染细胞处理后的冷冻电子显微镜和断层扫描 用STF-3577检测发现病毒诱导的细胞内膜重排受损 和相关的新生病毒粒子，以及相应积累的可能的前体小个体 膜泡。重要的是，在SARS-CoV-2感染细胞中加入STF-3577抑制了基因组 在测试的最高浓度(20微米)下，EC50为803 nm，对细胞活力无影响。不是 在STF-3577靶向的NSP4 AH中观察到了自然突变。STF-3577具有高度的口服力 生物利用度，在7天重复给药中耐受性良好，在SARS中只需两剂即可降低病毒肺滴度。 与SARS-CoV-2蛋白水解酶抑制剂具有较强的体外协同作用。我们假设 这是：1)STF-3577是一种有吸引力的领先分子，可用于IND研究；2)一种聚焦的 药物化学策略可以识别下一代/支持更强大的STF-3577类似物；3) 脂泡聚集抑制试验是指导临床用药的理想生化方法 效价努力的化学优化；4)与来自其他病毒的候选NSP4多肽的类似分析可以 用于指导针对其他引起大流行的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的理想结合伙伴 药物(例如，蛋白酶抑制剂)；以及6)对STF-的抗性可能存在很高的障碍 3577号。我们将通过以下方法验证这些假设：1)优化STF3577‘S抗SARS-CoV-2的效力和 药代动力学；2)优化的NSP4抑制剂体内抗SARS-CoV-2活性的测定 小鼠和仓鼠；3)扩大病毒学数据包；4)提名nsp4抑制剂IND候选；以及 5)探索在其他具有大流行潜力的RNA病毒中靶向NSP4功能。成功的成就 其中将产生一种令人兴奋的新类别的抗病毒药物来治疗门诊感染的SARS-CoV-2，因为两者都是 单一或协同联合疗法，以及其他具有大流行潜力的RNA病毒。