The main protease (MPro) of SARS-CoV-2, the causative agent of COVID-19, is a pivotal nonstructural protein critical for viral replication and pathogenesis. Its protease function relies on three ...active site pockets for substrate recognition and a catalytic cysteine for enzymatic activity. To develop potential SARS-CoV-2 antivirals, we successfully synthesized a diverse range of azapeptide inhibitors with various covalent warheads to target MPro's catalytic cysteine. Our characterization identified potent MPro inhibitors, including MPI89 that features an aza-2,2-dichloroacetyl warhead with a remarkable EC50 value of 10 nM against SARS-CoV-2 infection in ACE2+ A549 cells and a selective index of 875. MPI89 is also remarkably selective and shows no potency against SARS-CoV-2 papain-like protease and several human proteases. Crystallography analyses demonstrated that these inhibitors covalently engaged the catalytic cysteine and used the aza-amide carbonyl oxygen to bind to the oxyanion hole. MPI89 stands as one of the most potent MPro inhibitors, suggesting the potential for further exploration of azapeptides and the aza-2,2-dichloroacetyl warhead for developing effective therapeutics against COVID-19.
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•Azapeptides are a viable scaffold for the design of SARS-CoV-2 main protease inhibitors.•Diverse covalent warheads can be readily attached to azapeptides.•MPI89 with an aza-2,2-dichloroacetyl warhead has an EC50 value of 10 nM against SARS-CoV-2.•Crystallography analysis indicates most inhibitors form a covalent bound with the MPro active site cysteine.•MPI89 maintains high potency against the E166V/L50F mutant of MPro.
Abstract
Using the amber suppression approach, N
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‐(4‐azidobenzoxycarbonyl)‐δ,ϵ‐dehydrolysine, an allysine precursor is genetically encoded in E. coli. Its genetic incorporation followed by two ...sequential biocompatible reactions allows convenient synthesis of proteins with site‐specific lysine dimethylation. Using this approach, dimethyl‐histone H3 and p53 proteins have been synthesized and used to probe functions of epigenetic enzymes including histone demethylase LSD1 and histone acetyltransferase Tip60. We confirmed that LSD1 is catalytically active toward H3K4me2 and H3K9me2 but inert toward H3K36me2, and methylation at p53 K372 directly activates Tip60 for its catalyzed acetylation at p53 K120.
The maturity of neomucosa growing on a serosal surface for the treatment of short bowel syndrome still is questionable. The aim of this study was to evaluate the intestinal neomucosa to assess its ...histologic maturity.
A 6-cm-long isolated ileal segment (IS) was prepared in 8 Wistar albino-type rats. The IS was divided from the antimesenteric side, and 2 intestinal tubes were established, which shared a common wall and a common pedicle. After ileal biopsy sampling for the control group (CG), the IS was fashioned into a mucous fistula. Eight weeks later, all the rats were killed, and the ISs were investigated for neomucosal growth. Sections were prepared with periodic acid shift (PAS) and H & E staining for light microscopy. They also were evaluated by transmission electron microscopy. The microscopic morphology of the 2 groups was evaluated. Immunohistochemical staining was performed to show the expression of the tissue β1, α3 and α2β1 integrin subunits of both the neomucosa (NS) and control group (CG) segments.
Sections of the NS showed a well-arranged columnar epithelial cell layer with goblet cells that were generally located superficially and with a complete basement membrane. Under the electron microscope, the sections from the NS group showed an epithelial cell layer with proper microvilli of the same height, although they were shorter than those of the CG, and tight intercellular junctions between the epithelial cells. Significant differences between the NS and CG groups were found in the measurements of villus width at base, microvillus surface, and microvillus height. The lamina propria consisted of rich collagen fibers and active fibroblasts in the NS group. In the immunohistochemical staining, although β1 integrine showed a dense distribution (+++) in the lamina propria, particularly localizing at the depth of the tunica mucosa layer, α3 integrin was observed to have a less dense immunoreactivity (++) in both groups. The expression of α2β1 integrin showed slight and dispersed (+) staining.
The NS showed histologic maturity and ultimate structural similarity with the native small bowel mucosa, which provides strong indirect evidence for the proper functioning of the neomucosa.
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