Development of a fast CE method for high throughput screening of ecto‐5′‐nucleotidase inhibitors

Abstract

Overexpression of ecto-5'-nucleotidase is found to be linked to cancer progression and other diseases which makes screening of its inhibitors on demand. Unfortunately, there is scarcity of reported ecto-5'-nucleotidase inhibitors because of unavailability of simple, fast and efficient methods to study its inhibition. In this study, we have developed a very fast, reverse polarity capillary electrophoresis based method for screening of ecto-5'-nucleotidase inhibitors. Both EOF and electrophoretic mobility were maintained in same direction by dynamically coating lining of capillary and applying negative voltage, resulting in appearance of both adenosine monophosphate (AMP) and adenosine peaks in less than 2 min. Separations were performed in reverse polarity using -20 kV. Hexadimethrine bromide (HDB) was used as a buffer additive (0.025% in 40 mM borate buffer). An online sample stacking was attained by a special sweeping effect which enabled a long (65 s) injection time without compromise in peak shape. Using this newly developed method, both AMP and adenosine peaks were obtained with a baseline resolution, in less than two minutes. Method was validated by measuring k , and V values of human ecto-5'-nucleotidas and K value of standard inhibitor quercitin against this enzyme. Method is simple and fast and can be conveniently optimized for high throughput screening of ecto-5'-nucleotidase inhibitors.

Challenging Bioanalyses with Capillary Electrophoresis

“…The evaluation of K m with online reactions is simpler and preferable to off-line reactions. In the period under review, K m values were quantified online with CE-UV absorbance detection for a variety of enzymes including ecto-5-nucleotidase, human recombinant matrix metalloproteinase, human neutrophil elastase, bovine testicular hyaluronidase, histone deacetylase, thrombin, and human nucleoside/nucleotide kinases . In other reports, K m values were quantified online with CE-fluorescence detection for d -amino acid oxidase and BCR-ABL .…”

“…CE has been used to compare inhibitors of native and recombinant enzymes . In the period under review, inhibitors of enzymes in free solution have been characterized with UV detection for ecto-5-nucleotidase, thrombin, histone deacetylase, glucosamine-6 phosphate synthase, and human acetyl-CoA carboxylase 2 and with laser-induced fluorescence for d -amino acid oxidase and BCR-ABL . Further, inhibition studies have been done for immobilized enzymes including acetylcholinesterase, , alanine aminotransferase, β-glucosidase, and sulfotransferase 1A1 using UV detection.…”

Single-step Trypsin Inhibitor Assay on a Microchannel Array Device Immobilizing Enzymes and Fluorescent Substrates by Inkjet Printing

“…However, electrophoretic separation-based CE allows high-efficiency separation; multiple enzyme inhibitor assay methods using CE have thus been reported. 1,3,14,[18][19][20][21] However, CE requires large equipment and involves multi-step reactions.…”

Advances in screening enzyme inhibitors by capillary electrophoresis

“…Capillary electrophoresis (CE) as a simple, low sample consumption, low solvent volume, high resolution, and high speed analytical technology has attracted lots of attention. Owing to these advantages, it was well matched with the purpose of trace or ultratrace sample analysis and has been widely used in enzyme inhibitor screening . There are two main operation modes on enzyme inhibitor screening: off‐line (precapillary enzyme assays) in which process CE was used as an analytical tool; online (incapillary enzyme assays) which combined the sample injection, mix, reaction, separation, and detection within a single run.…”