In-Capillary Screening of Matrix Metalloproteinase Inhibitors by Electrophoretically Mediated Microanalysis with Fluorescence Detection

Abstract

A capillary electrophoresis-based method with enzymatic reaction inside the capillary for the screening of matrix metalloproteinase (MMP) inhibitors has been developed. MMP-2 and MMP-9, which have been considered as promising targets for cancer therapy, were selected as the model enzymes. The hydrolysis of a fluorogenic substrate catalyzed by MMPs was determined by measuring the increase in fluorescence. For high-throughput screening, the short-end injection was employed. The enzyme, substrate containing inhibitors, and enzyme solutions were injected from the outlet of the capillary via the sandwich mode. They were mixed by alternating the potential at positive and negative polarities. Online hydrolysis, separation, and detection were achieved in 70 s with approximately 0.87 fmol of MMP required for each assay. The applicability of electrophoretically mediated microanalysis (EMMA) with fluorescence detection to estimate the inhibitory mechanism and to determine the IC(50) values was evaluated for two natural inhibitors, epigallocatechin gallate and oleic acid. A few other natural compounds such as resveratrol, quercetin, caffeic acid, glucosamine, and doxycycline were also screened to test their inhibitory potency. The results obtained were compared with those obtained by offline enzyme assay and confirm the effectiveness of the present method. A rapid, cost-effective, and fully automated method for MMP inhibitor screening is proposed.

Human protein kinase inhibitor screening by capillary electrophoresis using transverse diffusion of laminar flow profiles for reactant mixing

“…However, mixing more than two reactants by EMMA is difficult [8,25,26,28]. In this work, even when combining EMMA to a zero potential amplification step [8,9,26,27] the ADP produced was below the LOQ (about 0.20 M).…”

Advances in Capillary Electrophoresis-Based Enzyme Assays

“…An alternative for sensitive assays is the use fluorescence-labeled substrates in combination with laser-induced fluorescence (LIF) detection [22][23][24][25][26][27]. When using substrates without UV chromophores, derivatization of the products and/or substrates can be performed.…”

“…Enzyme kinetic data and the IC 50 value of the argatroban obtained by the EMMA assay were in good agreement with the respective data obtained by a pre-capillary as well as a spectrophotometric assay. Rapid polarity switching for plug mixing was also used in a fluorometric EMMA assay for matrix metalloproteinase (MMP) inhibitors [24]. The substrate was a heptapeptide containing methoxycoumarin as fluorophore and a dinitrophenyl group as quencher.…”

“…Assay sensitivity was increased by field-amplified sample injection techniques in CE methods for sirtuin enzymes [19,21] and flavin-containing monooxygenases [22]. Furthermore, the reversed electrode polarity stacking mode was employed in a MEKC method for the monitoring of purine nucleoside phosphorylase and adenosine deaminase activity [23].An alternative for sensitive assays is the use fluorescence-labeled substrates in combination with laser-induced fluorescence (LIF) detection [22][23][24][25][26][27]. When using substrates without UV chromophores, derivatization of the products and/or substrates can be performed.…”

Screening of mammalian target of rapamycin inhibitors in natural product extracts by capillary electrophoresis in combination with high performance liquid chromatography–tandem mass spectrometry

“…The separation and detection of specific substrate peptide and product peptide could be achieved by CE-based method [28,29]. Moreover, CEbased screening method has several other advantages, such as minute requirement of reagents and test compounds, automation, and short analysis time [30][31][32][33]. To date, several CE methods have been reported for studying kinase inhibition or screening of kinase inhibitors [14,21,[34][35][36].…”