Inactivation Of Jack Bean Urease By Scutellarin:Elucidation Of Inhibitory Efficacy And Mechanism

ObjectiveScutellarin (SL), a naturally occurring plant flavonoid, is the major active component of Erigeron breviscapus and its extract breviscapine. SL is customarily applied in the treatment of kidney disease, liver damage, glaucoma and cardiovascular diseases. SL proved to possess pharmacological activities including cardiovascular protective effects, anti-inflammation, anti-neoplasm and optic nerve protective effects. Though it has been demonstrated that several flavonoids possessed inhibitory activity against urease and their activity correlated to the number and position of phenolic hydroxyl groups, there are still no studies about the inhibitory effect against urease and mechanisms of SL.The purpose of this research is to investigate the inhibitory effect of SL on jack bean urease. Firstly, molecular docking was performed to investigate the possible inhibitiory effect of SL against jack bean urease. Second, attempts are made to measure the IC50and illuminate the kinetics and mechanism of inhibition by SL. The present schedule should provide novel insight into further development and application of SL, and also contribute to exploration of urease inhibitors. Methods1Molecular docking between SL and jack bean urease was carried out by the Auto-Dock version4.0program, with the purpose of exploring the possibility of its inhibition against urease.2The modified Berthelot (phenol-hypochlorite) method, a quantitative analysis method for ammoniacal nitrogen, was used to determine the inhibitory effect of SL on jack bean urease.3The inhibitory efficacy of SL on jack bean urease was measured by preincubation study and the half inhibitory concentration (IC50) experiment.4The kinetic parameters-the Michaelis constant KM and the maximum velocity Vmax were determined from the Lineweavere-Burk plots and, the reaction progress curves were applied to explore the inhibitory type of SL on urease, as well as the inhibitory constants. 5Protection experiment of SL-inhibitory urease, SL-thiol-urease interaction and reactivation of SL-inhibitory urease were carried out to illustrate the possible mechanism of inhibition by SL.Results1Molecular dockingSL formed eight hydrogen bonds with amino acid residues in the flap area of jack bean urease, especially sulfhydryl group of CME-592. This result indicated that SL possibly inhibited the enzymatic activity by influencing the active comformational form of the Flap area.2Urease inhibition assay and preincubation study(1) The urease activity made a rapidly decrease initially until the balance was reached between urease, SL and the urease-SL complex. With the preincubation time over20min, the inhibitory effect of SL on jack bean urease was relatively less.(2) The IC50of SL was1.35±0.15mM. Compared with the strong inhibitor quercetin (IC50=0.617±0.019mM), the inhibitory potency of SL was inferior. However, SL was still a excellent jack bean urease inhibitor.3Progress curves analysisIn the absence of SL, Km and Vmax were found to be7.10±0.10mM and1.15±0.04mM/min respectively. From the reaction progress curves, we can realized that the allegro formation of the transient SL-urease complex with an inhibition constant of Ki=5.37×10-2mM and then isomerizate slowly into the stable complex with the overall inhibition constant of Ki*=3.49×10-3mM. In general, the progress curves analysis and preincubation studies proved that the inhibitory action of SL against urease was in accord with the slow-binding manner.4Inhibitory site of urease by SL(1) SL highly possible reduce the enzymatic activity through tightly combining to the thiol residues of urease.(2) The SL-thiol-urease interaction proved that SL reacted more easily with-SH group in thoils compound than those from urease.(3) The inhibition of urease by SL was demonstrated to be invertible as urease could be recovered by dithiothreitol. ConclusionIn this work, molecular docking program was used to explore the possible inhibitory activity and binding sites of SL against jack bean urease. On the basis of the results of molecular docking, a systematic study was carried out to illustrate the inhibitory kinetics and possible mechanism of SL against urease activity. The experimental results indicated that SL reduced jack bean urease activity in a slow-binding manner, which possibly binded to the Flap area of jack bean urease to restrict its active conformational form. Therefore, SL could be regarded as a potential urease inhibitor in various fields.