Inhibition of human thrombin by the constituents of licorice: inhibition kinetics and mechanistic insights through in vitro and in silico studies

Abstract

Thrombin inhibition therapy is a practical strategy to reduce thrombotic and cardiovascular risks via blocking the formation of blood clots. This study aimed to identify naturally occurring thrombin inhibitors from licorice (one of the most popular edible herbs), as well as to investigate their inhibitory mechanisms. Among all tested licorice constituents, licochalcone A was found as the most efficacious agent against human thrombin (IC₅₀ = 7.96 μM). Inhibition kinetic analyses demonstrated that licochalcone A was a mixed inhibitor against thrombin-mediated Z-Gly-Gly-Arg-AMC acetate hydrolysis, with a K _i value of 12.23 μM. Furthermore, mass spectrometry-based chemoproteomic assays and molecular docking simulations revealed that licochalcone A could bind to human thrombin at both exosite I and the catalytic site. In summary, our findings demonstrated that the chalcones isolated from licorice were a new class of direct thrombin inhibitors, also suggesting that licochalcone A was a promising lead compound for developing novel anti-thrombotic agents.

Fig. 1. The chemical structures of major constituents in licorice root.

Fig. 2. Inhibitory effects of the major constituents in licorice on human thrombin at three different concentrations (0 μM, 1 μM, 10 μM, 100 μM). All inhibition experiments were performed in triplicate (n = 3) and the values are expressed as mean ± SD.

Fig. 3. The dose–response curves of components from licorice on human thrombin; isoliquiritin (A), isoliquiritigenin (B), licochalcone A (C), licochalcone B (D), licochalcone C (E), echinatin (F), neoisoliquiritin (G) and isoliquiritin apioside (H). All inhibition experiments were performed in triplicate (n = 3) and the values are expressed as mean ± SD.

Fig. 4. The inhibitory behavior of licochalcone A on human thrombin. Left: the Lineweaver–Burk plot of licochalcone A (A) against thrombin-mediated Z-GGRAMC acetate hydrolysis. Right: the secondary plot from the Lineweaver–Burk plot for human thrombin inhibition by licochalcone A (B). All inhibition experiments were performed in triplicate (n = 3) and the values are expressed as mean ± SD.

Fig. 5. A stereo view of the crystal structure of thrombin and a stereodiagram of Z-GGRAMC acetate docked with licochalcone A. Inhibitor could bind to the active site (A), exosite I (C) of human thrombin. The detailed view represents the interactions between licochalcone A and the amino acids in the active site (B) and exosite I (D) of human thrombin.