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).
The robot, named ADAM, was developed by scientists at Aberystwyth and Cambridge Universities. Rather than a humanoid robot like Honda’s ASIMO, scientist ADAM is actually a fully automated robotic laboratory that can formulate hypotheses and then design experiments to test them. ADAM was set to work to identify genes responsible for producing orphan enzymes in yeast. Using existing knowledge and algorithms programmed by human scientists, ADAM came up with hypotheses about the genes encoding the enzymes and, without any further intervention, selected yeast mutants to carry out cell growth experiments to test his theories. ADAM was able to confirm 12 out of 20 hypotheses and trace the previously unknown genes responsible for producing the yeast enzymes. One of the orphan enzymes is involved in the synthesis of the amino acid lysine and, although it has been suggested as a target for fungicides, no one had previously identified the gene. Adam was able to suggest three possible candidate genes and all were found to produce an enzyme capable of catalysing the correct reaction.