High-throughput screening (HTS) of large compound collections typically results in numerous small molecule hits that must be carefully evaluated to identify valid drug leads. Although several filtering mechanisms and other tools exist that can assist the chemist in this process, it is often the case that costly synthetic resources are expended in pursuing false positives. We report here a rapid and reliable NMR-based method for identifying reactive false positives including those that oxidize or alkylate a protein target. Importantly, the reactive species need not be the parent compound, as both reactive impurities and breakdown products can be detected. The assay is called ALARM NMR (a La assay to detect reactive molecules by nuclear magnetic resonance) and is based on monitoring DTT-dependent (13)C chemical shift changes of the human La antigen in the presence of a test compound or mixture. Extensive validation has been performed to demonstrate the reliability and utility of using ALARM NMR to assess thiol reactivity. This included comparing ALARM NMR to a glutathione-based fluorescence assay, as well as testing a collection of more than 3500 compounds containing HTS hits from 23 drug targets. The data show that current in silico filtering tools fail to identify more than half of the compounds that can act via reactive mechanisms. Significantly, we show how ALARM NMR data has been critical in identifying reactive compounds that would otherwise have been prioritized for lead optimization. In addition, a new filtering tool has been developed on the basis of the ALARM NMR data that can augment current in silico programs for identifying nuisance compounds and improving the process of hit triage.

As the epidemic outbreak of 2019 coronavirus disease , general population may experience psychological distress. Evidence has suggested that negative coping styles may be related to subsequent mental illness. Therefore, we investigate the general population's psychological distress and coping styles in the early stages of the COVID-19 outbreak. A cross-sectional battery of surveys was conducted from February 1-4, 2020. The Kessler 6 psychological distress scale, the simplified coping style questionnaire and a general information questionnaire were administered on-line to a convenience sample of 1599 in China. A multiple linear regression analysis was performed to identify the influence factors of psychological distress. General population's psychological distress were significant differences based on age, marriage, epidemic contact characteristics, concern with media reports, and perceived impacts of the epidemic outbreak (all p <0.001) except gender (p = 0.316). The population with younger age (F = 102.04), unmarried (t = 15.28), with history of visiting Wuhan in the past month (t = -40.86), with history of epidemics occurring in the community (t = -10.25), more concern with media reports (F = 21.84), perceived more impacts of the epidemic outbreak (changes over living situations, F = 331.71; emotional control, F = 1863.07; epidemic-related dreams, F = 1642.78) and negative coping style (t = 37.41) had higher level of psychological distress. Multivariate analysis found that marriage, epidemic contact characteristics, perceived impacts of the epidemic and coping style were the influence factors of psychological distress (all p <0.001). Epidemic of COVID-19 caused high level of psychological distress. The general mainland Chinese population with unmarried, history of visiting Wuhan in the past month, perceived more impacts of the epidemic and negative coping style had higher level of psychological distress in the early stages of COVID-19 PLOS ONE

Background: The purpose of this study was to investigate the psychological status of the general population in mainland China during the outbreak of coronavirus disease 2019 (COVID-19), and to explore the factors influencing psychological distress, in order to provide the basis for further psychological intervention programs. Methods: We administered three questionnaires on-line to a convenience sample of the general population from different regions of mainland China from February 1 to February 4, 2020. We used the Mandarin versions of the sixitem Kessler psychological distress scale (K6), the Simplified Coping Style Questionnaire (SCSQ), and the Social Support Rating Scale (SSRS). We also collected demographic data and other information related to the COVID-19 outbreak. Multivariate binary logistic regression analysis was used to identify factors influencing psychological distress. Results: Of 1607 respondents, 1588 returned valid questionnaires and were included in the analysis. Nearly one quarter (22.8%) had high levels of psychological distress (K6 score ≥ 13). Individuals with higher psychological distress were more likely to be unmarried, spend more than 6 h per day searching for information about COVID-19, more frequently adopt a passive coping style, and report less social support than those with lower psychological distress. Conclusions: The COVID-19 outbreak in China has a great impact on the mental health status of the general population. Active coping strategies and increased social support are significantly correlated with decreased psychological distress, and may serve as the basis for psychological interventions.

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So far, the physiology of Saccharomyces cerevisiae at near-zero growth rates has been studied in retentostat cultures with a growth-limiting supply of the carbon and energy source. Despite its relevance in nature and industry, the near-zero growth physiology of S. cerevisiae under conditions where growth is limited by the supply of non-energy substrates remains largely unexplored. This study analyzes the physiology of S. cerevisiae in aerobic chemostat and retentostat cultures grown under either ammonium or phosphate limitation. To compensate for loss of extracellular nitrogen- or phosphorus-containing compounds, establishing near-zero growth rates (μ < 0.002 h−1) in these retentostats required addition of low concentrations of ammonium or phosphate to reservoir media. In chemostats as well as in retentostats, strongly reduced cellular contents of the growth-limiting element (nitrogen or phosphorus) and high accumulation levels of storage carbohydrates were observed. Even at near-zero growth rates, culture viability in non-energy-limited retentostats remained above 80% and ATP synthesis was still sufficient to maintain an adequate energy status and keep cells in a metabolically active state. Compared to similar glucose-limited retentostat cultures, the nitrogen- and phosphate-limited cultures showed aerobic fermentation and a partial uncoupling of catabolism and anabolism. The possibility to achieve stable, near-zero growth cultures of S. cerevisiae under nitrogen or phosphorus limitation offers interesting prospects for high-yield production of bio-based chemicals. IMPORTANCE The yeast Saccharomyces cerevisiae is a commonly used microbial host for production of various biochemical compounds. From a physiological perspective, biosynthesis of these compounds competes with biomass formation in terms of carbon and/or energy equivalents. Fermentation processes functioning at extremely low or near-zero growth rates would prevent loss of feedstock to biomass production. Establishing S. cerevisiae cultures in which growth is restricted by the limited supply of a non-energy substrate therefore could have a wide range of industrial applications but remains largely unexplored. In this work we accomplished near-zero growth of S. cerevisiae through limited supply of a non-energy nutrient, namely, the nitrogen or phosphorus source, and carried out a quantitative physiological study of the cells under these conditions. The possibility to achieve near-zero-growth S. cerevisiae cultures through limited supply of a non-energy nutrient may offer interesting prospects to develop novel fermentation processes for high-yield production of bio-based chemicals.

We studied the synthesis, cleavage rates, and oral administration of prodrugs of the HIV protease inhibitors (PIs) lopinavir and ritonavir. Phosphate esters attached directly to the central hydroxyl groups of these PIs did not demonstrate enzyme-mediated cleavage in vitro and did not provide measurable plasma levels of the parent drugs in vivo. However, oxymethylphosphate (OMP) and oxyethylphosphate (OEP) prodrugs provided improved rates of cleavage, high levels of aqueous solubility, and high plasma levels of the parent drugs when dosed orally in rats and dogs. Dosing unformulated capsules containing the solid prodrugs led to plasma levels equivalent to those observed for dosing formulated solutions of the parent drugs. A direct synthetic process for the preparation of OMP and OEP prodrugs was developed, and the improved synthetic method may be applicable to the preparation of analogous soluble prodrugs of other drug classes with limited solubility.

Little is known about the mechanism of HCV polymerase-catalyzed nucleotide incorporation and the individual steps employed by this enzyme during a catalytic cycle. In this paper, we applied various biochemical tools and examined the mechanism of polymerase catalysis. We found that formation of a productive RNA-enzyme complex is the slowest step followed by RNA dissociation and initiation of primer strand synthesis. Various groups have reported several classes of small molecule inhibitors of hepatitis C virus NS5B polymerase; however, the mechanism of inhibition for many of these inhibitors is not clear. We undertook a series of detailed mechanistic studies to characterize the mechanisms of inhibition for these HCV polymerase inhibitors. We found that the diketoacid derivatives competitively bind to the elongation NTP pocket in the active site and inhibit both the initiation and elongation steps of polymerization. While both benzimidazoles and benzothiadiazines are noncompetitive with respect to the active site elongation NTP pocket, benzothiadiazine compounds competitively bind to the initiation pocket in the active site and inhibit only the initiation step of de novo RNA polymerization. The benzimidazoles bind to the thumb allosteric pocket and inhibit the conformational changes during RNA synthesis. We also observed a cross interaction between the thumb allosteric pocket and the initiation pocket using inhibitor-inhibitor cross competition studies. This information will be very important in designing combination therapies using two small molecule drugs to treat hepatitis C virus.