On-chip assay for determining the inhibitory effects and modes of action of drugs against xanthine oxidase

Xanthine oxidase (XO) is a key enzyme that can catalyze the conversion of xanthine to uric acid, causing various diseases in humans. We have developed a high-throughput chip-based assay that uses a photodiode array (PDA) microchip system to explore the inhibitory effects of drug analogs on XO. Inhibitory activities of cyclosporin A, aminoglutethimide, dithranol and naringenin against XO were assessed using this chip-based xanthine assay in the presence or absence of the antioxidant enzyme, superoxide dismutase (SOD). In addition, the mechanism of drug action was also disclosed by monitoring the combined effect of respective drug analogs and SOD on XO in the assay. The assessment was based on the red light absorption property of nitroblue tetrazolium (NBT) formazan, formed by free radical-mediated NBT reduction. Compared to naringenin (50 and 100 μM; a known XO inhibitor), cyclosporin A (5 and 10 μM) exhibited similar XO inhibitory activity, whereas dithranol (1 and 3 μM) and aminoglutethimide (2.5 and 5 mM) showed minimum XO inhibition. Low standard deviation obtained during the assay demonstrates the preciseness and accuracy of the developed approach. Compared to the existing methods, the developed approach is advantageous due to its simplicity and compatibility with high-throughput screening procedures. Furthermore, this approach can be applied to the early phase of drug discovery screening to explore various drug analogs for their XO inhibitory activities.