COVID-19: Identification and Development of Clinical Candidates to Treat SARS-CoV-2

我们与NIH内外的专家合作开发了8种检测方法，用于重新利用已批准的药物集合的筛选，以鉴定对SARS-CoV-2有活性的化合物。从这些筛选中鉴定出的化合物有可能作为单一药物或药物组合用于临床试验，以治疗COVID-19患者。 1.与加里惠特克（康奈尔大学）合作的SARS-CoV-2假型颗粒（PP）进入试验。用含有MLV gag-pol、CoV刺突和荧光素酶报告基因w/病毒包装信号的三种质粒转染系统生成PP。在测定过程中，PP通过刺突介导的细胞进入将荧光素酶报告RNA递送至宿主细胞。该方法已用于筛选SARS-CoV-2进入抑制剂以及从其他方法中鉴定的其他化合物的机制研究。最近，我们已经将该测定应用于几种刺突突变的变体，以监测对当前疗法的敏感性的潜在变化。 2.与加里惠特克（康奈尔大学）合作进行的SARS假型颗粒（PP）进入试验和MERS进入试验。这两种检测方法均针对1536孔筛选进行了优化，并用于批准药物采集的药物再利用筛选。在用活SARS-CoV-2感染的细胞病变效应测定（Southern Research Institute）中进一步评价鉴定的命中物，以鉴定对冠状病毒具有广泛活性的抗SARS-CoV 2化合物。 3.与南方研究所（SRI）合作在Vero 6细胞中进行的SARS-CoV-2细胞病变效应试验。在SRI的BSL-3实验室中，使用NCATS制备的试验就绪板筛选了约10，000种批准和研究药物、临床前候选药物和生物活性化合物的几个小集合。该测定也已用于评价从NCATSS BSL-2测定中鉴定的抗SARS-CoV-2化合物。 4. SARS-CoV-2 3CL蛋白酶（也称为主蛋白酶）测定。这种病毒蛋白酶在SARS-CoV-2病毒复制中发挥着关键作用。我们已经开发并优化了这种酶测定法，并筛选了大约10，000种化合物。目前，整个NCATS化合物收集的虚拟建模屏幕正在使用来自这个重点库屏幕的数据作为训练集。先导化合物将用于进一步药物开发的化学优化。 5. SARS-CoV-2 RNA依赖性RNA聚合酶（RdRP）检测。这种病毒RNA聚合酶对SARS-CoV-2病毒在宿主细胞中的复制至关重要。Remdesivir是一种最初为埃博拉病毒和其他病毒开发的RdRP抑制剂，已被FDA批准用于住院的COVID-19患者的紧急使用。这种药物的功效和效力对于SARS-CoV-2是有限的，因为它最初不是针对这种病毒开发的。Remdesivir目前仅通过静脉输注给药，因此无法进行门诊治疗。我们正在开发SARS-CoV-2 RdRP酶检测和基于细胞的RdRP检测，以支持新药开发的再利用筛选。 6. BSL-2实验室使用的SARS-CoV-2复制子检测。涉及SARS-CoV-2活病毒的研究需要BSL-3设施，这极大地限制了吞吐量。我们正在与三个实验室合作开发SARS-CoV-2的复制子类型检测，类似于我们以前对HCV的工作。该复制子测定将具有包含病毒复制机制的SARS-CoV-2基因组片段，而不具有病毒包膜蛋白，并且将包括微型基因组报告基因（荧光素酶）。我们将使用该测定筛选NCATS化合物集合用于药物发现和开发。 7. AlphaLISA检测SARS-CoV-2核衣壳（N）蛋白。目前，SARS-CoV-2的细胞病变效应（CPE）测定允许BSL-3实验室中的最大通量，但使用宿主细胞感染（细胞死亡）的替代读数。AlphaLISA N蛋白检测将允许BSL-3实验室直接测量宿主细胞中SARS-CoV-2感染后的病毒抗原产生。该检测试剂盒是同质的，适用于HTS。它的开发旨在帮助提高BSL 3实验室的吞吐量。 8. TR-FRET（HTRF）检测SARS-CoV-2核衣壳（N）蛋白。该检测与AlphaLISA N蛋白检测相似，但使用不同的读数。该试验可用作HTS的替代试验，或用作正交试验，以确认从上述AlphaLISA试验中鉴定的命中。 我们还开始了一些项目，以使候选人迅速进入SARS-CoV-2的临床试验。在一个项目中，我们的合作者最近完成了一种蛋白质天然产品作为抗艾滋病毒杀微生物剂的I期试验。其作用机制是选择性结合病毒包膜糖蛋白上的高甘露糖寡糖，与多种病原体相关，包括HIV、流感、埃博拉病毒和SARS冠状病毒。该候选物已显示出对几种冠状病毒的体外和体内活性，包括SARS-CoV和MERS-CoV。由于它与SARS-CoV上的刺突糖蛋白结合，因此很可能对所有冠状病毒株都具有活性，而不仅仅是驱动当前COVID-19大流行的SARS-CoV-2。 第二个项目涉及开发瑞德西韦（一种核苷类似物抗病毒药物）的代谢物作为口服疗法。目前，remdesivir用于治疗中度至晚期SARS-CoV-2感染，但需要在医院环境中静脉注射。目前的临床和临床前数据支持这一假设，即口服活性核苷类似物可能特别适用于治疗早期疾病，并且比医学上更复杂的静脉注射方法更适合全球分布。

We have worked in collaboration with experts in and outside of NIH on the development of 8 assays for use in repurposing screens of approved drug collections to identify compounds active against SARS-CoV-2. The compounds identified from such screens have potential for clinical trials as single agents or in drug combinations to treat COVID-19 patients. 1. A SARS-CoV-2 pseudotyped particle (PP) entry assay in collaboration with Gary Whittaker (Cornell University). The PPs were generated with three plasmid transfection system containing MLV gag-pol, CoV spike, and luciferase reporter gene w/ viral packaging signal. In the assay process, the PPs deliver luciferase reporter RNA to host cells via spike-mediated cell entry. This assay has been used for screen the inhibitors of SARS-CoV-2 entry as well as the mechanistic study of other compounds identified from other assays. Recently, we have applied this assay to several variants of spike mutations to monitor the potential changes of sensitivities to current therapeutics. 2. SARS pseudotyped particle (PP) entry assay and MERS entry assays in collaboration with Gary Whittaker (Cornell University). Both assays have been optimized for 1536-well screens and used for drug repurposing screens of approved drug collection. The identified hits were further evaluated in a cytopathic effect assay with live SARS-CoV-2 infection (Southern Research Institute) to identify anti-SARS-CoV2 compounds that have broad activity against coronaviruses. 3. A SARS-CoV-2 cytopathic effect assay in Vero 6 cells in collaboration with the Southern Research Institute (SRI). Several small collections of approximately 10,000 approved and investigational drugs, preclinical drug candidates, and bioactive compounds were screened using assay ready plates, prepared by NCATS, in the BSL-3 lab at SRI. This assay has also been used to evaluate anti-SARS-CoV-2 compounds identified from NCATSs BSL-2 assays. 4. A SARS-CoV-2 3CL protease (also named main protease) assay. This viral protease plays a critical role in SARS-CoV-2 viral replication. We have developed and optimized this enzyme assay and screened a collection of approximately 10,000 compounds. Currently, a virtual modeling screen of the entire NCATS compound collection is in progress using the data from this focused library screen as a training set. The lead compounds will be used for chemistry optimization for further drug development. 5. A SARS-CoV-2 RNA dependent RNA polymerase (RdRP) assay. This viral RNA polymerase is critical to SARS-CoV-2 viral replication in host cells. Remdesivir, an RdRP inhibitor originally developed for Ebola virus and other viruses, has been authorized by the FDA for emergency use in hospitalized COVID-19 patients. The efficacy and potency of this drug are limited for SARS-CoV-2, as it was not originally developed for this virus. Remdesivir is currently only administered by intravenous infusion that precludes it from outpatient treatment. We are developing a SARS-CoV-2 RdRP enzyme assay and a cell-based RdRP assay to support repurposing screens for new drug development. 6. A SARS-CoV-2 replicon assay for use BSL-2 laboratories. Research involving live SARS-CoV-2 virus requires BSL-3 facility, which greatly limits throughput. We are collaborating with three laboratories to develop a replicon type assay for SARS-CoV-2, similar to our previous work on HCV. This replicon assay will have a SARS-CoV-2 genomic fragment encompassing viral replication machinery, without the viral envelop proteins, and will include a mini-genome reporter gene (luciferase). We will use this assay to screen NCATS compound collections for drug discovery and development. 7. An AlphaLISA assay to detect the SARS-CoV-2 Nucleocapsid (N) protein. Currently, the cytopathic effect (CPE) assay of SARS-CoV-2 allows for the greatest throughput in BSL-3 laboratories, but uses a surrogate readout for host cell infection (cell death). The AlphaLISA N protein assay would allow BSL-3 labs to directly measure viral antigen production upon SARS-CoV-2 infection in host cells. This assay is homogenous and amenable to HTS. It was developed to help increase the throughput of BSL3 labs. 8. A TR-FRET (HTRF) assay to detect the SARS-CoV-2 Nucleocapsid (N) protein. This assay is similar to the AlphaLISA N protein assay, but uses a different readout. This assay could be used as an alternative assay for HTS, or as an orthogonal assay to confirm the hits identified from the above AlphaLISA assay. We also have begun projects to rapidly progress candidates into clinical trials for SARS-CoV-2. In one project, our collaborators recently completed phase I trials of a protein natural product for as a microbicide against HIV. Its mechanism of action, selectively binding to high-mannose oligosaccharides on viral envelope glycoproteins, is relevant to diverse pathogens, including HIV, Influenza, Ebolavirus, and SARS-CoV. The candidate has shown in vitro and in vivo activity against several coronaviruses, including SARS-CoV and MERS-CoV. Because it binds the spike glycoprotein on SARS-CoV, it is likely to have activity against all coronavirus strains, not only the SARS-CoV-2 driving the current COVID-19 pandemic. The second project involves developing a metabolite of remdesivir, a nucleoside analog antiviral, as an orally available therapy. Currently, remdesivir is used to treat moderate-to-advanced SARS-CoV-2 infection, but it requires intravenous administration in a hospital setting. Current clinical and preclinical data support the hypothesis that an orally active nucleoside analog may be especially useful for treating early-stage disease and would be more suitable for global distribution than the more medically complex intravenous approach.