Galloylated polyphenols represent a new class of antithrombotic agents with broad activity against thiol isomerases.

BACKGROUND

Both protein disulfide isomerase (PDI) and SARS-CoV-2 main protease (Mpro) are reliant on active-site cysteines stabilized by adjacent amino acids. We reasoned that redox-active compounds might interfere with both enzymes by acting in the vicinity of these reactive sites thus interfering with viral replication and thrombus formation. Our previous screen of 1019 flavonoids identified several compounds that inhibit SARS-CoV-2 Mpro.

OBJECTIVES

Our goal was to identify phytochemical inhibitors of SARS-CoV-2 Mpro that block thiol isomerases and are antithrombotic.

METHODS

PDI, ERp57, ERp5, ERp46, isolated domains of PDI, and PDI mutants were used to evaluate the effects of galloylated polyphenols and their analogs on thiol isomerase reductase activity. Laser-injury and ferric chloride models of thrombus formation and a tail snip assay were used to assess the effects on thrombosis and hemostasis.

RESULTS

Pinocembrin 7-O-(3''-galloyl-4'',6''-(S)-hexahydroxydiphenoyl)-β-D-glucose (PGHG) inhibited both PDI and SARS-CoV-2 Mpro. Evaluation of isolated PDI fragments and active-site cysteine mutants showed that PGHG acts at the catalytic domains. Structure-function studies showed that PGHG interacts with histidines within the Cys53-Gly54-His55-Cys56 motifs of PDI. PGHG was equally active against other thiol isomerases, including ERp57, ERp5, ERp72, and ERp46. Screening numerous galloylated polyphenols demonstrated a class effect on thiol isomerase inhibition. Structure-activity relationships indicated that the galloyl moieties within large galloylated polyphenols were important for their inhibitory activity. PGHG and punicalagin were antithrombotic in murine models of thrombus formation.

CONCLUSIONS

Galloylated polyphenols represent a large class of antithrombotic compounds with broad activity against thiol isomerases. Many of these compounds also inhibit SARS-CoV-2 Mpro and viral replication.