A HIGH-DENSITY SCREENING FORMAT FOR ENCODED COMBINATORIAL LIBRARIES -ASSAY MINIATURIZATION AND ITS APPLICATION TO ENZYMATIC-REACTIONS

A novel, miniaturized high-throughput screening format is described fo r assay of combinatorial libraries generated on beads. This approach, which is ideally suited to encoded libraries synthesized on beads, uti lizes the photolytic cleavage of individual compounds into a high-dens ity well array (>6500 wells within a standard 96-well microtiter plate footprint) with well volumes as low as 0.37 mu l. As a model study, a n encoded dipeptide library (324 members) acylated with isobutyl succi nate was assayed using this format to search for potential inhibitors of matrilysin, a member of the matrix metalloproteinase superfamily. I n situ release of compounds from solid support was accomplished by pho tochemical cleavage after beads and enzyme were distributed to the wel ls. After the addition of a fluorogenic substrate to the array, the ex tent of enzyme inhibition and identification of active compounds was q uantitated by imaging of the fluorescence emission upon uv irradiation . The structure-activity relationship data generated from the identifi ed inhibitors in this study corroborate previous findings, thus valida ting the utility of this approach as a means of high-throughput screen ing of bead-based libraries. (C) 1997 Academic Press.