A 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 allowed for rapid movement of 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 on the spike protein. TMPRSS2 has a protease domain capable of cleaving the two cut sites; 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.

Preclinical Pharmacokinetics and In Vitro Properties of GS-441524, A Potential Oral Drug Candidate for COVID-19 Treatment

Preclinical pharmacokinetics (PK) and in vitro ADME properties of GS-441524, a potential oral agent for the treatment of Covid-19, were studied. GS-441524 was stable in vitro in liver microsomes, cytosols, and hepatocytes of mice, rats, monkeys, dogs, and humans. The plasma free fractions of GS-441524 were 62-78% across all studied species. The in vitro transporter study results showed that GS-441524 was a substrate of MDR1, BCRP, CNT3, ENT1, and ENT2; but not a substrate of CNT1, CNT2, and ENT4. GS-441524 had a low to moderate plasma clearance (CLp), ranging from 4.1 mL/min/kg in dogs to 26 mL/min/kg in mice; the steady state volume distribution (Vdss) ranged from 0.9 L/kg in dogs to 2.2 L/kg in mice after IV administration. Urinary excretion appeared to be the major elimination process for GS-441524. Following oral administration, the oral bioavailability was 8.3% in monkeys, 33% in rats, 39% in mice, and 85% in dogs. The PK and ADME properties of GS-441524 support its further development as an oral drug candidate.

Proof of concept continuous event logging in living cells

Cells must detect and respond to molecular events such as the presence or absence of specific small molecules. To accomplish this, cells have evolved methods to measure the presence and concentration of these small molecules in their environment and enact changes in gene expression or behavior. However, cells dont usually change their DNA in response to such outside stimuli. In this work, we have engineered a genetic circuit that can enact specific and controlled genetic changes in response to changing small molecule concentrations. Known DNA sequences can be repeatedly integrated into a genomic array such that their identity and order encodes information about past small molecule concentrations that the cell has experienced. To accomplish this, we use catalytically inactive CRISPR-Cas9 (dCas9) to bind to and block attachment sites for the integrase Bxb1. Therefore, through the co-expression of dCas9 and guide RNA, Bxb1 can be directed to integrate one of two engineered plasmids, which correspond to two orthogonal small molecule inducers that can be recorded with this system. We identified the optimal location of guide RNA binding to the Bxb1 attP integrase attachment site, and characterized the detection limits of the system by measuring the minimal small molecule concentration and shortest induction time necessary to produce measurable differences in array composition as read out by Oxford Nanopore long read sequencing technology.

Camel nanobodies broadly neutralize SARS-CoV-2 variants

With the emergence of SARS-CoV-2 variants, there is urgent need to develop broadly neutralizing antibodies. Here, we isolate two VHH nanobodies (7A3 and 8A2) from dromedary camels by phage display, which have high affinity for the receptor-binding domain (RBD) and broad neutralization activities against SARS-CoV-2 and its emerging variants. Cryo-EM complex structures reveal that 8A2 binds the RBD in its up mode and 7A3 inhibits receptor binding by uniquely targeting a highly conserved and deeply buried site in the spike regardless of the RBD conformational state. 7A3 at a dose of [≥]5 mg/kg efficiently protects K18-hACE2 transgenic mice from the lethal challenge of B.1.351 or B.1.617.2, suggesting that the nanobody has promising therapeutic potentials to curb the COVID-19 surge with emerging SARS-CoV-2 variants. One-Sentence SummaryDromedary camel (Camelus dromedarius) VHH phage libraries were built for isolation of the nanobodies that broadly neutralize SARS-CoV-2 variants.

The humanized nanobody RBD-1-2G tolerates the spike N501Y mutation to neutralize SARS-CoV-2

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.

Mitoxantrone modulates a glycosaminoglycan-spike complex to inhibit SARS-CoV-2 infection

Spike-mediated entry of SARS-CoV-2 into human airway epithelial cells is an attractive therapeutic target for COVID-19. In addition to protein receptors, the SARS-CoV-2 spike (S) protein also interacts with heparan sulfate, a negatively charged glycosaminoglycan (GAG) attached to certain membrane proteins on the cell surface. This interaction facilitates the engagement of spike with a downstream receptor to promote viral entry. Here, we show that Mitoxantrone, an FDA-approved topoisomerase inhibitor, targets a spike-GAG complex to compromise the fusogenic function of spike in viral entry. As a single agent, Mitoxantrone inhibits the infection of an authentic SARS-CoV-2 strain in a cell-based model and in human lung EpiAirway 3D tissues. Gene expression profiling supports the plasma membrane as a major target of Mitoxantrone but also underscores an undesired activity targeting nucleosome dynamics. We propose that Mitoxantrone analogs bearing similar GAG-binding activities but with reduced affinity for DNA topoisomerase may offer an alternative therapy to overcome breakthrough infections in the post-vaccine era.

A high throughput screening assay for inhibitors of SARS-CoV-2 pseudotyped particle entry

Effective small molecule therapies to combat the SARS-CoV-2 infection are still lacking as the COVID-19 pandemic continues globally. High throughput screening assays are needed for lead discovery and optimization of small molecule SARS-CoV-2 inhibitors. In this work, we have applied viral pseudotyping to establish a cell-based SARS-CoV-2 entry assay. Here, the pseudotyped particles (PP) contain SARS-CoV-2 spike in a membrane enveloping both the murine leukemia virus (MLV) gag-pol polyprotein and luciferase reporter RNA. Upon addition of PP to HEK293-ACE2 cells, the SARS-CoV-2 spike protein binds to the ACE2 receptor on the cell surface, resulting in priming by host proteases to trigger endocytosis of these particles, and membrane fusion between the particle envelope and the cell membrane. The internalized luciferase reporter gene is then expressed in cells, resulting in a luminescent readout as a surrogate for spike-mediated entry into cells. This SARS-CoV-2 PP entry assay can be executed in a biosafety level 2 containment lab for high throughput screening. From a collection of 5,158 approved drugs and drug candidates, our screening efforts identified 7 active compounds that inhibited the SARS-CoV-2-S PP entry. Of these seven, six compounds were active against live replicating SARS-CoV-2 virus in a cytopathic effect assay. Our results demonstrated the utility of this assay in the discovery and development of SARS-CoV-2 entry inhibitors as well as the mechanistic study of anti-SARS-CoV-2 compounds. Additionally, particles pseudotyped with spike proteins from SARS-CoV-2 B.1.1.7 and B.1.351 variants were prepared and used to evaluate the therapeutic effects of viral entry inhibitors.

A SARS-CoV-2 nucleocapsid protein TR-FRET assay amenable to high-throughput screening

Drug development for specific antiviral agents against coronavirus disease 2019 (COVID-19) is still an unmet medical need as the pandemic continues to spread globally. Although huge efforts for drug repurposing and compound screens have put forth, only few compounds remain in late stage clinical trials. New approaches and assays are needed to accelerate COVID-19 drug discovery and development. Here we report a time-resolved fluorescence resonance energy transfer-based assay that detects the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (NP) produced in infected cells. It uses two specific anti-NP monoclonal antibodies (MAbs) conjugated to donor and acceptor fluorophores that produces a robust ratiometric signal for high throughput screening of large compound collections. Using this assay, we measured a half maximal inhibitory concentration (IC50) for Remdesivir of 9.3 M against infection with SARS-CoV-2 USA/WA1/2020 (WA-1). The assay also detected SARS-CoV-2 South African (Beta, {beta}), Brazilian/Japanese variant P.1 (Gamma, {gamma}), and Californian (Epsilon, {varepsilon}), variants of concern or interest (VoC). Therefore, this homogeneous SARS-CoV-2 NP detection assay can be used for accelerating lead compound discovery for drug development and for evaluating drug efficacy against emerging SARS-CoV-2 VoC.

Meta-analytical evidence on mental disorder symptoms during the COVID-19 pandemic in Latin America

ObjectiveThere is a lack of evidence related to the prevalence of mental disorder symptoms as well as their heterogeneities during the COVID-19 pandemic in Latin America, a continent across the equators. The current study aims to provide meta-analytical evidence on mental disorder symptoms during COVID-19 among frontline healthcare workers, general healthcare workers, the general population, and university students in Latin America. MethodsBibliographical databases, such as PubMed, Embase, Web of Sciences, PsycINFO, and medRxiv, were systematically searched to identify pertinent studies up to Februry 6, 2021. Two coders performed the screening using predefined eligibility criteria. Studies were assigned quality scores using the Mixed Methods Appraisal Tool. The double data extraction method was used to minimize data entry errors. ResultsA total of 33 studies with 101,772 participants in Latin America were identified. The pooled prevalence of anxiety, depression, distress, and insomnia was 32%, 27%, 32%, and 35%, respectively. There was a higher prevalence of mental health symptoms in South America compared to Central America (33% vs. 27%, p <0.001). The pooled prevalence of mental health symptoms in the general population, general healthcare workers, frontline healthcare workers, and students in Latin America was 33%, 31%, 37%, and 36%, respectively. ConclusionThe high yet heterogenous level of prevalence of mental disorder symptoms emphasizes the need for appropriate identification of psychological interventions in Latin America.

Meta-Analytic Evidence of Depression and Anxiety in Eastern Europe during the COVID-19 Pandemic

ObjectiveTo perform a systematic and meta-analysis on the prevalence rates of mental health symptoms including anxiety and depression during the COVID-19 pandemic in the general population in Eastern Europe, as well as three select sub-populations: students, general healthcare workers, and frontline healthcare workers. Data sourcesStudies in PubMed, Embase, Web of Science, Psycinfo, and medRxiv up to February 6, 2021. Eligibility criteria and data analysisPrevalence rates of mental health symptoms in the general population and key sub-populations during the COVID-19 pandemic in Eastern Europe. Data were pooled using a random-effects meta-analysis to estimate the prevalence rates of anxiety and depression. ResultsThe meta-analysis identifies and includes 21 studies and 26 independent samples in Eastern Europe. Poland (n=4), Serbia (n=4), Russia (n=3), and Croatia (n=3) had the greatest number of studies. To our knowledge, no studies have been conducted in eleven Eastern European countries including Hungary, Slovakia, and Slovenia. The pooled prevalence of anxiety in 18 studies with 22 samples was 30% (95% CI: 24% - 37%) and pooled prevalence of depression in 18 studies with 23 samples was 27% (95% CI: 21% - 34%). ImplicationsThe cumulative evidence from the meta-analysis reveals high prevalence rates of clinically significant symptoms during the COVID-19 pandemic in Eastern Europe. The findings suggest evidence of a potential mental health crisis in Eastern Europe during the ongoing COVID-19 pandemic. Our synthesis also reveals a relative lack of studies in certain Eastern European countries as well as high heterogeneities among the existing studies, calling for more effort to achieve evidence-based mental healthcare in Eastern Europe. HighlightsO_LIThe pooled prevalence of anxiety and depression were 30% and 27% in Eastern Europe, respectively. C_LI Trial registrationCRD42020224458

A Systematic Review and Meta-Analysis of the Mental Health Symptoms during the Covid-19 Pandemic in Southeast Asia

AimsThe Covid-19 pandemic has had a substantial impact on the mental health of the general public and high-risk groups worldwide. Due to its proximity and close links to China, Southeast Asia was one of the first regions to be affected by the outbreak. The aim of this systematic review was to evaluate the prevalence of anxiety, depression and insomnia in the general adult population and healthcare workers (HCWs) in Southeast Asia during the course of the first year of the pandemic. MethodsSeveral literature databases were systemically searched for articles published up to February 2021 and two reviewers independently evaluated all relevant studies using pre-determined criteria. The prevalence rates of mental health symptoms were calculated using a random-effect meta-analysis model. ResultsIn total, 32 samples from 25 studies with 20,352 participants were included. Anxiety was assessed in all 25 studies and depression in 15 studies with pooled prevalence rates of 22% and 16% respectively. Only two studies assessed insomnia, which was estimated at 19%. The prevalence of anxiety and depression was similar amongst frontline HCWs (18%), general HCWs (17%), and students (20%) whilst being noticeably higher in the general population (27%). ConclusionsThis is the first systematic review to investigate the mental health impact of the Covid-19 pandemic in Southeast Asia. A considerable proportion of the general population and HCWs reported mild to moderate symptoms of anxiety and depression; the pooled prevalence rater, however, remain significantly lower than those reported in other areas such as China and Europe.

A 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 cytophatic 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 M). Systematic clustering analysis of active compounds revealed three promising chemotypes which have not been previously identified as inhibitors of SARS-CoV-2 infection. Further analysis identified allosteric binders to host receptor angiotensin-converting enzyme 2, which were able 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.

The Mental Health Under the COVID-19 Crisis in Africa: A Systematic Review and Meta-Analysis

ObjectiveIn this paper, we aim to provide a systematic review and meta-analysis on the prevalence rates of mental health symptoms of anxiety, depression, and insomnia among the major populations during the COVID-19 pandemic in Africa. DesignA systematic review and meta-analysis. Data sourcesWe search and include articles using PubMed, Embase, Web of Science, PsycINFO, and medRxiv databases between Feb 202 and Feb 6th, 2021. Eligibility criteria and data analysisThe meta-analysis targets the prevalence rates of mental health symptoms of major populations including frontline/general healthcare workers (HCWs), the general adult population, and medical students during the COVID-19 pandemic in Africa. To estimate the prevalence rates of anxiety, depression, and insomnia, we pooled data using random-effects meta-analyses. ResultsIn this meta-analysis, we identify and include 28 studies and 32 independent samples from 12 countries with a total of 15,072 participants in Africa. Ethiopia (7) and Egypt (6) had the largest number of studies. While many countries including, but not limited to, Algeria, Kenya, and Ghana had a high number of COVID-19 cases, as many as three quarters of African countries have no studies. The pooled prevalence of anxiety in 27 studies was 37% (95%CI: 31-43%, I2 = 99.0%) and that of depression in 24 studies was 45% (95%CI: 36-51%, I2 = 99.5%) and that of insomnia in 9 studies was 28% (95%CI: 20-41%, I2 = 99.2%). The pooled prevalence rates of anxiety, depression, and insomnia in North Africa (44%, 55%, and 31%, respectively) are higher than the rates in Sub-Saharan Africa (31%, 30%, and 24%, respectively). Our analysis indicated high heterogeneity and varying prevalence rates of mental health symptoms during the COVID-19 pandemic in Africa. DiscussionWe discuss our findings that a) a scarcity of studies in several African countries with a high number of COVID-19 cases, b) high heterogeneity among the studies, c) the extent of prevalence of mental health symptoms in Africa to be high, and d) the pattern of mental health symptoms in Africa differs from elsewhere, i.e., more African adults suffer from depression rather than anxiety and insomnia during COVID 19 compared to adult population in other countries or regions. Hence, our findings carry crucial implications for healthcare organizations and future research to enable evidence-based medicine in Africa. Our findings also call for increased scholarly attention on Africa, the least studied continent with a limited amount of research on mental health symptoms under the COVID 19 pandemic. Trial registrationCRD42020224458

A Systematic Review and Meta-Analysis on Mental Illness Symptoms in Spain in the COVID-19 Crisis

ObjectiveThis paper systematically reviews and assesses the prevalence of anxiety, depression, and insomnia symptoms in the general population, frontline healthcare workers (HCWs), and adult students in Spain during the COVID-19 crisis. Data sourcesArticles in PubMed, Embase, Web of Science, PsycINFO, and medRxiv from March 2020 to February 6, 2021. ResultsThe pooled prevalence of anxiety symptoms in 23 studies comprising a total sample of 85,560 was 20% (95% CI: 15% - 25%, I2 = 99.9%), that of depression symptoms in 23 articles with a total sample comprising of 86,469 individuals was 23% (95% CI: 18% - 28%, I2 = 99.8%), and that of insomnia symptoms in 4 articles with a total sample of 915 were 52% (95% CI: 42-64%, I2 = 88.9%). The overall prevalence of mental illness symptoms in frontline HCWs, general population, and students in Spain are 42%, 19%, and 50%, respectively. DiscussionThe accumulative evidence from the meta-analysis reveals that adults in Spain suffered higher prevalence rates of mental illness symptoms during the COVID-19 crisis with a significantly higher rate relative to other countries such as China. Our synthesis reveals high heterogeneity, varying prevalence rates and a relative lack of studies in frontline and general HCWs in Spain, calling future research and interventions to pay attention to those gaps to help inform evidence-based mental health policymaking and practice in Spain during the continuing COVID-19 crisis. The high prevalence rates call for preventative and prioritization measures of the mental illness symptoms during the Covid-19 pandemic.

SARS-CoV-2 Shedding Dynamics Across the Respiratory Tract, Sex, and Disease Severity for Adult and Pediatric COVID-19

BackgroundSARS-CoV-2 shedding dynamics in the upper (URT) and lower respiratory tract (LRT) remain unclear. ObjectiveTo analyze SARS-CoV-2 shedding dynamics across COVID-19 severity, the respiratory tract, sex and age cohorts (aged 0 to 17 years, 18 to 59 years, and 60 years or older). DesignSystematic review and pooled analyses. SettingMEDLINE, EMBASE, CENTRAL, Web of Science Core Collection, medRxiv and bioRxiv were searched up to 20 November 2020. ParticipantsThe systematic dataset included 1,266 adults and 136 children with COVID-19. MeasurementsCase characteristics (COVID-19 severity, age and sex) and quantitative respiratory viral loads (rVLs). ResultsIn the URT, adults with severe COVID-19 had higher rVLs at 1 DFSO than adults (P = 0.005) or children (P = 0.017) with nonsevere illness. Between 1-10 DFSO, severe adults had comparable rates of SARS-CoV-2 clearance from the URT as nonsevere adults (P = 0.479) and nonsevere children (P = 0.863). In the LRT, severe adults showed higher post-symptom-onset rVLs than nonsevere adults (P = 0.006). In the analyzed period (4-10 DFSO), severely affected adults had no significant trend in SARS-CoV-2 clearance from LRT (P = 0.105), whereas nonsevere adults showed a clear trend (P < 0.001). After stratifying for disease severity, sex and age (including child vs. adult) were not predictive of the duration of respiratory shedding. LimitationLimited data on case comorbidities and few samples in some cohorts. ConclusionHigh, persistent LRT shedding of SARS-CoV-2 characterized severe COVID-19 in adults. After symptom onset, severe cases tended to have higher URT shedding than their nonsevere counterparts. Disease severity, rather than age or sex, predicted SARS-CoV-2 kinetics. LRT specimens should more accurately prognosticate COVID-19 severity than URT specimens. Primary Funding SourceNatural Sciences and Engineering Research Council.

One Year of Evidence on Mental Health in the COVID-19 Crisis - A Systematic Review and Meta-Analysis

This paper provides a systematic review and meta-analysis on the prevalence rate of mental health issues of general population, general and frontline healthcare workers (HCWs) in China over one year of the COVID-19 crisis. We systematically searched PubMed, Embase, Web of Science, and Medrxiv at November 16th, 2020, pooled data using random-effects meta-analyses to estimate the prevalence rates, and ran meta-regression to tease out the heterogeneity. The meta-regression results uncovered several predictors of the prevalence rates, including severity, type of mental issues, population, sampling location, and study quality. Pooled prevalence rates are significantly different from, yet largely between, the findings of previous meta-analyses, suggesting the results of our larger study are consistent with yet more accurate than the findings of the smaller, previous meta-analyses. The prevalence rates of distress and insomnia and those of frontline HCWs are higher suggest future research and interventions should pay more attention to those mental outcomes and populations. Our findings suggest a need to examine the prevalence rates at varying levels of severity. The one-year cumulative evidence on sampling locations (Wuhan vs. non-Wuhan) corroborates the typhoon eye effect theory. Trial registrationCRD4202022059

Teleophthalmology and its evolving role in a COVID-19 pandemic: A scoping review

INTRODUCTION@#Teleophthalmology may assist the healthcare sector in adapting to limitations imposed on clinical practice by a viral pandemic. A scoping review is performed in this study to assess the current applications of teleophthalmology for its suitability to diagnose, monitor or manage ophthalmological conditions with accuracy.@*METHODS@#A search of PubMed was conducted for teleophthalmology-related articles published from 1 January 2018 to 4 May 2020. Only articles that focused on the use of teleophthalmology in terms of diagnosis and management, as well as its benefits and detriments, were included. The Mixed Methods Appraisal Tool (MMAT) was used to assess the quality of the included articles.@*RESULTS@#A total of 38 articles were assessed at the full-text level. There were 2 qualitative studies and 1 quantitative randomised controlled trial, while the majority were either quantitative descriptive studies (19, 50.0%) or quantitative non-randomised studies (16, 42.1%). Overall, 8 studies described reducing manpower requirements, 4 described reducing direct patient-doctor contact, 17 described storage of medical imaging and clinical data, and 9 described real-time teleconferencing. The MMAT analysis revealed limitations in appropriate sampling strategy in both quantitative non-randomised studies (9 of 16, 56.3%) and quantitative descriptive studies (9 of 19, 47.4%). Cost-effectiveness of teleophthalmology was not performed in any included study.@*CONCLUSION@#This current review of the various aspects of teleophthalmology describes how it may potentially assist the healthcare sector to cope with the limitations imposed by a viral pandemic through technology. Further research is required to evaluate the cost-effectiveness of the various strategies.

Non-covalent TMPRSS2 inhibitors identified from virtual screening

The 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 MERS. Starting with comparative structural modeling and 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 (PP) entry assay. A number of novel inhibitors were identified, providing starting points for further development of drug candidates for the treatment of COVID-19.

Heterogeneity in transmissibility and shedding SARS-CoV-2 via droplets and aerosols

Which virological factors mediate overdispersion in the transmissibility of emerging viruses remains a longstanding question in infectious disease epidemiology. Here, we use systematic review to develop a comprehensive dataset of respiratory viral loads (rVLs) of SARS-CoV-2, SARS-CoV-1 and influenza A(H1N1)pdm09. We then comparatively meta-analyze the data and model individual infectiousness by shedding viable virus via respiratory droplets and aerosols. Our analyses indicate heterogeneity in rVL as an intrinsic virological factor facilitating greater overdispersion for SARS-CoV-2 in the COVID-19 pandemic than A(H1N1)pdm09 in the 2009 influenza pandemic. For COVID-19, case heterogeneity remains broad throughout the infectious period, including for pediatric and asymptomatic infections. Hence, many COVID-19 cases inherently present minimal transmission risk, whereas highly infectious individuals shed tens to thousands of SARS-CoV-2 virions/min via droplets and aerosols while breathing, talking and singing. Coughing increases the contagiousness, especially in close contact, of symptomatic cases relative to asymptomatic ones. Infectiousness tends to be elevated between 1-5 days post-symptom onset. Our findings show how individual case variations influence virus transmissibility and present considerations for disease control in the COVID-19 pandemic. Significance StatementFor some emerging infectious diseases, including COVID-19, few cases cause most secondary infections. Others, like influenza A(H1N1)pdm09, spread more homogenously. The virological factors that mediate such distinctions in transmissibility remain unelucidated, prohibiting the development of specific disease control measures. We find that intrinsic case variation in respiratory viral load (rVL) facilitates overdispersion, and superspreading, for COVID-19 but more homogeneous transmission for A(H1N1)pdm09. We interpret the influence of heterogeneity in rVL on individual infectiousness by modelling likelihoods of shedding viable virus via respiratory droplets and aerosols. We analyze the distribution and kinetics of SARS-CoV-2 rVL, including across age and symptomatology subgroups. Our findings compare individual infectiousness across COVID-19 and A(H1N1)pdm09 cases and present quantitative guidance on triaging COVID-19 contact tracing.

Development of a high-throughput homogeneous AlphaLISA drug screening assay for the detection of SARS-CoV-2 Nucleocapsid

The coronavirus disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is in urgent need of therapeutic options. High-throughput screening (HTS) offers the research field an opportunity to rapidly identify such compounds. In this work, we have developed a homogeneous cell-based HTS system using AlphaLISA detection technology for the SARS-CoV-2 nucleocapsid protein (NP). Our assay measures both recombinant NP and endogenous NP from viral lysates and tissue culture supernatants (TCS) in a sandwich-based format using two monoclonal antibodies against the NP analyte. Viral NP was detected and quantified in both tissue culture supernatants and cell lysates, with large differences observed between 24 hours and 48 hours of infection. We simulated the viral infection by spiking in recombinant NP into 384-well plates with live Vero-E6 cells and were able to detect the NP with high sensitivity and a large dynamic range. Anti-viral agents that inhibit either viral cell entry or replication will decrease the AlphaLISA NP signal. Thus, this assay can be used for high-throughput screening of small molecules and biologics in the fight against the COVID-19 pandemic.