Hinokiflavone from Platycladi cacumen as a potent broad-spectrum inhibitor of gut microbial Loop-1 β-glucuronidases: Inhibition kinetics and molecular simulation

Abstract

Gut microbial Loop-1 β-glucuronidases (gmGUS) played an important role in irinotecan-induced gastrointestinal toxicity by regulating the level of its active metabolite SN38 through enterohepatic recirculation. gmGUS inhibition has emerged as a promising approach to relieve its dose-limiting intestinal toxicity and improve its medication efficacy. This study aims to investigate the inhibitory effects and mechanisms of Platycladi cacumen and its main constituent hinokiflavone against four different types of Loop-1 gmGUS (EeGUS, SaGUS, CpGUS and EcGUS). Our results showed that the ethanol extract of Platycladi cacumen displayed strong broad-spectrum inhibition against four gmGUS, and hinokiflavone could potently inhibit EeGUS, SaGUS, CpGUS and EcGUS with IC₅₀ values of 0.09 ± 0.01 μM, 0.44 ± 0.01 μM, 0.20 ± 0.01 μM and 0.69 ± 0.10 μM, respectively. Inhibition kinetic analyses demonstrated that hinokiflavone acted as a strong competitive inhibitor of EeGUS with K_i value of 0.13 μM, while it displayed non-competitive inhibition against SaGUS, CpGUS and EcGUS, with the K_i values of 0.43 μM, 0.33 μM and 0.76 μM, respectively. Docking simulations revealed that hinokiflavone could tightly bind with Tyr-485 and Glu-516 in catalytic sites of EeGUS, as well it created strong interactions with amino acids in loop structures of SaGUS (Asn-362), CpGUS (Phe-363, Met-364, Ala-365 and Arg-375) and EcGUS (Leu-361) to interfere the substrate entry into the catalytic pocket. Collectively, these results confirmed that hinokiflavone from Platycladi cacumen is a potent naturally occurring inhibitor of gmGUS with broad efficiency, suggesting hinokiflavone will be helpful for alleviating intestinal toxicity in irinotecan therapy.