Applying pulse UV irradiation-induced chemiluminescence approach for high-throughput screening assay of tyrosinase inhibitors

Tyrosinase is an enzyme that metabolizes L-tyrosine and is found in various organisms. Its overactivity can lead to health issues in humans, such as hyperpigmentation, and can adversely affect human skin, leading to skin cancers. This has heightened the significance of tyrosinase inhibitors in pharmaceuticals and cosmetics, particularly skin-whitening formulations. In this study, we developed a high-throughput screening assay for identifying tyrosinase inhibitors. This assay leverages the strong chemiluminescence signal emitted by L-tyrosine upon nanosecond UV irradiation in the presence of L-012 chemiluminescence dye, which is based on the formation of reactive oxygen species (ROS). We measured the decrease in chemiluminescence signal induced by tyrosinase enzyme, which converts chemiluminescent L-tyrosine into non-chemiluminescent L-DOPA. The addition of tyrosinase inhibitors prevents this conversion, leading to recovery in chemiluminescence of L-tyrosine. However, the reliability of the assay can be compromised by the ROS-scavenging activity and phenolic nature of certain enzyme inhibitors. To mitigate potential false results caused by some inhibitors, tyrosinase was immobilized on the microplate surface, and the inhibitors were incubated with the fixed enzyme, then, the enzyme activity was assessed after washing away the inhibitors. The proposed assay successfully facilitated high-throughput screening (less than 1 min per sample) of numerous tyrosinase inhibitor candidates from various pharmacological classes. The percentage inhibition of tyrosinase activity determined by our assay was statistically compared with results from a previously reported assay, revealing comparable outcomes and confirming the reliability of our approach. In addition, we evaluated the environmental impact and applicability of the assay using two recent metrics, yielding promising results.