Epigallocatechin-3-gallate potently inhibits the in vitro activity of hydroxy-3-methyl-glutaryl-CoA reductase

Abstract

Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is the rate-controlling enzyme of cholesterol synthesis, and owing to its biological and pharmacological relevance, researchers have investigated several compounds capable of modulating its activity with the hope of developing new hypocholesterolemic drugs. In particular, polyphenol-rich extracts were extensively tested for their cholesterol-lowering effect as alternatives, or adjuvants, to the conventional statin therapies, but a full understanding of the mechanism of their action has yet to be reached. Our work reports on a detailed kinetic and equilibrium study on the modulation of HMGR by the most-abundant catechin in green tea, epigallocatechin-3-gallate (EGCG). Using a concerted approach involving spectrophotometric, optical biosensor, and chromatographic analyses, molecular docking, and site-directed mutagenesis on the cofactor site of HMGR, we have demonstrated that EGCG potently inhibits the in vitro activity of HMGR (K(i) in the nanomolar range) by competitively binding to the cofactor site of the reductase. Finally, we evaluated the effect of combined EGCG-statin administration.

Fig.1.

Schematic representation of the mechanism of synergistic inhibition of HMGR (M) by EGCG (Y) and pravastatin (X). The phenomenological description of the multiple equilibria consists of the formation of two complexes (MX and MY) upon binding of the statin at the substrate site and of EGCG at the cofactor site, both with the ability of binding to the other ligand in a ternary complex (MXY).

Fig.2.

Molecular docking of EGCG in the NADPH binding site of HMG-CoA reductase (A: surface representation; B: cartoon and stick representation with h-bonds formed) and the same representation of the complex NADPH/reductase (C, D). Reductase chain A and chain B are colored in yellow and light blue, respectively.

Fig.3.

Inhibition of human HMG-CoA activity by EGCG. HMG-CoA reductase residual activities versus tea catechin concentration measured at four different NADPH concentrations: the curves and related standard deviations are global fits of the data to equations 3 and 4 and yield the derivation of the apparent equilibrium dissociation constant (K_d,app) at different cofactor concentrations. Moreover, Marquardt-Levenberg nonlinear least squares fitting procedure is used to obtain the equilibrium dissociation constant K_d of the complex HMG-CoA/EGCG. ▴: 30 µM NADPH(A); ■: 90 µM NADPH(B); ¨: 150 µM NADPH(C); •: 270 µM NADPH(D). E: Linear fit to equation 4 of K_d,app values calculated for the EGCG-HMGR interaction versus cofactor concentrations. F: Time courses for the inhibition of HMGR activity by EGCG at different preincubation periods.

Fig.4.

Enzyme kinetic constants for the inhibition of HMGR by EGCG. The dependences of V_max, K_m, K_cat, and K_cat/K_m from EGCG concentrations were fitted to the proper equations for a competitive inhibition model.

Fig.5.

Overlay of association and dissociation kinetics of soluble EGCG to CMD-bound HMG-CoA reductase (A); dependence of the extent of binding on ligand concentration. Nonlinear fit (solid line) and 95% confidence-bound (dashed lines) are reported. Each experimental point was the average of five replicates (B).

Fig.6.

Chromatographic analysis of HMGR modulation by EGCG. The superimposition of elution profiles of the species involved in the equilibrium (CoA:mevalonate stoichiometric ratio = 1:1) at different EGCG concentrations is presented. Peak area corresponding to CoA produced in the enzyme-free assay was each time subtracted from corresponding peaks in each sample (A). Residual activity plots of both bacterial expressed (■) and microsomal liver HMGR (¨), and HMGR mutant (▴) measured monitoring CoA/mevalonate production (B). Raw data were fitted to equation 3.

Fig.7.

Residual activity plot of HMGR in the presence of EGCG (■), pravastatin (▴), and pravastatin and EGCG (•). Raw data for the HMGR inhibition by pravastatin in the presence of EGCG were fitted to equation 7.