Tyrosinase (TYR, E.C. 1.14.18.1), a critical enzyme participating in melanogenesis, catalyzes the first two steps in melanin biosynthesis including the ortho-hydroxylation of L-tyrosine and the oxidation of L-DOPA. Previous pharmacological investigations have revealed that an abnormal level of TYR is tightly associated with various dermatoses, including albinism, age spots, and malignant melanoma. TYR inhibitors can partially block the formation of pigment, which are always used for improving skin tone and treating dermatoses. The practical and reliable assays for monitoring TYR activity levels are very useful for both disease diagnosis and drug discovery. This review comprehensively summarizes structural and enzymatic characteristics, catalytic mechanism and substrate preference of TYR, as well as the recent advances in biochemical assays for sensing TYR activity and their biomedical applications. The design strategies of various TYR substrates, alongside with several lists of all reported biochemical assays for sensing TYR including analytical conditions and kinetic parameters, are presented for the first time. Additionally, the biomedical applications and future perspectives of these optical assays are also highlighted. The information and knowledge presented in this review offer a group of practical and reliable assays and imaging tools for sensing TYR activities in complex biological systems, which strongly facilitates high-throughput screening TYR inhibitors and further investigations on the relevance of TYR to human diseases.

Human carboxylesterase 1 (hCE1), plays pivotal roles in endobiotics homeostasis and xenobiotic metabolism. The plasma level of hCE1 could serve as a useful serologic biomarker for several hepatic diseases, such as hepatocellular carcinoma. However, no probe reaction has been fruitfully used to determine the activity of trace hCE1 in human plasma. This study aims to design and develop a highly selective marker reaction for measuring the enzymatic activities of hCE1 in complex biological samples including human plasma. N-(4-Methyl butyrate)-4-hydroxy-1,8-naphthalimide (NMHN), which contains a small alcohol group and a large acyl moiety, was intentionally designed based on the substrate preference of hCE1. NMHN could be easily hydrolyzed by hCE1 with very high catalytic efficacy under physiological conditions, while both reaction-phenotyping assays and chemical inhibition assays demonstrated that this reaction exhibited super selectivity towards hCE1 over other human hydrolases. Furthermore, the marker reaction possessed ideal kinetic behaviour (classic Michaelis–Menten) with high intrinsic clearance (5.8 mL min⁻¹ mg⁻¹). Based on this probe, a rapid hCE1 quantification method was developed and fully validated, which was successfully applied to determine the real activities of hCE1 in various biological samples including human plasma. Our findings afforded a promising tool for measuring the real activity of hCE1 and laid a solid foundation for further investigations on the biological functions of hCE1 in complex biological samples.

Human catechol-O-methyltransferase (hCOMT) is considered a therapeutic target due to its crucial roles in the metabolic inactivation of endogenous neurotransmitters and xenobiotic drugs. There are nevertheless few safe and effective COMT inhibitors and there lacks a diversity in structure. To discover novel safe and effective hCOMT inhibitors from herbal products, in this study, 53 herbal products were collected and their inhibitory effects against hCOMT were investigated. Among them, Scutellariae radix (SR) displayed the most potent inhibitory effect on hCOMT with an IC₅₀ value of 0.75 μg mL⁻¹. To further determine specific chemicals as COMT inhibitors, an affinity ultrafiltration coupled with liquid chromatography-mass spectrometry method was developed and successfully applied to identify COMT inhibitors from SR extract. The results demonstrated that scutellarein 2, baicalein 9 and oroxylin A 12 were potent COMT inhibitors, showing a high binding index (>3) and very low IC₅₀ values (32.9 ± 3.43 nM, 37.3 ± 4.32 nM and 18.3 ± 2.96 nM). The results of inhibition kinetics assays and docking simulations showed that compounds 2, 9 and 12 were potent competitive inhibitors against COMT-mediated 3-BTD methylation, and they could stably bind to the active site of COMT. These findings suggested that affinity ultrafiltration allows a rapid identification of natural COMT inhibitors from a complex plant extract matrix. Furthermore, scutellarein 2, baicalein 9 and oroxylin A 12 are potent inhibitors of hCOMT in SR, which could be used as promising lead compounds to develop more efficacious non-nitrocatechol COMT inhibitors for biomedical applications.