Screening Of Xanthine Oxidase Inhibitors Based On Molecular Interactions And Their Uric Acid-lowering Effects

Objective Hyperuricemia(HUA)is a chronic metabolic disorder caused by an impairment in the metabolism of purines in the body,which manifests itself clinically as high serum uric acid levels.Prolonged high uric acid levels are closely associated with diseases such as HUA and gout.Uric acid lowering therapy(ULT)is effective in the prevention and treatment of hyperuricemia and gout,and numerous guidelines recommend Xanthine oxidase inhibitor(XOI)as the first-line ULT drugs for the treatment of chronic gout with hyperuricemia.However,the existing drugs are associated with many clinical adverse effects.In this study,XOIs with uric acid-lowering activity were screened from the relevant components of medicine and food homology based on molecular interactions,with a view to providing a molecular structural basis for the development of new natural XOIs.Methods By collecting and integrating 101 traditional Chinese medicines,including clove,and the list of items that can be used in health food,a total of 189 food medicines were listed in the "Catalogue of substances that are both food and herbal medicine according to tradition"(consultation draft)adopted by the former Health and Family Planning Commission in 2015.The TCMSP database,Pub Chem database and specialized chemical databases were used to obtain information and three-dimensional structures of small molecule-related components of medicine and food homology,and to construct a library of small molecule ligands of medicine and food homology.The three-dimensional structure of Xanthine oxidase(XO)was used as the receptor protein target,and the XO molybdenum pterin active site and surrounding hydrophobic channels were used as docking pockets for molecular docking using the CDOCKER module of the Shenzhen supercomputing platform Discovery Studio v4.0 software,and sorted according to binding energy.A model of hyperuricemia Hep G2 cells was constructed by inducing a large amount of uric acid production in the hepatocyte line Hep G2 with medium containing 2.5 m M adenosine.The cells were randomly divided into blank control group,model group,allopurinol group and homoeopathic group.Except for the blank group,the model group,the allopurinol group and the medicine and food homology group were respectively incubated with adenosine,allopurinol(containing adenosine)and different concentrations of medicine and food homology small molecule(containing adenosine)for 24 h,and then each group was incubated with xanthine oxidase at a final concentration of 0.005 U/m L for 12 h,the uric acid-lowering activity of small molecule in Hep G2 cells was measured using the uric acid micro-assay kit.The IC50 values of the medicine and food homology small molecules on xanthine oxidase were calculated,and the toxic effects of different concentrations of the medicine and food homology small molecules on Hep G2 cells were detected using the CCK-8 method.ResultsUsing XO as the target,the CDOCKER module of Discovery Studio v4.0 software was used to screen 36 medicine and food homology small molecule from an independent constructed library of medicine and food homologous small molecule ligands.Tested in vitro,kaempferide,kaempferol,isorhamnetin,quercetin,caffeic acid,protocatechuic acid,luteolin and diosmetin all inhibited uric acid production in Hep G2 cells in a dose-dependent manner(P < 0.05),their IC50 values for xanthine oxidase were 45.92,28.27,69.33,86.96,60.44,29.11,12.68,4.97 μM.The uric acid-lowering effect of 100 μM protocatechuic acid was not statistically different from that of allopurinol(P = 0.1382);100 μM luteolin(P = 0.0196)and diosmetin(P < 0.001)had a better uric acidlowering effect than allopurinol.The results of CCK-8 assay showed that the above eight small molecules of Chinese medicine had no significant impact on the proliferation ability of Hep G2 cells at different concentrations(P > 0.05).The results of intermolecular interaction analysis showed that all the above eight small molecules of Chinese medicine had hydrogen bonding or hydrophobic interaction with xanthine oxidase Glu 801.Conclusions Kaempferide,kaempferol,isorhamnetin,quercetin,caffeic acid,protocatechuic acid,luteolin and diosmetin small molecule components derived from medicine and food homology,can effectively reduce the high production of uric acid in the Hep G2 cell model of hyperuricemia.Protocatechuic acid,luteolin and diosmetin were comparable to allopurinol in terms of their uric acid-lowering activity,providing a reference direction and structural basis for the design and development of future uric acid-lowering drugs.Caffeic acid and protocatechuic acid exert their uric acid-lowering effects by binding to amino acids around the molybdopterin site of xanthine oxidase;kaempferide,kaempferol,isorhamnetin,quercetin,luteolin and diosmetin exert their uric acidlowering effects by binding to amino acids associated with hydrophobic channels around molybdopterin.The formation of hydrogen bonds or hydrophobic interactions with Glu 801 in xanthine oxidase is the key to the uric acid-lowering activity of the small molecules,but the exact mechanism of its action needs to be further verified.