Steroid Drugs Inhibit Bacterial Respiratory Oxidases and Are Lethal Toward Methicillin-Resistant Staphylococcus aureus.

BACKGROUND: Cytochrome bd complexes are respiratory oxidases found exclusively in prokaryotes that are important during infection for numerous bacterial pathogens. METHODS: In silico docking was employed to screen approved drugs for their ability to bind to the quinol site of Escherichia coli cytochrome bd-I. Respiratory inhibition was assessed with oxygen electrodes using membranes isolated from E. coli and methicillin-resistant Staphylococcus aureus strains expressing single respiratory oxidases (ie, cytochromes bd, bo', or aa3). Growth/viability assays were used to measure bacteriostatic and bactericidal effects. RESULTS: The steroid drugs ethinylestradiol and quinestrol inhibited E. coli bd-I activity with median inhibitory concentration (IC50) values of 47 ± 28.9 µg/mL (158 ± 97.2 µM) and 0.2 ± 0.04 µg/mL (0.5 ± 0.1 µM), respectively. Quinestrol inhibited growth of an E. coli "bd-I only" strain with an IC50 of 0.06 ± 0.02 µg/mL (0.2 ± 0.07 µM). Growth of an S. aureus "bd only" strain was inhibited by quinestrol with an IC50 of 2.2 ± 0.43 µg/mL (6.0 ± 1.2 µM). Quinestrol exhibited potent bactericidal effects against S. aureus but not E. coli. CONCLUSIONS: Quinestrol inhibits cytochrome bd in E. coli and S. aureus membranes and inhibits the growth of both species, yet is only bactericidal toward S. aureus.

3DLigandSite: structure-based prediction of protein-ligand binding sites.

3DLigandSite is a web tool for the prediction of ligand-binding sites in proteins. Here, we report a significant update since the first release of 3DLigandSite in 2010. The overall methodology remains the same, with candidate binding sites in proteins inferred using known binding sites in related protein structures as templates. However, the initial structural modelling step now uses the newly available structures from the AlphaFold database or alternatively Phyre2 when AlphaFold structures are not available. Further, a sequence-based search using HHSearch has been introduced to identify template structures with bound ligands that are used to infer the ligand-binding residues in the query protein. Finally, we introduced a machine learning element as the final prediction step, which improves the accuracy of predictions and provides a confidence score for each residue predicted to be part of a binding site. Validation of 3DLigandSite on a set of 6416 binding sites obtained 92% recall at 75% precision for non-metal binding sites and 52% recall at 75% precision for metal binding sites. 3DLigandSite is available at https://www.wass-michaelislab.org/3dligandsite. Users submit either a protein sequence or structure. Results are displayed in multiple formats including an interactive Mol* molecular visualization of the protein and the predicted binding sites.

Identification of sequence changes in myosin II that adjust muscle contraction velocity.

The speed of muscle contraction is related to body size; muscles in larger species contract at slower rates. Since contraction speed is a property of the myosin isoform expressed in a muscle, we investigated how sequence changes in a range of muscle myosin II isoforms enable this slower rate of muscle contraction. We considered 798 sequences from 13 mammalian myosin II isoforms to identify any adaptation to increasing body mass. We identified a correlation between body mass and sequence divergence for the motor domain of the 4 major adult myosin II isoforms (ß/Type I, IIa, IIb, and IIx), suggesting that these isoforms have adapted to increasing body mass. In contrast, the non-muscle and developmental isoforms show no correlation of sequence divergence with body mass. Analysis of the motor domain sequence of ß-myosin (predominant myosin in Type I/slow and cardiac muscle) from 67 mammals from 2 distinct clades identifies 16 sites, out of 800, associated with body mass (padj < 0.05) but not with the clade (padj > 0.05). Both clades change the same small set of amino acids, in the same order from small to large mammals, suggesting a limited number of ways in which contraction velocity can be successfully manipulated. To test this relationship, the 9 sites that differ between human and rat were mutated in the human ß-myosin to match the rat sequence. Biochemical analysis revealed that the rat-human ß-myosin chimera functioned like the native rat myosin with a 2-fold increase in both motility and in the rate of ADP release from the actin-myosin crossbridge (the step that limits contraction velocity). Thus, these sequence changes indicate adaptation of ß-myosin as species mass increased to enable a reduced contraction velocity and heart rate.

Differentially conserved amino acid positions may reflect differences in SARS-CoV-2 and SARS-CoV behaviour.

MOTIVATION: SARS-CoV-2 is a novel coronavirus currently causing a pandemic. Here, we performed a combined in-silico and cell culture comparison of SARS-CoV-2 and the closely related SARS-CoV. RESULTS: Many amino acid positions are differentially conserved between SARS-CoV-2 and SARS-CoV, which reflects the discrepancies in virus behaviour, i.e. more effective human-to-human transmission of SARS-CoV-2 and higher mortality associated with SARS-CoV. Variations in the S protein (mediates virus entry) were associated with differences in its interaction with ACE2 (cellular S receptor) and sensitivity to TMPRSS2 (enables virus entry via S cleavage) inhibition. Anti-ACE2 antibodies more strongly inhibited SARS-CoV than SARS-CoV-2 infection, probably due to a stronger SARS-CoV-2 S-ACE2 affinity relative to SARS-CoV S. Moreover, SARS-CoV-2 and SARS-CoV displayed differences in cell tropism. Cellular ACE2 and TMPRSS2 levels did not indicate susceptibility to SARS-CoV-2. In conclusion, we identified genomic variation between SARS-CoV-2 and SARS-CoV that may reflect the differences in their clinical and biological behaviour. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Aprotinin Inhibits SARS-CoV-2 Replication.

Severe acute respiratory syndrome virus 2 (SARS-CoV-2) is the cause of the current coronavirus disease 19 (COVID-19) pandemic. Protease inhibitors are under consideration as virus entry inhibitors that prevent the cleavage of the coronavirus spike (S) protein by cellular proteases. Herein, we showed that the protease inhibitor aprotinin (but not the protease inhibitor SERPINA1/alpha-1 antitrypsin) inhibited SARS-CoV-2 replication in therapeutically achievable concentrations. An analysis of proteomics and translatome data indicated that SARS-CoV-2 replication is associated with a downregulation of host cell protease inhibitors. Hence, aprotinin may compensate for downregulated host cell proteases during later virus replication cycles. Aprotinin displayed anti-SARS-CoV-2 activity in different cell types (Caco2, Calu-3, and primary bronchial epithelial cell air-liquid interface cultures) and against four virus isolates. In conclusion, therapeutic aprotinin concentrations exert anti-SARS-CoV-2 activity. An approved aprotinin aerosol may have potential for the early local control of SARS-CoV-2 replication and the prevention of COVID-19 progression to a severe, systemic disease.

Is the Bombali virus pathogenic in humans?

MOTIVATION: The potential of the Bombali virus, a novel Ebolavirus, to cause disease in humans remains unknown. We have previously identified potential determinants of Ebolavirus pathogenicity in humans by analysing the amino acid positions that are differentially conserved (specificity determining positions; SDPs) between human pathogenic Ebolaviruses and the non-pathogenic Reston virus. Here, we include the many Ebolavirus genome sequences that have since become available into our analysis and investigate the amino acid sequence of the Bombali virus proteins at the SDPs that discriminate between human pathogenic and non-human pathogenic Ebolaviruses. RESULTS: The use of 1408 Ebolavirus genomes (196 in the original analysis) resulted in a set of 166 SDPs (reduced from 180), 146 (88%) of which were retained from the original analysis. This indicates the robustness of our approach and refines the set of SDPs that distinguish human pathogenic Ebolaviruses from Reston virus. At SDPs, Bombali virus shared the majority of amino acids with the human pathogenic Ebolaviruses (63.25%). However, for two SDPs in VP24 (M136L, R139S) that have been proposed to be critical for the lack of Reston virus human pathogenicity because they alter the VP24-karyopherin interaction, the Bombali virus amino acids match those of Reston virus. Thus, Bombali virus may not be pathogenic in humans. Supporting this, no Bombali virus-associated disease outbreaks have been reported, although Bombali virus was isolated from fruit bats cohabitating in close contact with humans, and anti-Ebolavirus antibodies that may indicate contact with Bombali virus have been detected in humans. AVAILABILITY AND IMPLEMENTATION: Data files are available from https://github.com/wasslab/EbolavirusSDPsBioinformatics2019. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.