Anthraquinones from Cassiae semen as thrombin inhibitors: in vitro and in silico studies

Thrombin inhibitor therapy is one of the most effective therapeutic strategies for the prevention and treatment of cardiovascular and thrombotic diseases. Although several marketed direct thrombin inhibitors (DTIs) have been widely used in clinic, the potentially serious complications of these DTIs prompted the researchers to find more DTIs with improved safety profiles. Herein, we report that natural anthraquinones in Cassiae semen (the seed of Cassia obtusifolia L. or C. tora L.), including obtusifolin, obtusin, aurantio-obtusin and chryso-obtusin, display strong to moderate inhibition on human thrombin, with the IC₅₀ values ranging from 9.08 μM to 27.88 μM. Further investigation on the inhibition kinetics demonstrates that these anthraquinones are mixed inhibitors against thrombin-mediated Z-GGRAMC acetate hydrolysis, while obtusifolin and aurantio-obtusin show strong thrombin inhibition capacity, with the K_i values of 9.63 μM and 10.30 μM, respectively. Docking simulations demonstrate that both obtusifolin and aurantio-obtusin can simultaneously bind on the catalytic cavity and the two anion binding exosites (ABE1 and ABE2), while the hydroxyl group at the C-7 site and the methoxyl group at the C-8 site can create key interactions with the amino acids surrounding the catalytic cavity via hydrogen bonding. All these findings suggest that obtusifolin and aurantio-obtusin are strong thrombin inhibitors possessing a unique anthraquinone skeleton, and could be used as lead compounds for the development of new thrombin inhibitors with improved properties.