Screening And Structure-inhibitory Activity Relationships Of Natural Inhibitors Against Escherichia Coli ?-glucuronidase

Bacterial ?-glucuronidase(GUS)plays crucial roles in the deconjugation of a variety of endogenous and drug glucuronides in the gut,which leads to the gastrointestinal toxicity of certain drugs.Inhibiting gut microbial GUS activity can alleviate the toxicity or side effects of Irinotecan and NSAIDs.Edible plants and herbal medicines have been recognized as important sources of ?-glucuronidase inhibitors due to their safety and friendliness.E.coli ?-glucuronidase(Ec GUS)was widely used for GUS inhibitor screening because it was frequently observed in mammalian gut and can be easily prepared.Objective and significance:This project aims to screen for Ec GUS inhibitors from iridoids and cinnamic acid derivatives and explore the structure-inhibitory activity relationships(SAR)of discovered inhibitors,which would provide theoretical basis for the design and development of more potent Ec GUS inhibitors from the two types of compounds.Methods:(1)The chemical components in baobab fruits were purified by extraction,preparative chromatography,and then identified by 1D and 2D NMR spectroscopy and other methods.(2)The reaction system of the inhibitory activity assays was established with recombinant Ec GUS as the enzyme and PNPG as the substrate.The inhibitory effects and structure-inhibitory relationships of the two series of compounds were analyzed.The inhibitory kinetics of potential compounds were characterized to obtain the inhibition type.(3)Molecular docking study was conducted to explore the binding interactions between the inhibitors and Ec GUS.Results:(1)Three new compounds were isolated and determined,including 3?-O-n-butyl Verminoside(22),3?-O-n-butyl Verminoside(23),7?-O-n-butyl Verminoside(24).(2)6-O-(E)-caffeoylajugol(9)and 6-O-(E)-feruloylajugol(10)were potent mixed type inhibitors against Ec GUS-mediated PNPG hydrolysis among all iridoids tested.Molecular docking study suggested that 6-O-(E)-caffeoylajugol(9)bound at the entrance of Ec GUS through hydrogen bond interactions with His330,Leu361,and Glu413.Binding mode of 6-O-(E)-feruloylajugol(10)with Ec GUS was similar to that of 6-O-(E)-caffeoylajugol(9),but more hydrogen bond interactions,such as interactions with Asp163,Ser360,Asn566 and Lys568,were also found.The SAR study revealed that both the presence of iridoid and a group,including the coumaroyl,feruloyl,and caffeoyl groups,in the C-6 position of the iridoids were essential for Ec GUS inhibition.(3)The acteoside(5)and caffeic acid ethyl ester(17)were potent mixed type and competitive inhibitors against Ec GUS,respectively.Acteoside(5)docked at the entrance of Ec GUS,while the caffeic acid ethyl ester(17)could be well-docked into the active pocket through the interactions with Asp163,Tyr468,and Glu504.The SAR study revealed that the presence of phenylpropionic acid skeleton was essential for Ec GUS inhibition.On contrast,the glucosyl and arabinosyl groups at C-6?? position of cinnamic acid derivatives with phenylpropanol glycoside skeleton were not preferable.Conclusion: The mechanism and the structure-inhibitory activity relationships of iridoids and cinnamic acid derivatives toward Ec GUS were explored through the Ec GUS inhibitory activity study and molecular docking studies,which provides theoretical basis for the design and development of more potent Ec GUS inhibitors from the two types of compounds.