Surrogate Ligand Competition High-Throughput Screen for Inhibitors of 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase by

Dxr and devised a high-throughputscreen(HTS)forcompoundsthatbindtothisenzymeatafunctionalsite.Evidenceispresentedthatthesurrogateligand directly binds or allosterically affects both the D-1-deoxyxylulose 5-phosphate (DXP) and NADPH binding sites.Compounds that bind at either or both sites that compete for binding with the surrogate ligand register as hits. The time-resolved fluorescence-based assay represents an improvement over the Dxr enzyme assay that relies on relatively insensitivemeasurements of NADPH oxidation. Screening 32,000 compounds from a diverse historical library, the authors obtained 89potent inhibitors in the surrogate ligand competition assay. The results presented here suggest that peptide surrogate ligandsmay be useful in formatting HTS for proteins with difficult biochemical assays or targets of unknown function. (