A fluorescence lifetime-based binding assay for acetylpolyamine amidohydrolases from Pseudomonas aeruginosa using a [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) ligand probe.

摘要: High-throughput assays for drug screening applications have to fulfill particular specifications. Besides the capability identify even compounds with low potency, one of major issues is minimize number false-positive hits in a campaign order reduce logistic effort subsequent cherry picking and confirmation procedure. In this respect, fluorescence lifetime (FLT) appears as an ideal readout parameter that supposed be robust against autofluorescent light-absorbing compounds, most common source systematic false positives. The extraordinary features recently discovered [1,3]dioxolo[4,5-f][1,3] benzodioxole dyes were exploited develop FLT-based binding assay exceptionally readout. setup was comprehensively validated shown comply not only all requirements powerful high-throughput but also suitable determine accurate constants inhibitors enzymes histone deacetylase family. Using described assay, first three members enzyme family from Pseudomonas aeruginosa identified. characterized terms potency selectivity profile. novel ligand probe should applicable other homologues are inhibited by N-hydroxy-N′-phenyloctandiamide.