High-throughput screening (HTS) to identify small-molecule enzyme inhibitors is standard practice, but the substrate-based assays that are typically used are not available for poorly characterised enzymes. Writing in the journal Nature Biotechnology, scientists at the Scripps Institute have now described a substrate-free method for identifying inhibitors of such enzymes.
The new technique combines Activity-Based Protein Profiling (ABPP) with Fluorescence Polarisation (FP) to provide a system compatible with HTS methodology. ABPP utilises reactive chemical probes to covalently modify the active sites of enzymes, exploiting catalytic or recognition features of the site. By using ABPP in competitive mode, small molecules can be screened to identify those able to block the covalent modification of the enzyme by the probe. Since the probes are generally applicable to mechanistically related target proteins, parallel screening can be carried out to simultaneously identify selective inhibitors. The downside of ABPP is the low throughput nature, since readouts are typically based on one-dimensional SDS-PAGE gels. However, by combining ABPP with fluorescence polarisation technology (fluopol-ABPP), the Scripps team has greatly increased throughput.
The team reports application of fluopol-ABPP to identify the alkaloid emetine as a selective inhibitor of the hydrolytic enzyme retinoblastoma-binding protein-9 (RBBP9) and electrophilic inhibitors of glutathione S-transferase omega 1 (GSTO1).