A High-throughput Fluorescent Turn-on Assay for Hydrolases of Amino Acid Esters

Biphenyl hydrolase-like (BPHL) and retinoblastoma-binding protein 9 (RBBP9) are human serine hydrolases with valacyclovirase activity, and both are involved in amino acid ester prodrug activation as well as tumorigenesis. These functional associations warrant further biochemical investigation of both enzymes; however, convenient activity assays for these two enzymes, especially BPHL, remain limited. Here, we developed a fluorescent turn-on assay based on amino acid ester-derived umbelliferone probes. This assay enables real-time, continuous, and microplate-compatible measurement of enzyme activity, steady-state kinetic characterization, substrate preference profiling, and inhibitor evaluation. The utility of this assay in high-throughput screening has also been demonstrated, yielding an excellent screening window coefficient (or Z' factor) for both BPHL and RBBP9. In a pilot screen against BPHL, thonningianin A was identified as a micromolar inhibitor, providing a potential starting point for developing tool compounds to explore the biological functions of BPHL. Collectively, this fluorescent turn-on assay provides a convenient platform for studying BPHL, RBBP9, and potentially other amino acid ester hydrolases.