title: Human neutrophil elastase inhibition studied by capillary electrophoresis with laser induced fluorescence detection and microscale thermophoresis.
abstract: Capillary electrophoresis-laser induced fluorescence (CZE-LIF) and microscale thermophoresis (MST) were used for the first time to study the inhibition of human neutrophil elastase (HNE). We recently studied HNE kinetics (Km and Vmax) by developing an in-capillary CZE-LIF assay based on transverse diffusion of laminar flow profiles (TDLFP) for reactant mixing. In this work, the former assay was adapted to monitor HNE inhibition. Two natural well known HNE inhibitors from the triterpene family, ursolic acid and oleanolic acid, were tested to validate the developed assay. Since the solubility of pentacyclic triterpenes in aqueous media where the enzymatic reaction will take place is limited, the effect of DMSO and ethanol on HNE was studied using microscale thermophoresis (MST). An agglomeration of the enzyme was revealed when preparing the inhibitor in 5% (v/v) DMSO. This phenomenon did not occur in the presence of ethanol. Therefore, ethanol was used as inhibitor solvent, at a limited percentage of 20% (v/v). In these conditions and after optimization of the TDLFP approach, the repeatability (RSD on migration times and peak-areas inferior to 2.2%) of the CZE-LIF assay and the sensitivity (LOQ of few nM) were found to be satisfactory for conducting inhibition assays. IC50 values for ursolic and oleanolic acid were successfully determined. They were respectively equal to 5.62±0.10μM (r(2)=0.9807; n=3) and to 8.21±0.23μM (r(2)=0.9887; n=3). Excellent agreement was found between the results obtained by CE and those reported in literature which validates the developed method. Particularly, the CE-based assay is able to rank HNE inhibitors relative to each other. Furthermore, MST technique was used for evaluating HNE interaction with the ursolic acid. Up to 16 capillaries were automatically processed to obtain in one titration experiment the dissociation constant for the HNE-ursolic acid complex. Ki was found to be 2.72±0.66μM (n=3) which is in excellent agreement with the value determined by CE enzyme inhibition studies (Ki=2.81μM) confirming the reliability of the developed CE assay and the competitive inhibition mode of ursolic acid.
authors.affiliation: Institut de Chimie Organique et Analytique, Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
authors.forename: Fayad
authors.initials: F
authors.lastname: Syntia
authors.affiliation: Institut de Chimie Organique et Analytique, Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France. Electronic address: reine.nehme@univ-orleans.fr.
authors.forename: Reine
authors.initials: R
authors.lastname: Nehmé
authors.affiliation: Institut de Chimie Organique et Analytique, Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
authors.forename: Bérengère
authors.initials: B
authors.lastname: Claude
authors.affiliation: Institut de Chimie Organique et Analytique, Université d'Orléans, CNRS FR 2708, UMR 7311, Orléans, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Morin
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004058:Diffusion; D019075:Electrophoresis, Capillary; D004789:Enzyme Activation / Q000187:drug effects; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D007834:Lasers; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors*; D009828:Oleanolic Acid / Q000494:pharmacology; D053978:Pentacyclic Triterpenes / Q000737:chemistry; D015203:Reproducibility of Results; D015843:Serpins / Q000378:metabolism*; D014315:Triterpenes / Q000737:chemistry / Q000494:pharmacology; D000097245:Ursolic Acid
chemical_list: D019272:Leukocyte Elastase; D009828:Oleanolic Acid; D053978:Pentacyclic Triterpenes; D015843:Serpins; D014315:Triterpenes; D000097245:Ursolic Acid; C075177:SERPINB1 protein, human
keywords: Enzyme; Inhibition; Laser induced fluorescence; Microscale thermophoresis; Transverse diffusion of laminar flow profiles