Plant flavonoid inhibition of SARS-CoV-2 main protease and viral replication.

Plant-based flavonoids have been evaluated as inhibitors of ß-coronavirus replication and as therapies for COVID-19 on the basis of their safety profile and widespread availability. The SARS-CoV-2 main protease (Mpro) has been implicated as a target for flavonoids in silico. Yet no comprehensive in vitro testing of flavonoid activity against SARS-CoV-2 Mpro has heretofore been performed. We screened 1,019 diverse flavonoids for their ability to inhibit SARS-CoV-2 Mpro. Multiple structure-activity relationships were identified among active compounds such as enrichment of galloylated flavonoids and biflavones, including multiple biflavone analogs of apigenin. In a cell-based SARS-CoV-2 replication assay, the most potent inhibitors were apigenin and the galloylated pinocembrin analog, pinocembrin 7-O-(3''-galloyl-4'',6''-(S)-hexahydroxydiphenoyl)-beta-D-glucose (PGHG). Molecular dynamic simulations predicted that PGHG occludes the S1 binding site via a galloyl group and induces a conformational change in Mpro. These studies will advance the development of plant-based flavonoids-including widely available natural products-to target ß-coronaviruses.

Sulfenylation links oxidative stress to protein disulfide isomerase oxidase activity and thrombus formation.

BACKGROUND: Oxidative stress contributes to thrombosis in atherosclerosis, inflammation, infection, aging, and malignancy. Oxidant-induced cysteine modifications, including sulfenylation, can act as a redox-sensitive switch that controls protein function. Protein disulfide isomerase (PDI) is a prothrombotic enzyme with exquisitely redox-sensitive active-site cysteines. OBJECTIVES: We hypothesized that PDI is sulfenylated during oxidative stress, contributing to the prothrombotic potential of PDI. METHODS: Biochemical and enzymatic assays using purified proteins, platelet and endothelial cell assays, and in vivo murine thrombosis studies were used to evaluate the role of oxidative stress in PDI sulfenylation and prothrombotic activity. RESULTS: PDI exposure to oxidants resulted in the loss of PDI reductase activity and simultaneously promoted sulfenylated PDI generation. Following exposure to oxidants, sulfenylated PDI spontaneously converted to disulfided PDI. PDI oxidized in this manner was able to transfer disulfides to protein substrates. Inhibition of sulfenylation impaired disulfide formation by oxidants, indicating that sulfenylation is an intermediate during PDI oxidation. Agonist-induced activation of platelets and endothelium resulted in the release of sulfenylated PDI. PDI was also sulfenylated by oxidized low-density lipoprotein (oxLDL). In an in vivo model of thrombus formation, oxLDL markedly promoted platelet accumulation following an arteriolar injury. PDI oxidoreductase inhibition blocked oxLDL-mediated augmentation of thrombosis. CONCLUSION: PDI sulfenylation is a critical posttranslational modification that is an intermediate during disulfide PDI formation in the setting of oxidative stress. Oxidants generated by vascular cells during activation promote PDI sulfenylation, and interference with PDI during oxidative stress impairs thrombus formation.

Route exploration and synthesis of the reported pyridone-based PDI inhibitor STK076545.

The enzyme protein disulfide isomerase (PDI) is essential for the correct folding of proteins and the activation of certain cell surface receptors, and is a promising target for the treatment of cancer and thrombotic conditions. A previous high-throughput screen identified the commercial compound STK076545 as a promising PDI inhibitor. To confirm its activity and support further biological studies, a resynthesis was pursued of the reported ß-keto-amide with an N-alkylated pyridone at the α-position. Numerous conventional approaches were complicated by undesired fragmentations or rearrangements. However, a successful 5-step synthetic route was achieved using an aldol reaction with an α-pyridone allyl ester as a key step. An X-ray crystal structure of the final compound confirmed that the reported structure of STK076545 was achieved, however its lack of PDI activity and inconsistent spectral data suggest that the commercial structure was misassigned.