β-coronavirus (CoVs) alone has been responsible for three major global outbreaks in the 21st century. The current crisis has led to an urgent requirement to develop therapeutics. Even though a number of vaccines are available, alternative strategies targeting essential viral components are required as a backup against the emergence of lethal viral variants. One such target is the main protease (M pro ) that plays an indispensable role in viral replication. The availability of over 270 M pro X-ray structures in complex with inhibitors provides unique insights into ligand–protein interactions. Herein, we provide a comprehensive comparison of all nonredundant ligand-binding sites available for SARS-CoV2, SARS-CoV, and MERS-CoV M pro . Extensive adaptive sampling has been used to investigate structural conservation of ligand-binding sites using Markov state models (MSMs) and compare conformational dynamics employing convolutional variational auto-encoder-based deep learning. Our results indicate that not all ligand-binding sites are dynamically conserved despite high sequence and structural conservation across β-CoV homologs. This highlights the complexity in targeting all three M pro enzymes with a single pan inhibitor.

Background Patients with chronic obstructive pulmonary disease (COPD) are more likely to be readmitted than patients with other chronic medical conditions, yet knowledge regarding such readmissions is limited. We aimed to determine factors associated with readmission within 30 days of a COPD hospitalization or death with an emphasis on examining aspects of socioeconomic status and specific comorbidities. Methods A population-based cohort study was conducted using health administrative data from Ontario, Canada. All hospitalizations for COPD between 2004 and 2014 were considered. The primary exposures were socioeconomic status as measured by residential instability (an ecologic variable), and comorbidities such as cardiovascular disease and cancer. Other domains of socioeconomic status were considered as secondary exposures. Logistic regression with generalized estimating equations was used to examine the effect of exposures, adjusting for other patient factors, on 30-day readmission or death. Results There were 126,013 patients contributing to 252,756 index COPD hospitalizations from 168 Ontario hospitals. Of these hospitalizations, 19.4% resulted in a readmission and 2.8% resulted in death within 30 days. After adjusting for other factors, readmissions or death were modestly more likely among people with the highest residential instability compared to the lowest (OR 1.05, 95% CI 1.01–1.09). Comorbidities such as cardiovascular disease and cancer, as well as other aspects of low socioeconomic status also increased readmission or death risk. Interpretation Socioeconomic status, measured in various ways, and many comorbidities predict 30-day readmission or death in patients hospitalized for COPD. Strategies that address these factors may help reduce readmissions and death.

Highlights d The phosphatase PPP6C promotes sensitivity of melanoma cells to MEK inhibitors d PPP6C loss causes ERK pathway hyperactivation in cells harboring BRAF and RAS mutations d PP6 regulatory subunits recruit MEK for dephosphorylation by PPP6C d Recurrent melanoma-associated PPP6C mutants lead to elevated MEK and ERK activity

The metalloproteinase anthrax lethal factor (LF) is secreted by Bacillus anthracis to promote disease virulence through disruption of host signaling pathways. LF is a highly specific protease, exclusively cleaving mitogen-activated protein kinase kinases (MKKs) and rodent NLRP1B (NACHT leucine-rich repeat and pyrin domain-containing protein 1B). How LF achieves such restricted substrate specificity is not understood. Previous studies have suggested the existence of an exosite interaction between LF and MKKs that promotes cleavage efficiency and specificity. Through a combination of in silico prediction and site-directed mutagenesis, we have mapped an exosite to a non-catalytic region of LF. Mutations within this site selectively impair proteolysis of full-length MKKs yet have no impact on cleavage of short peptide substrates. Although this region appears important for cleaving all LF protein substrates, we found that mutation of specific residues within the exosite differentially affects MKK and NLRP1B cleavage in vitro and in cultured cells. One residue in particular, Trp-271, is essential for cleavage of MKK3, MKK4, and MKK6 but dispensable for targeting of MEK1, MEK2, and NLRP1B. Analysis of chimeric substrates suggests that this residue interacts with the MKK catalytic domain. We found that LF-W271A blocked ERK phosphorylation and growth in a melanoma cell line, suggesting that it may provide a highly selective inhibitor of MEK1/2 for use as a cancer therapeutic. These findings provide insight into how a bacterial toxin functions to specifically impair host signaling pathways and suggest a general strategy for mapping protease exosite interactions.

Staphylococcus aureus is a leading cause of significant morbidity and mortality and an enormous economic burden to public health worldwide. Infections caused by methicillin-resistant S. aureus (MRSA) pose a major threat as MRSA strains are becoming increasingly prevalent and multi-drug resistant. To this date, vaccines targeting surface-bound antigens demonstrated promising results in preclinical testing but have failed in clinical trials. S. aureus pathogenesis is in large part driven by immune destructive and immune modulating toxins and thus represent promising vaccine targets. Hence, the objective of this study was to evaluate the safety and immunogenicity of a staphylococcal 4-component vaccine targeting secreted bi-component pore-forming toxins (BCPFTs) and superantigens (SAgs) in non-human primates (NHPs). The 4-component vaccine proved to be safe, even when repeated vaccinations were given at a dose that is 5 to 10- fold higher than the proposed human dose. Vaccinated rhesus macaques did not exhibit clinical signs, weight loss, or changes in hematology or serum chemistry parameters related to the administration of the vaccine. No acute, vaccine-related elevation of serum cytokine levels was observed after vaccine administration, confirming the toxoid components lacked superantigenicity. Immunized animals demonstrated high level of toxin-specific total and neutralizing antibodies toward target antigens of the 4-component vaccine as well as cross-neutralizing activity toward staphylococcal BCPFTs and SAgs that are not direct targets of the vaccine. Cross-neutralization was also observed toward the heterologous streptococcal pyogenic exotoxin B. Ex vivo stimulation of PBMCs with individual vaccine components demonstrated an overall increase in several T cell cytokines measured in supernatants. Immunophenotyping of CD4 T cells ex vivo showed an increase in Ag-specific polyfunctional CD4 T cells in response to antigen stimulation. Taken together, we demonstrate that the 4-component vaccine is well-tolerated and immunogenic in NHPs generating both humoral and cellular immune responses. Targeting secreted toxin antigens could be the next-generation vaccine approach for staphylococcal vaccines if also proven to provide efficacy in humans.