Small molecule antiviral compound collection (SMACC): A comprehensive, highly curated database to support the discovery of broad-spectrum antiviral drug molecules.

Diseases caused by new viruses cost thousands if not millions of human lives and trillions of dollars. We have identified, collected, curated, and integrated all chemogenomics data from ChEMBL for 13 emerging viruses that hold the greatest potential threat to global human health. By identifying and solving several challenges related to data annotation accuracy, we developed a highly curated and thoroughly annotated database of compounds tested in both phenotypic and target-based assays for these viruses that we dubbed SMACC (Small Molecule Antiviral Compound Collection). The pilot version of the SMACC database contains over 32,500 entries for 13 viruses. By analyzing data in SMACC, we have identified ∼50 compounds with polyviral inhibition profile, mostly covering flavi- and coronaviruses. The SMACC database may serve as a reference for virologists and medicinal chemists working on the development of novel BSA agents in preparation for future viral outbreaks. SMACC is publicly available at https://smacc.mml.unc.edu.

A humanized nanobody phage display library yields potent binders of SARS CoV-2 spike.

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

Allosteric Binders of ACE2 Are Promising Anti-SARS-CoV-2 Agents.

The COVID-19 pandemic has had enormous health, economic, and social consequences. Vaccines have been successful in reducing rates of infection and hospitalization, but there is still a need for acute treatment of the disease. We investigate whether compounds that bind the human angiotensin-converting enzyme 2 (ACE2) protein can decrease SARS-CoV-2 replication without impacting ACE2's natural enzymatic function. Initial screening of a diversity library resulted in hit compounds active in an ACE2-binding assay, which showed little inhibition of ACE2 enzymatic activity (116 actives, success rate ∼4%), suggesting they were allosteric binders. Subsequent application of in silico techniques boosted success rates to ∼14% and resulted in 73 novel confirmed ACE2 binders with K d values as low as 6 nM. A subsequent SARS-CoV-2 assay revealed that five of these compounds inhibit the viral life cycle in human cells. Further effort is required to completely elucidate the antiviral mechanism of these ACE2-binders, but they present a valuable starting point for both the development of acute treatments for COVID-19 and research into the host-directed therapy.

Suite of TMPRSS2 Assays for Screening Drug Repurposing Candidates as Potential Treatments of COVID-19.

SARS-CoV-2 is the causative viral pathogen driving the COVID-19 pandemic that prompted an immediate global response to the development of vaccines and antiviral therapeutics. For antiviral therapeutics, drug repurposing allows for rapid movement of the existing clinical candidates and therapies into human clinical trials to be tested as COVID-19 therapies. One effective antiviral treatment strategy used early in symptom onset is to prevent viral entry. SARS-CoV-2 enters ACE2-expressing cells when the receptor-binding domain of the spike protein on the surface of SARS-CoV-2 binds to ACE2 followed by cleavage at two cut sites by TMPRSS2. Therefore, a molecule capable of inhibiting the protease activity of TMPRSS2 could be a valuable antiviral therapy. Initially, we used a fluorogenic high-throughput screening assay for the biochemical screening of 6030 compounds in NCATS annotated libraries. Then, we developed an orthogonal biochemical assay that uses mass spectrometry detection of product formation to ensure that hits from the primary screen are not assay artifacts from the fluorescent detection of product formation. Finally, we assessed the hits from the biochemical screening in a cell-based SARS-CoV-2 pseudotyped particle entry assay. Of the six molecules advanced for further studies, two are approved drugs in Japan (camostat and nafamostat), two have entered clinical trials (PCI-27483 and otamixaban), while the other two molecules are peptidomimetic inhibitors of TMPRSS2 taken from the literature that have not advanced into clinical trials (compounds 92 and 114). This work demonstrates a suite of assays for the discovery and development of new inhibitors of TMPRSS2.

Discovery of Synergistic and Antagonistic Drug Combinations against SARS-CoV-2 In Vitro

COVID-19 is undoubtedly the most impactful viral disease of the current century, afflicting millions worldwide. As yet, there is not an approved vaccine, as well as limited options from existing drugs for treating this disease. We hypothesized that combining drugs with independent mechanisms of action could result in synergy against SARS-CoV-2. Using in silico approaches, we prioritized 73 combinations of 32 drugs with potential activity against SARS-CoV-2 and then tested them in vitro . Overall, we identified 16 synergistic and 8 antagonistic combinations, 4 of which were both synergistic and antagonistic in a dose-dependent manner. Among the 16 synergistic cases, combinations of nitazoxanide with three other compounds (remdesivir, amodiaquine and umifenovir) were the most notable, all exhibiting significant synergy against SARS-CoV-2. The combination of nitazoxanide, an FDA-approved drug, and remdesivir, FDA emergency use authorization for the treatment of COVID-19, demonstrate a strong synergistic interaction. Notably, the combination of remdesivir and hydroxychloroquine demonstrated strong antagonism. Overall, our results emphasize the importance of both drug repurposing and preclinical testing of drug combinations for potential therapeutic use against SARS-CoV-2 infections.

Hybrid In Silico Approach Reveals Novel Inhibitors of Multiple SARS-CoV-2 Variants.

The National Center for Advancing Translational Sciences (NCATS) has been actively generating SARS-CoV-2 high-throughput screening data and disseminates it through the OpenData Portal (https://opendata.ncats.nih.gov/covid19/). Here, we provide a hybrid approach that utilizes NCATS screening data from the SARS-CoV-2 cytopathic effect reduction assay to build predictive models, using both machine learning and pharmacophore-based modeling. Optimized models were used to perform two iterative rounds of virtual screening to predict small molecules active against SARS-CoV-2. Experimental testing with live virus provided 100 (∼16% of predicted hits) active compounds (efficacy > 30%, IC50 ≤ 15 µM). Systematic clustering analysis of active compounds revealed three promising chemotypes which have not been previously identified as inhibitors of SARS-CoV-2 infection. Further investigation resulted in the identification of allosteric binders to host receptor angiotensin-converting enzyme 2; these compounds were then shown to inhibit the entry of pseudoparticles bearing spike protein of wild-type SARS-CoV-2, as well as South African B.1.351 and UK B.1.1.7 variants.

Inhibiting SARS-CoV-2 infection with lysosomal alkalizers

Understanding the SARS-CoV-2 virus? routes of infection, virus?host?protein interactions, and mechanisms of virus-induced cytopathic effects will greatly aid in the discovery and design of new therapeutics to treat COVID-19. Chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including alkalizing lysosomes and blocking autophagy as well as exhibiting dose-limiting toxicities in patients. To identify an alternative lysosome-based drug repurposing opportunity we evaluated additional lysosomotropic compounds . We found that six of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero E6 cells with half-maximal effective concentration (EC50) values ranging from 2.0 to 13 ?M and selectivity indices (SIs;SI = CC50/EC50) ranging from 1.5- to >10-fold. We demonstrate how the compounds (1) blocked lysosome functioning and autophagy, (2) prevented pseudotyped particle entry, (3) increased lysosomal pH, and (4) that ROC-325 reduced viral titers in the EpiAirway 3D tissue model. Consistent with these findings, the siRNA knockdown of ATP6V0D1 blocked the HCoV-NL63 cytopathic effect in LLC-MK2 cells. Moreover, an analysis of SARS-CoV-2 infected Vero E6 cell lysate revealed significant dysregulation of autophagy and lysosomal function, suggesting a contribution of the lysosome to the life cycle of SARS-CoV-2. Our findings support targeting the lysosome to combat SARS-CoV-2 infections and inhibitors of lysosomal function could become an important component of drug combination therapies aimed at improving treatment and outcomes for COVID-19.

Discovery of TMPRSS2 Inhibitors from Virtual Screening as a Potential Treatment of COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has prompted researchers to pivot their efforts to finding antiviral compounds and vaccines. In this study, we focused on the human host cell transmembrane protease serine 2 (TMPRSS2), which plays an important role in the viral life cycle by cleaving the spike protein to initiate membrane fusion. TMPRSS2 is an attractive target and has received attention for the development of drugs against SARS and Middle East respiratory syndrome. Starting with comparative structural modeling and a binding model analysis, we developed an efficient pharmacophore-based approach and applied a large-scale in silico database screening for small-molecule inhibitors against TMPRSS2. The hits were evaluated in the TMPRSS2 biochemical assay and the SARS-CoV-2 pseudotyped particle entry assay. A number of novel inhibitors were identified, providing starting points for the further development of drug candidates for the treatment of coronavirus disease 2019.

Mining of high throughput screening database reveals AP-1 and autophagy pathways as potential targets for COVID-19 therapeutics.

The recent global pandemic of the Coronavirus disease 2019 (COVID-19) caused by the new coronavirus SARS-CoV-2 presents an urgent need for the development of new therapeutic candidates. Many efforts have been devoted to screening existing drug libraries with the hope to repurpose approved drugs as potential treatments for COVID-19. However, the antiviral mechanisms of action of the drugs found active in these phenotypic screens remain largely unknown. In an effort to deconvolute the viral targets in pursuit of more effective anti-COVID-19 drug development, we mined our in-house database of approved drug screens against 994 assays and compared their activity profiles with the drug activity profile in a cytopathic effect (CPE) assay of SARS-CoV-2. We found that the autophagy and AP-1 signaling pathway activity profiles are significantly correlated with the anti-SARS-CoV-2 activity profile. In addition, a class of neurology/psychiatry drugs was found to be significantly enriched with anti-SARS-CoV-2 activity. Taken together, these results provide new insights into SARS-CoV-2 infection and potential targets for COVID-19 therapeutics, which can be further validated by in vivo animal studies and human clinical trials.

Application of niclosamide and analogs as small molecule inhibitors of Zika virus and SARS-CoV-2 infection.

Zika virus has emerged as a potential threat to human health globally. A previous drug repurposing screen identified the approved anthelminthic drug niclosamide as a small molecule inhibitor of Zika virus infection. However, as antihelminthic drugs are generally designed to have low absorption when dosed orally, the very limited bioavailability of niclosamide will likely hinder its potential direct repurposing as an antiviral medication. Here, we conducted SAR studies focusing on the anilide and salicylic acid regions of niclosamide to improve physicochemical properties such as microsomal metabolic stability, permeability and solubility. We found that the 5-bromo substitution in the salicylic acid region retains potency while providing better drug-like properties. Other modifications in the anilide region with 2'-OMe and 2'-H substitutions were also advantageous. We found that the 4'-NO2 substituent can be replaced with a 4'-CN or 4'-CF3 substituents. Together, these modifications provide a basis for optimizing the structure of niclosamide to improve systemic exposure for application of niclosamide analogs as drug lead candidates for treating Zika and other viral infections. Indeed, key analogs were also able to rescue cells from the cytopathic effect of SARS-CoV-2 infection, indicating relevance for therapeutic strategies targeting the COVID-19 pandemic.

Biological activity-based modeling identifies antiviral leads against SARS-CoV-2.

Computational approaches for drug discovery, such as quantitative structure-activity relationship, rely on structural similarities of small molecules to infer biological activity but are often limited to identifying new drug candidates in the chemical spaces close to known ligands. Here we report a biological activity-based modeling (BABM) approach, in which compound activity profiles established across multiple assays are used as signatures to predict compound activity in other assays or against a new target. This approach was validated by identifying candidate antivirals for Zika and Ebola viruses based on high-throughput screening data. BABM models were then applied to predict 311 compounds with potential activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of the predicted compounds, 32% had antiviral activity in a cell culture live virus assay, the most potent compounds showing a half-maximal inhibitory concentration in the nanomolar range. Most of the confirmed anti-SARS-CoV-2 compounds were found to be viral entry inhibitors and/or autophagy modulators. The confirmed compounds have the potential to be further developed into anti-SARS-CoV-2 therapies.

Drug Repurposing Screen for Compounds Inhibiting the Cytopathic Effect of SARS-CoV-2.

Drug repurposing is a rapid approach to identify therapeutics for the treatment of emerging infectious diseases such as COVID-19. To address the urgent need for treatment options, we carried out a quantitative high-throughput screen using a SARS-CoV-2 cytopathic assay with a compound collection of 8,810 approved and investigational drugs, mechanism-based bioactive compounds, and natural products. Three hundred and nineteen compounds with anti-SARS-CoV-2 activities were identified and confirmed, including 91 approved drugs and 49 investigational drugs. The anti-SARS-CoV-2 activities of 230 of these confirmed compounds, of which 38 are approved drugs, have not been previously reported. Chlorprothixene, methotrimeprazine, and piperacetazine were the three most potent FDA-approved drugs with anti-SARS-CoV-2 activities. These three compounds have not been previously reported to have anti-SARS-CoV-2 activities, although their antiviral activities against SARS-CoV and Ebola virus have been reported. These results demonstrate that this comprehensive data set is a useful resource for drug repurposing efforts, including design of new drug combinations for clinical trials for SARS-CoV-2.

Synergistic and Antagonistic Drug Combinations against SARS-CoV-2.

Antiviral drug development for coronavirus disease 2019 (COVID-19) is occurring at an unprecedented pace, yet there are still limited therapeutic options for treating this disease. We hypothesized that combining drugs with independent mechanisms of action could result in synergy against SARS-CoV-2, thus generating better antiviral efficacy. Using in silico approaches, we prioritized 73 combinations of 32 drugs with potential activity against SARS-CoV-2 and then tested them in vitro. Sixteen synergistic and eight antagonistic combinations were identified; among 16 synergistic cases, combinations of the US Food and Drug Administration (FDA)-approved drug nitazoxanide with remdesivir, amodiaquine, or umifenovir were most notable, all exhibiting significant synergy against SARS-CoV-2 in a cell model. However, the combination of remdesivir and lysosomotropic drugs, such as hydroxychloroquine, demonstrated strong antagonism. Overall, these results highlight the utility of drug repurposing and preclinical testing of drug combinations for discovering potential therapies to treat COVID-19.

The SARS-CoV-2 Cytopathic Effect Is Blocked by Lysosome Alkalizing Small Molecules.

Understanding the SARS-CoV-2 virus' pathways of infection, virus-host-protein interactions, and mechanisms of virus-induced cytopathic effects will greatly aid in the discovery and design of new therapeutics to treat COVID-19. Chloroquine and hydroxychloroquine, extensively explored as clinical agents for COVID-19, have multiple cellular effects including alkalizing lysosomes and blocking autophagy as well as exhibiting dose-limiting toxicities in patients. Therefore, we evaluated additional lysosomotropic compounds to identify an alternative lysosome-based drug repurposing opportunity. We found that six of these compounds blocked the cytopathic effect of SARS-CoV-2 in Vero E6 cells with half-maximal effective concentration (EC50) values ranging from 2.0 to 13 µM and selectivity indices (SIs; SI = CC50/EC50) ranging from 1.5- to >10-fold. The compounds (1) blocked lysosome functioning and autophagy, (2) prevented pseudotyped particle entry, (3) increased lysosomal pH, and (4) reduced (ROC-325) viral titers in the EpiAirway 3D tissue model. Consistent with these findings, the siRNA knockdown of ATP6V0D1 blocked the HCoV-NL63 cytopathic effect in LLC-MK2 cells. Moreover, an analysis of SARS-CoV-2 infected Vero E6 cell lysate revealed significant dysregulation of autophagy and lysosomal function, suggesting a contribution of the lysosome to the life cycle of SARS-CoV-2. Our findings suggest the lysosome as a potential host cell target to combat SARS-CoV-2 infections and inhibitors of lysosomal function could become an important component of drug combination therapies aimed at improving treatment and outcomes for COVID-19.

An Enzymatic TMPRSS2 Assay for Assessment of Clinical Candidates and Discovery of Inhibitors as Potential Treatment of COVID-19.

SARS-CoV-2 is the viral pathogen causing the COVID19 global pandemic. Consequently, much research has gone into the development of preclinical assays for the discovery of new or repurposing of FDA-approved therapies. Preventing viral entry into a host cell would be an effective antiviral strategy. One mechanism for SARS-CoV-2 entry occurs when the spike protein on the surface of SARS-CoV-2 binds to an ACE2 receptor followed by cleavage at two cut sites ("priming") that causes a conformational change allowing for viral and host membrane fusion. TMPRSS2 has an extracellular protease domain capable of cleaving the spike protein to initiate membrane fusion. A validated inhibitor of TMPRSS2 protease activity would be a valuable tool for studying the impact TMPRSS2 has in viral entry and potentially be an effective antiviral therapeutic. To enable inhibitor discovery and profiling of FDA-approved therapeutics, we describe an assay for the biochemical screening of recombinant TMPRSS2 suitable for high throughput application. We demonstrate effectiveness to quantify inhibition down to subnanomolar concentrations by assessing the inhibition of camostat, nafamostat, and gabexate, clinically approved agents in Japan. Also, we profiled a camostat metabolite, FOY-251, and bromhexine hydrochloride, an FDA-approved mucolytic cough suppressant. The rank order potency for the compounds tested are nafamostat (IC50 = 0.27 nM), camostat (IC50 = 6.2 nM), FOY-251 (IC50 = 33.3 nM), and gabexate (IC50 = 130 nM). Bromhexine hydrochloride showed no inhibition of TMPRSS2. Further profiling of camostat, nafamostat, and gabexate against a panel of recombinant proteases provides insight into selectivity and potency.

Robotic High-Throughput Biomanufacturing and Functional Differentiation of Human Pluripotent Stem Cells.

Efficient translation of human induced pluripotent stem cells (hiPSCs) depends on implementing scalable cell manufacturing strategies that ensure optimal self-renewal and functional differentiation. Currently, manual culture of hiPSCs is highly variable and labor-intensive posing significant challenges for high-throughput applications. Here, we established a robotic platform and automated all essential steps of hiPSC culture and differentiation under chemically defined conditions. This streamlined approach allowed rapid and standardized manufacturing of billions of hiPSCs that can be produced in parallel from up to 90 different patient-and disease-specific cell lines. Moreover, we established automated multi-lineage differentiation to generate primary embryonic germ layers and more mature phenotypes such as neurons, cardiomyocytes, and hepatocytes. To validate our approach, we carefully compared robotic and manual cell culture and performed molecular and functional cell characterizations (e.g. bulk culture and single-cell transcriptomics, mass cytometry, metabolism, electrophysiology, Zika virus experiments) in order to benchmark industrial-scale cell culture operations towards building an integrated platform for efficient cell manufacturing for disease modeling, drug screening, and cell therapy. Combining stem cell-based models and non-stop robotic cell culture may become a powerful strategy to increase scientific rigor and productivity, which are particularly important during public health emergencies (e.g. opioid crisis, COVID-19 pandemic).

Fruitful Neutralizing Antibody Pipeline Brings Hope To Defeat SARS-Cov-2.

With the recent spread of severe acute respiratory syndrome coronavirus (SARS-CoV-2)_ infecting >16 million people worldwide as of 28 July 2020, causing >650 000 deaths, there is a desperate need for therapeutic agents and vaccines. Building on knowledge of previous outbreaks of SARS-CoV-1 and Middle East respiratory syndrome (MERS), the development of therapeutic antibodies and vaccines against coronavirus disease 2019 (COVID-19) is taking place at an unprecedented speed. Current efforts towards the development of neutralizing antibodies against COVID-19 are summarized. We also highlight the importance of a fruitful antibody development pipeline to combat the potential escape plans of SARS-CoV-2, including somatic mutations and antibody-dependent enhancement (ADE).

Discovery of Synergistic and Antagonistic Drug Combinations against SARS-CoV-2 In Vitro.

COVID-19 is undoubtedly the most impactful viral disease of the current century, afflicting millions worldwide. As yet, there is not an approved vaccine, as well as limited options from existing drugs for treating this disease. We hypothesized that combining drugs with independent mechanisms of action could result in synergy against SARS-CoV-2. Using in silico approaches, we prioritized 73 combinations of 32 drugs with potential activity against SARS-CoV-2 and then tested them in vitro . Overall, we identified 16 synergistic and 8 antagonistic combinations, 4 of which were both synergistic and antagonistic in a dose-dependent manner. Among the 16 synergistic cases, combinations of nitazoxanide with three other compounds (remdesivir, amodiaquine and umifenovir) were the most notable, all exhibiting significant synergy against SARS-CoV-2. The combination of nitazoxanide, an FDA-approved drug, and remdesivir, FDA emergency use authorization for the treatment of COVID-19, demonstrate a strong synergistic interaction. Notably, the combination of remdesivir and hydroxychloroquine demonstrated strong antagonism. Overall, our results emphasize the importance of both drug repurposing and preclinical testing of drug combinations for potential therapeutic use against SARS-CoV-2 infections.

Correction to Remdesivir: A Review of Its Discovery and Development Leading to Human Clinical Trials for Treatment of COVID-19.

[This corrects the article DOI: 10.1021/acscentsci.0c00489.].

Remdesivir: A Review of Its Discovery and Development Leading to Emergency Use Authorization for Treatment of COVID-19.

The global pandemic of SARS-CoV-2, the causative viral pathogen of COVID-19, has driven the biomedical community to action-to uncover and develop antiviral interventions. One potential therapeutic approach currently being evaluated in numerous clinical trials is the agent remdesivir, which has endured a long and winding developmental path. Remdesivir is a nucleotide analogue prodrug that perturbs viral replication, originally evaluated in clinical trials to thwart the Ebola outbreak in 2014. Subsequent evaluation by numerous virology laboratories demonstrated the ability of remdesivir to inhibit coronavirus replication, including SARS-CoV-2. Here, we provide an overview of remdesivir's discovery, mechanism of action, and the current studies exploring its clinical effectiveness.