Fluorescent parallel electrophoresis assay of enzyme inhibition

Enzyme inhibitors comprise the largest class of pharmaceutical compounds. The discovery and development of new enzyme inhibitor drug candidates depends on sensitive tools to quantify inhibition constants, Ki, for the most promising candidates. A high throughput, automated, and miniaturized approach to measure inhibition is reported. In this technique enzyme inhibition occurs within a 16 nL nanogel reaction zone that is integrated into a capillary. The reaction and electrophoresis separation are completed in under 10 min. The nanoliter enzyme reaction zones are easily positioned inside a standard separation capillary by pseudo-immobilizing enzymes within a thermally reversible nanogel. This report optimizes and validates a capillary nanogel electrophoresis reaction and separation with a multi-capillary array instrument. Inhibitor constants are determined for the neuraminidase enzyme to quantify the effect of the transition state analog, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA), as well as the inhibitor Siastatin B. With the multi-capillary array assay replicate Ki values are determined to be 5.6 ± 0.3 μM (n = 3) and 9.2 ± 0.3 μM (n = 3) for DANA and Siastatin B, respectively. The enzyme reaction in each separation capillary converts the substrate to a product in real time. The nanogel is used under suppressed electroosmotic flow, sustains enzyme function, and is easily filled and replaced by changing the capillary temperature. Using laser-induced fluorescence allows the determination to be achieved with substrate concentrations well below the Michaelis-Menten constant, making the method independent of the substrate concentration and therefore a more easily implemented assay. A lower measurement cost is realized when the reaction volume is miniaturized because the amounts of enzyme, substrate and inhibitor are reduced. Fast enzyme reactions are possible because of the small reaction volume. With a multi-capillary array, the inhibition assay is achieved in a fraction of the time required for traditional methods. The separation-based assay can even be applied to labeled substrates not cleaned up following the labeling reaction.