A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors

Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for high-throughput screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified four molecules, including the broad-spectrum antiviral merimepodib/VX-497, which show anti-nsp5 activity and inhibit SARS-CoV-2 replication in A549-ACE2 cells with IC50 values in the 4-21 {micro}M range. The here described assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.

An Endogenously activated antiviral state restricts SARS-CoV-2 infection in differentiated primary airway epithelial cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the coronavirus disease-19 (COVID-19) pandemic, was identified in late 2019 and went on to cause over 3.3 million deaths in 15 months. To date, targeted antiviral interventions against COVID-19 are limited. The spectrum of SARS-CoV-2 infection ranges from asymptomatic to fatal disease. However, the reasons for varying outcomes to SARS-CoV-2 infection are yet to be elucidated. Here we show that an endogenously activated interferon lambda (IFN{lambda}) pathway leads to resistance against SARS-CoV-2 infection. Using a well-differentiated primary nasal epithelial cell (WD-PNEC) model from multiple adult donors, we discovered that susceptibility to SARS-CoV-2 infection, but not respiratory syncytial virus (RSV) infection, varied. One of four donors was resistant to SARS-CoV-2 infection. High baseline IFN{lambda} expression levels and associated interferon stimulated genes correlated with resistance to SARS-CoV-2 infection. Inhibition of the JAK/STAT pathway in WD-PNECs with high endogenous IFN{lambda} secretion resulted in higher SARS-CoV-2 titres. Conversely, prophylactic IFN{lambda} treatment of WD-PNECs susceptible to infection resulted in reduced viral titres. An endogenously activated IFN{lambda} response, possibly due to genetic differences, may be one explanation for the differences in susceptibility to SARS-CoV-2 infection in humans. Importantly, our work supports the continued exploration of IFN{lambda} as a potential pharmaceutical against SARS-CoV-2 infection.

Gender Differences and Predictors of Practice Ownership in a Sample of Ontario Dentists.

PURPOSE: To examine the influence of gender on practice ownership among Ontario dentists. METHODS: In 2012, a 52-item survey was sent to a random sample of 3000 Ontario dentists (1500 men and 1500 women) to collect information on personal, professional and sociodemographic characteristics. The resulting data were analyzed using descriptive statistics and linear regression modeling. RESULTS: The 867 respondents included 463 men, 401 women and 3 people whose gender was unreported, yielding a response rate of 29%. Univariate regression analyses revealed that male dentists were 2.2 times more likely to be practice owners than female dentists. Dentists > 60 years were less likely to be owners than younger dentists. The odds of practice ownership were lower for dentists who preferred the ability to secure part-time work. A higher level of confidence in one's business acumen significantly predicted practice ownership. Dentists making concessions in their career to pursue family life were more likely to be associates, and those who perceived that their partners made concessions to aid in their career were more likely to be owners. In multivariate analyses, the effect of gender on practice ownership became insignificant, yet the influence of age, preference for career aspects, confidence in business skills and perceptions regarding concessions by self and partner persisted. Gender-stratified analyses revealed that familial factors significantly predicted ownership for female dentists, but not for male dentists. CONCLUSION: Gender appears to be linked to practice ownership, but when other factors are considered such as age, preference for part-time work, higher levels of confidence in business skills and perceptions of career concessions, the relationship does not remain.

Gender Differences and Predictors of Work Hours in a Sample of Ontario Dentists.

PURPOSE: To determine the influence of gender on weekly work hours of Ontario dentists. METHODS: In 2012, a 52-item survey was sent to a random sample of 3000 Ontario dentists (1500 men and 1500 women) to collect information on personal, professional and sociodemographic characteristics. The resulting data were analyzed using descriptive statistics and linear regression modeling. RESULTS: The 867 respondents included 463 men, 401 women and 3 people whose gender was unreported, yielding a response rate of 29%.Most dentists worked full-time, with men working, on average, 2 h/week longer than women. Younger dentists worked more than older dentists. Practice ownership increased weekly work hours, and men reported ownership more often than women. Canadian-trained women worked significantly fewer hours than those trained internationally. Women were more likely than men to work part time and take parental leave and more often reported being primary caregivers and solely responsible for household chores. Women with partner support for such tasks worked more hours than those who were solely responsible. Dentists with children ≤ 3 years of age worked fewer hours than those without children; however, after controlling for spousal responsibility for caregiver duties, this effect was eliminated. More women than men reported making concessions in their career to devote time to family. CONCLUSION: Gender, age, practice ownership, training location and degree of spousal support for household and caregiving responsibilities were predictors of weekly work hours. For women specifically, training location and household and caregiving responsibilities predicted weekly work hours.