Influence of EGCG oxidation on inhibitory activity against the SARS-CoV-2 main protease.

SARS-CoV-2 main protease (Mpro) is a well-recognized target for COVID-19 therapy. Green tea (−)-epigallocatechin-3-gallate (EGCG) possesses Mpro-inhibitory activity; however, the influence of EGCG oxidation on its inhibition activity remains obscure, given its high oxidation propensity. This study reveals that prolonged EGCG oxidation in the presence of Mpro dramatically increases its inhibitory activity with an IC 50 of 0.26 μM. The inhibitory mechanism is that EGCG-quinone preferentially binds the active site Mpro-Cys145-SH, which forms a quinoprotein. Though Mpro is present in the cell lysate, EGCG preferentially depletes its thiols. Non-cytotoxic EGCG effectively generates a quinoprotein in living cells, thus EGCG might selectively inhibit Mpro in SARS-CoV-2 infected cells. Chlorogenic acid facilitates EGCG oxidation. Together, they synergistically deplete multiple Mpro thiols though this is not more beneficial than EGCG alone. By contrast, excessive EGCG oxidation prior to incubation with Mpro largely compromises its inhibitory activity. Overall, the low IC 50 and the high selectivity imply that EGCG is a promising dietary Mpro inhibitor. While EGCG oxidation in the presence of Mpro has a pivotal role in inhibition, enhancing EGCG oxidation by chlorogenic acid no longer increases its inhibitory potential. EGCG oxidation in the absence of Mpro should be avoided to maximize its Mpro-inhibitory activity. [Display omitted] • Prolonged EGCG oxidation in the presence of Mpro greatly increases its inhibitory activity. • Mpro inhibition by EGCG is closely linked to quinoprotein formation. • Among the 12 cysteinyl thiols in Mpro, EGCG preferentially binds the active site Cys145-SH. • EGCG and its oxidation catalyst, CGA, synergistically reduce Mpro thiols but not Mpro activity. • Prolonged EGCG oxidation prior to incubation with Mpro reduces inhibitory capacity. [ABSTRACT FROM AUTHOR]