Potential alternative therapeutic strategies for immune-mediated disorders are being increasingly recognized and are studied extensively. We previously reported the therapeutic potential of Brugia malayi derived recombinant cystatin (rBmaCys) in attenuating clinical symptoms of experimental colitis. The aim of this study was to elucidate the mechanisms involved in the rBmaCys-induced suppression of inflammation in the colon. Our results show that, the frequency of CD4+CD25+FoxP3+ regulatory T-cells was elevated in the colon and mesenteric lymph nodes. Similarly, the peritoneal macrophages recovered from the rBmaCys-treated colitis mice were alternatively activated and displayed reduced expression of TNF-α and IL-6. Another finding was significant increases in IgM+B1a-cells in the peritoneal cavity of mice following rBmaCys-treatment. These findings suggested that the regulatory cell network promoted by the rBmaCys in the colon and associated lymphoid tissues is important for its anti-inflammatory activity in the dextran sulfate sodium (DSS)-induced colitis mice.
To date, there are no approved oral antiviral therapies that can be administered early in the course of COVID‐19 to suppress progression of the disease or for prophylaxis. EDP‐235 is a potent and selective inhibitor of SARS‐CoV‐2 3C‐like protease (3CLpro). EDP‐235 inhibits SARS‐CoV‐2 3CLpro protease activity with an IC50 of 5.8 ± 3.7 nM and retains its activity against variant 3CLpro proteins from multiple SARS‐CoV‐2 lineages (IC50range of 2.8–5.8 nM). 3CLpro protease activity progress curves showed significant curvature in a time‐ and EDP‐235‐concentration‐dependent manner indicative of slow‐onset inhibition. Slow reversal of inhibition of SARS‐CoV‐2 3CLpro enzyme activity was observed in a jump dilution experiment. Michaelis‐Menten kinetic studies with a FRET peptide substrate in the presence of EDP‐235 indicated that EDP‐235 is a substrate‐competitive inhibitor of SARS‐CoV‐2 3CLpro with an overall dissociation constant Kiof 3.0 ± 1.6 nM. SARS‐CoV‐2 3CLpro was crystallized bound to a close analog of EDP‐235 and structure elucidation revealed that the ligand bound at the active site and interacted with side chains of conserved residues Cys‐145, His‐163, and Glu‐166. EDP‐235 also potently inhibits 3CLpro enzymes from other α‐coronaviruses (IC50range of 2–4 nM) and β‐coronaviruses (SARS‐CoV IC50 of 5.4 nM, MERS‐CoV IC50 of 70 nM) which cause disease in humans to date. EDP‐235 resistance mutations in HCoV‐229E map to the active site of 3CLpro close to the predicted binding site and offer additional support to the mechanism of inhibition. EDP‐235 also showed a favorable selectivity profile (>300 selectivity index) when tested against a panel of 30 mammalian proteases. In summary, EDP‐235 acts as a slow‐onset, slow‐reversible, substrate‐competitive inhibitor of SARS‐CoV‐2 3CLpro. The outstanding preclinical profile of EDP‐235 supports its further evaluation as an oral therapeutic for the management of COVID‐19.
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