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：我们的总体目标是促进NSP4介导的临床口服小分子抑制剂 SARS-COV-2和其他大流行关注的RNA病毒的膜相关复制。积极链 RNA病毒与细胞内膜或新型膜结构复制其基因组 由特定的病毒非结构（NS）蛋白诱导。 SARS-COV-2还诱导细胞内膜 支持其复制及其NSP4蛋白的结构最近与此过程有关。检查 NSP4显示了N末端两亲螺旋（AH）。在体外添加后者在脂质蔬菜中特异性地添加 诱导了它们的聚集，表明该细分市场可能介导NSP4膜改变活性的一部分。 令人兴奋的是，STF-3577，一种对抑制剂的优化类似物，我们先前针对相似的功能确定 由丙型肝炎病毒的NS4B介导，可防止NSP4 AH介导的脂质囊泡聚集在剂量依赖性中 IC50为480nm的时尚。 SARS-COV-2感染细胞的冷冻电子显微镜和断层扫描 使用STF-3577显示出特征性病毒引起的细胞内膜重排的损害 和相关的新生病毒，以及可能的前体的相应积累 膜蔬菜。重要的是，在SARS-COV-2感染细胞中添加STF-3577抑制了基因组 以803nm为803nm的EC50复制对最高浓度测试的细胞活力没有影响（20 um）。不 在STF-3577靶向的NSP4 AH中已经观察到了自然突变。 STF-3577口头高 生物利用度在7天重复给药中的耐受性良好，只有两剂降低了病毒肺滴度> 3 log> 3 log in sars- COV-2感染的小鼠，它与SARS-COV-2蛋白酶抑制剂具有强大的体外协同作用。我们假设 那：1）STF-3577代表了进入辅助研究的有吸引力的铅分子； 2）集中精力 药物化学策略可以识别下一代/备份STF-3577的潜在类似物； 3） 抑制脂质囊泡聚集测定法代表了理想的生化测定法，以帮助指导医学 化学优化效力努力； 4）与其他病毒的候选NSP4肽的类似测定可以 用于指导靶向其他大流行关注的RNA病毒的抑制剂的发展； 5）STF- 3577及其优化的类似物代表其他直接作用抗Sars-Cov-2的理想组合伙伴 药物（例如蛋白酶抑制剂）； 6）可能存在对STF的抵抗力发展的高障碍 3577。我们将通过：1）优化STF-3577的抗SARS-COV-2效力和 药代动力学； 2）确定优化的NSP4抑制剂针对SARS-COV-2的体内活性 老鼠和仓鼠； 3）扩展病毒学数据包； 4）提名NSP4抑制剂IND候选者；和 5）在其他大流行潜力的RNA病毒中探索靶向NSP4功能。成功的成就 上述将产生令人兴奋的新类抗病毒药，以治疗SARS-COV-2的门诊感染，这是 单或协同组合疗法以及其他大流行潜力的RNA病毒。