title: Andrographolide sodium bisulphite-induced inactivation of urease: inhibitory potency, kinetics and mechanism.
abstract: BACKGROUND
  The inhibitory effect of andrographolide sodium bisulphite (ASB) on jack bean urease (JBU) and Helicobacter pylori urease (HPU) was performed to elucidate the inhibitory potency, kinetics and mechanism of inhibition in 20 mM phosphate buffer, pH 7.0, 2 mM EDTA, 25 °C.
  
  
  METHODS
  The ammonia formations, indicator of urease activity, were examined using modified spectrophotometric Berthelot (phenol-hypochlorite) method. The inhibitory effect of ASB was characterized with IC50 values. Lineweaver-Burk and Dixon plots for JBU inhibition of ASB was constructed from the kinetic data. SH-blocking reagents and competitive active site Ni2+ binding inhibitors were employed for mechanism study. Molecular docking technique was used to provide some information on binding conformations as well as confirm the inhibition mode.
  
  
  RESULTS
  The IC50 of ASB against JBU and HPU was 3.28±0.13 mM and 3.17±0.34 mM, respectively. The inhibition proved to be competitive and concentration- dependent in a slow-binding progress. The rapid formation of initial ASB-JBU complex with an inhibition constant of Ki=2.86×10(-3) mM was followed by a slow isomerization into the final complex with an overall inhibition constant of Ki*=1.33×10(-4) mM. The protective experiment proved that the urease active site is involved in the binding of ASB. Thiol reagents (L-cysteine and dithiothreithol) strongly protect the enzyme from the loss of enzymatic activity, while boric acid and fluoride show weaker protection, indicating that the active-site sulfhydryl group of JBU was potentially involved in the blocking process. Moreover, inhibition of ASB proved to be reversible since ASB-inactivated JBU could be reactivated by dithiothreitol application. Molecular docking assay suggested that ASB made contacts with the important sulfhydryl group Cys-592 residue and restricted the mobility of the active-site flap.
  
  
  CONCLUSIONS
  ASB was a competitive inhibitor targeting thiol groups of urease in a slow-binding manner both reversibly and concentration-dependently, serving as a promising urease inhibitor for the treatment of urease-related diseases.
authors.affiliation: Guangdong Provincial Key Laboratory of of New Chinese Medicinals Development and Research, Guangzhou University of Chinese Medicine, Guangzhou, 510006, P. R. China. mozhizhun@hotmail.com.
authors.forename: Zhi-Zhun
authors.initials: ZZ
authors.lastname: Mo
authors.affiliation: Guangdong Provincial Key Laboratory of of New Chinese Medicinals Development and Research, Guangzhou University of Chinese Medicine, Guangzhou, 510006, P. R. China. 851702307@qq.com.
authors.forename: Xiu-Fen
authors.initials: XF
authors.lastname: Wang
authors.affiliation: Guangdong Provincial Key Laboratory of of New Chinese Medicinals Development and Research, Guangzhou University of Chinese Medicine, Guangzhou, 510006, P. R. China. 985958620@qq.com.
authors.forename: Xie
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Guangdong Provincial Key Laboratory of of New Chinese Medicinals Development and Research, Guangzhou University of Chinese Medicine, Guangzhou, 510006, P. R. China. 42107660@qq.com.
authors.forename: Ji-Yan
authors.initials: JY
authors.lastname: Su
authors.affiliation: Guangdong Provincial Key Laboratory of of New Chinese Medicinals Development and Research, Guangzhou University of Chinese Medicine, Guangzhou, 510006, P. R. China. 451001258@qq.com.
authors.forename: Hai-Ming
authors.initials: HM
authors.lastname: Chen
authors.affiliation: Guangdong Provincial Key Laboratory of of New Chinese Medicinals Development and Research, Guangzhou University of Chinese Medicine, Guangzhou, 510006, P. R. China. 519390982@qq.com.
authors.forename: Yu-Hong
authors.initials: YH
authors.lastname: Liu
authors.affiliation: Guangdong Provincial Key Laboratory of of New Chinese Medicinals Development and Research, Guangzhou University of Chinese Medicine, Guangzhou, 510006, P. R. China. 519114819@qq.com.
authors.forename: Zhen-Biao
authors.initials: ZB
authors.lastname: Zhang
authors.affiliation: Guangdong Provincial Key Laboratory of Clinical Research on Traditional Chinese Medicine Syndrome, The Second Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou, 510120, P. R. China. 629016410@qq.com.
authors.forename: Jian-Hui
authors.initials: JH
authors.lastname: Xie
authors.affiliation: Guangdong Provincial Key Laboratory of of New Chinese Medicinals Development and Research, Guangzhou University of Chinese Medicine, Guangzhou, 510006, P. R. China. suziren@126.com.|Dongguan Mathematical Engineering Academy of Chinese Medicine, Guangzhou University of Chinese Medicine, Dongguan, 523000, P. R. China. suziren@126.com.
authors.forename: Zi-Ren
authors.initials: ZR
authors.lastname: Su
mesh_terms: D032449:Canavalia / Q000201:enzymology; D004224:Diterpenes / Q000494:pharmacology*; D007700:Kinetics; D062105:Molecular Docking Simulation; D013447:Sulfites / Q000494:pharmacology*; D014510:Urease / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism
chemical_list: D004224:Diterpenes; D013447:Sulfites; C030419:andrographolide; D014510:Urease; C009279:sodium bisulfite