title: Competitive binding experiments can reduce the false positive results of affinity-based ultrafiltration-HPLC: A case study for identification of potent xanthine oxidase inhibitors from Perilla frutescens extract.
abstract: The purpose of this study was to assess the possibility of using competitive binding experiments with ultrafiltration-HPLC analysis to identify potent xanthine oxidase (XO) inhibitors from the Perilla frutescens extract as an attempt to reduce the number of false positive results. To isolate the enzyme-ligand complex from unbound compounds, the P. frutescens extract was either incubated in the absence of XO, in the presence of XO, or with the active site blocked XO before the ultrafiltration was performed. Allopurinaol was used as the XO active site blocker. The unbound compounds were subjected to HPLC analysis. The degree of total binding (TBD) and degree of specific binding (SBD) of each compound were calculated using the peak areas. TBD represents the binding affinities of compounds from the P. frutescens extract for the XO binding site. SBD represents the XO competitive binding between allopurinol and ligands from the extract samples. Two criteria were applied to select putative targets that could help avoid false positives. These include TBD>30% and SBD>10%. Using that approach, kaempferol-3-O-rutinoside, rosmarinic acid, methyl-rosmarinic acid, apigenin, and 4',5,7-trimethoxyflavone were identified, from total 11 compounds, as potent XO inhibitors. Finally, apigenin, 4',5,7-trimethoxyflavone, and luteolin were XO inhibitors verified through an XO inhibition assay and structural simulation of the complex. These results showed that the newly developed strategy has the advantage that the number of targets identified via ultrafiltration-HPLC can be narrowed from many false positives. However, not all false positives can be eliminated with this approach. Some potent inhibitors might also be excluded with the use of this method. The limitations of this method are also discussed herein.
authors.affiliation: Department of Food Science and Nutrition, Hallym University, Chuncheon 24252, Republic of Korea.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Institute of Natural Medicine, Hallym University, Chuncheon 24252, Republic of Korea.
authors.forename: Shin Hwa
authors.initials: SH
authors.lastname: Kwon
authors.affiliation: Department of Food Science and Nutrition, Hallym University, Chuncheon 24252, Republic of Korea.
authors.forename: Seung Hwan
authors.initials: SH
authors.lastname: Hwang
authors.affiliation: Department of Food Science and Nutrition, Hallym University, Chuncheon 24252, Republic of Korea; Institute of Korean Nutrition, Hallym University, Chuncheon 24252, Republic of Korea.
authors.forename: Young-Hee
authors.initials: YH
authors.lastname: Kang
authors.affiliation: Institute of Natural Medicine, Hallym University, Chuncheon 24252, Republic of Korea; Department of Biochemistry, School of Medicine, Hallym University, Chuncheon 24252, Republic of Korea.
authors.forename: Jae-Yong
authors.initials: JY
authors.lastname: Lee
authors.affiliation: Department of Food Science and Nutrition, Hallym University, Chuncheon 24252, Republic of Korea; Institute of Korean Nutrition, Hallym University, Chuncheon 24252, Republic of Korea; Institute of Natural Medicine, Hallym University, Chuncheon 24252, Republic of Korea. Electronic address: limss@hallym.ac.kr.
authors.forename: Soon Sung
authors.initials: SS
authors.lastname: Lim
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D055808:Drug Discovery / Q000379:methods; D004365:Drugs, Chinese Herbal / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D062105:Molecular Docking Simulation; D031344:Perilla frutescens / Q000737:chemistry*; D014462:Ultrafiltration; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
chemical_list: D004365:Drugs, Chinese Herbal; D004791:Enzyme Inhibitors; D014969:Xanthine Oxidase
keywords: Competitive binding experiment; False positive; Perilla frutescens; Ultrafiltration; Xanthine oxidase