Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries

Background: The rapidly expanding list of pharmacologically important targe ts has highlighted the need for ways to discover new inhibitors that are in dependent of functional assays. We have utilized peptides to detect inhibit ors of protein function. We hypothesized that most peptide ligands identifi ed by phage display would bind to regions of biological interaction in targ et proteins and that these peptides could be used as sensitive probes for d etecting low molecular weight inhibitors that bind to these sites. Results: We selected a broad range of enzymes as targets for phage display and isolated a series of peptides that bound specifically to each target. P eptide ligands for each target contained similar amino acid sequences and c ompetition analysis indicated that they bound one or two sites per target. Of 17 peptides tested, 13 were found to be specific inhibitors of enzyme fu nction. Finally, we used two peptides specific for Haemophilus influenzae t yrosyl-tRNA synthetase to show that a simple binding assay can be used to d etect small-molecule inhibitors with potencies in the micromolar to nanomol ar range. Conclusions: Peptidic surrogate ligands identified using phage display are preferentially targeted to a limited number of sites that inhibit enzyme fu nction. These peptides can be utilized in a binding assay as a rapid and se nsitive method to detect small-molecule inhibitors of target protein functi on. The binding assay can be used with a variety of detection systems and i s readily adaptable to automation, making this platform ideal for high-thro ughput screening of compound libraries for drug discovery.