Polyphenols Can Inhibit Furin In Vitro As A Result of the Reactivity of their Auto-oxidation Products to Proteins

J.      Zhu; W.J.M.      Van de Ven; T.      Verbiest; G.      Koeckelberghs; C.      Chen; Y.      Cui; A.J.M.      Vermorken

Abstract

Methods using fluorogenic peptide substrates have been proposed for screening of proprotein convertase (PC) inhibitors and they are attractive since they offer the advantage of being sensitive, cost-effective and susceptible to miniaturization. Several polyphenols, including epigallocatechin gallate ((-)EGCG), the main component of green tea, and quercetin, widely distributed in fruit and vegetables, however, led to false positive results when fluorogenic peptide substrates were used. Processing of genuine furin substrates was not inhibited by these polyphenols. In the present study, these discordant effects of (-)EGCG on the PC furin were studied. While quercetin can form aggregates in solution, aggregate-based promiscuous inhibition could be ruled out as underlying mechanism for (-)EGCG. Hydrogen peroxide production, from auto-oxidation, was too low to be a major factor but appeared associated to furin inhibition, suggesting a role for other auto-oxidation products. Since the instability of catechins is related to their electrophilic character, we tested the nucleophilic substance glutathione for stabilization. Indeed glutathione reduced furin inhibition and (-)EGCG binding to furin and serum albumin as shown by redox-cycling staining. Catechins, therefore, seem to form reactive compounds and this should be taken into account in screening assays. Adding glutathione to the detergent-based assay, as used in these studies to measure furin processing activity, strongly reduced inhibition by a number of polyphenols (catechins, gallic acid and quercetin), while the effect on the genuine inhibitor nona-D-arginine remained unchanged. In conclusion: the combined use of detergent and glutathione in the screening assay for furin inhibitors improves the predictive value.

Keywords: Polyphenols, (-)EGCG, proprotein convertase, furin-mediated substrate processing, promiscuous inhibitors, bovine serum albumin, screening, glutathione, hydrogen peroxide