Use of a cell-based, lawn format assay to rapidly screen a 442,368 bead-based peptide library

A cell-based, lawn format assay utilizing an in situ photocleavage method h as been developed that allows the rapid examination of large bead-based com pound libraries as discrete molecules. The format uses frog melanophore cel ls in a contiguous, adherent, confluent layer in small petri dishes covered with a 0.5-1-mm layer of agarose containing 130 micron diameter TentaGel b eads at a density of 2-20 beads/mm(2). Employing this technique a 9-mer, 44 2,368-member peptide library (designed around the 13 amino acid alpha-MSH p eptide sequence) made up of 12 separate pools of 36,864 peptides/pool was a ssayed. initially, a fraction (similar to 10%) of each pool was scanned (si milar to 3700 beads from each pool) in 60-mm petri dishes to identify the m ost active pools. Upon direct photocleavage of the beads with UV light (365 nm), each petri dish was photographed over a 60-min period with a CCD came ra to record changes in light intensity as an index of melanosome dispersio n. Active beads were those that were surrounded by a localized decrease in light transmittance indicating melanosome dispersed cells. Upon examination with a dissecting microscope, single beads centrally located to a circular array of dispersed cells were identified and removed from the agarose and sequenced by Edman degradation to determine the peptide sequence. Re-synthe sized pep tides were re-examined against ol-MSH receptor to confirm and qua ntify the activity. Several 9-mer peptides were identified with potencies s imilar to the natural 13-mer peptide. This method allows for the rapid scre ening of large bead-based photo-cleavable peptide libraries with the advant age that each compound is screened as a discrete molecule in a well-less fo rmat. (C) 2000 Elsevier Science Inc. All rights reserved.