Biological Evaluation And Molecular Docking Of Baicalin And Scutellarin As Helicobacter Pylori Urease Inhibitors

ObjectiveHelicobacter pylori infection is intimately associated with the human disease including peptic ulcer, gastritis, and even the gastric cancer. Currently, antibiotics-based drug treatment, the main part of therapy against H. pylori infection, followed by problems such as drug resistance and adverse effects, has become a rising concern. Therefore it is necessary to develop alternative medicine with relatively higher safety and satisfactory efficacy to treat H.pylori-induced gastric diseases. Since urease is the major colonization and virulence factor for H. pylori, strategies based on urease inhibition are now being one of the essential lines of treatment for H. pylori infection.In our previous investigation on Scutellaria baicalensis Georgi, its principal bioactive components, Baicalin (BA) and scutellarin (SL), predominantly existing in the root and the aerial part respectively, was demonstrated to effectively inhibit jack bean urease. Moreover, the inhibitory potential of SL toward jack bean urease was stronger than of BA. Considering H. pylori urease is differ from jack bean with regard to the enzyme structure as well as the the clinical implication on the major colonization and virulence factor for H. pylori, our research was to employ urease, shifting from jack bean urease to H. pylori urease, for exploration on the kinetics and mechanism underlying inhibitory action of BA and SL on H. pylori urease. The present work should not only give additional scientific support to the use of S. baicalensis in traditional Chinese medicine (TCM) to treat gastrointestinal disorders, but offer theoretical and experimental foundation for further development of urease inhibitors as well.Methods1. Determination of urease activityThe ammonia formations, indicator of urease activity, were examined using modified spectrophotometric Berthelot (phenol-hypochlorite) method.2. Urease inhibition by baicalin and scutellarinThe inhibitory effect of baicalin and scutellarin was characterized with IC50 values, compared to acetohydroxamic acid (AHA), a well known H. pylori urease inhibitor.3. Inhibition kinetic studyLineweaver-Burk and Dixon plots for the H. pylori urease inhibition of baicalin and scutellarin was constructed from the kinetic data. Michaelis constant (Km) and the maximum velocity (vmax) values were determined by means of Lineweaver-Burk plots. Inhibitory constant (Ki) value was calculated from the Dixon plot.4. Target of inhibtion analysisSH-blocking reagents and competitive active site Ni2+binding inhibitors were employed for analysising target of inhibition and mechanism study. This section included urease protection against BA or SL inactivation assay and reactivation of BA or SL-inactivated urease assay.5. Molecular docking studyMolecular docking technique by the Auto-Dock version 4.0 was used to provide some information on binding conformations as well as confirm the inhibition mode.Results1. H. pylori urease inhibition assayBaicalin and scutellarin effectively suppressed H. pylori urease in dose-dependent manner with IC50 of 0.82 ± 0.07 mM and 0.47 ± 0.04 mM, respectively. The IC50 of reference drug, AHA, was 0.14 ± 0.05 mM. Structure-activity relationship disclosed 4’-hydroxyl gave flavones an advantage to binding with H. pylori urease.2. Inhibition kinetic studyKinetic analysis revealed that the types of inhibition were non-competitive and reversible with inhibition constant Ki of 0.14 ± 0.01 mM and 0.18 ± 0.02 mM for baicalin and scutellarin, respectively.3. Target of inhibtion analysis(1) The mechanism of urease inhibition was considered to be blockage of the SH groups of H. pylori urease, since thiol reagents (L,D-dithiothreitol, L-Cysteine and glutathione) abolished the inhibitory action and competitive active site Ni2+ binding inhibitors (boric acid and sodium fluoride) carried invalid effect.(2) The inhibition of BA/SL was comfirmed to be reversible in consequence of the fact that there is reactivation when the inhibited-urease met dithiothreitol.4. Molecular docking studyMolecular docking study further supported the structure-activity analysis and indicated that baicalin and scutellarin interacted with the key residues Cys321 located on the mobile flap through S-H·π interaction, but did not interact with active site Ni2+. ConclusionBaicalin and scutellarin were non-competitive inhibitors targeting sulfhydryl groups especially Cys321 around the active site of H. pylori urease, representing potential to be good candidate for future research as urease inhibitor for treatment of H. pylori infection. Furthermore, our work gave additional scientific support to the use of S. baicalensis in traditional Chinese medicine (TCM) to treat gastrointestinal disorders.