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

我们与美国国立卫生研究院内外的专家合作，开发了8种检测方法，用于重新利用已批准的药物收集筛选，以确定对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进行临床试验，以治疗COVID-19患者。

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.