Development of off‐line and on‐line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

Abstract

Fast and convenient CE assays were developed for the screening of adenosine kinase (AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260 nm. An MEKC method using borate buffer (pH 9.5) containing 100 mM SDS (method A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH 7.5 or 8.5) was used and a constant current (95 microA) was applied (method B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10 min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method C). After hydrodynamic injection of a plug of reaction buffer (20 mM Tris-HCl, 0.2 mM MgCl2, pH 7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1 mM ATP, 100 microM adenosine, and 20 microM UMP as an internal standard (I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5 kV separation voltage (negative polarity) for 0.20 min to let the plugs interpenetrate. The voltage was turned off for 5 min (zero-potential amplification) and again turned on at a constant current of -60 microA to elute the products within 7 min. The method employing a polyacrylamide-coated capillary of 20 cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose-response curves and calculated K(i) values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay.

Enzymatic in‐capillary derivatization for glucose determination by electrophoresis with spectrophotometric detection

“…EMMA has been successfully applied mainly in the field of enzyme activity study, including such enzymes, as lactate dehydrogenase [5], catechol-O-methyltransferase [13], phenol sulfotransferase [14], glycosidases [15], haloalkane deha-logenase [16], amine oxidase [17], g-glutamyltransferase [18], and angiotensin [19], and a number of inhibition studies [20][21][22][23]. The figures basing on EMMA results were in good agreement with the data obtained using other methods.…”

Adenosine Kinase: Exploitation for Therapeutic Gain

“…To optimize ADK activity assays, capillary electrophoresis assays were developed, in which the enzymatic reaction was either performed in a test tube and subsequently Boison injected into the capillary or in which the enzymatic reaction was directly performed in the capillary (Iqbal et al, 2006). The latter approach led to further sampling size reductions and increased throughput (Iqbal et al, 2006). To date, several classes of ADK inhibitors have been developed and characterized, which broadly fall into the categories of nucleoside and nonnucleoside ADK inhibitors.…”

A highly sensitive CE‐UV method with dynamic coating of silica‐fused capillaries for monitoring of nucleotide pyrophosphatase/phosphodiesterase reactions

“…CE is a separation technique that has emerged as a fast, lowcost, and powerful separation technique for both charged and neutral analytes [23]. It has a high mass sensitivity in relation to the small (typically nanoliter) injection volume used.…”

“…Recently, we have developed CE-based enzyme assays for the determination of Michaelis-Menten constants (K m values), maximal velocities (V max ) for substrates, and inhibition constants (IC 50 or K i values) for enzyme inhibitors of various nucleotide and nucleoside-metabolizing enzymes, namely ecto-5 0 -nucleotidase [21], nucleoside triphosphate diphosphohydrolase (ecto-NTPDase) [22], adenosine kinase [23], and herpes simplex type 1 (HSV-1) thymidine kinase [24]. However, the previously established methods were not sensitive enough to quantify the reaction products of NPP reactions, because their detection limits for nucleotides such as AMP, the product of NPP-catalyzed hydrolysis of ATP, are only in the range of 1.6-2.3 mM.…”