title: Use of a cell-based, lawn format assay to rapidly screen a 442,368 bead-based peptide library.
abstract: A cell-based, lawn format assay utilizing an in situ photocleavage method has been developed that allows the rapid examination of large bead-based compound libraries as discrete molecules. The format uses frog melanophore cells 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 beads at a density of 2-20 beads/mm2. Employing this technique a 9-mer, 442,368-member peptide library (designed around the 13 amino acid alpha-MSH peptide sequence) made up of 12 separate pools of 36,864 peptides/pool was assayed. Initially, a fraction (approximately 10%) of each pool was scanned (approximately 3700 beads from each pool) in 60-mm petri dishes to identify the most 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 camera to record changes in light intensity as an index of melanosome dispersion. 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-synthesized peptides were re-examined against alpha-MSH receptor to confirm and quantify the activity. Several 9-mer peptides were identified with potencies similar to the natural 13-mer peptide. This method allows for the rapid screening of large bead-based photo-cleavable peptide libraries with the advantage that each compound is screened as a discrete molecule in a well-less format.
authors.affiliation: Department of Receptor Biochemistry, Glaxo Wellcome Research and Development, Research Triangle Park, NC 27709, USA. cjk10747@glaxowellcome.com
authors.forename: C K
authors.initials: CK
authors.lastname: Jayawickreme
authors.forename: H
authors.initials: H
authors.lastname: Sauls
authors.forename: N
authors.initials: N
authors.lastname: Bolio
authors.forename: J
authors.initials: J
authors.lastname: Ruan
authors.forename: M
authors.initials: M
authors.lastname: Moyer
authors.forename: W
authors.initials: W
authors.lastname: Burkhart
authors.forename: B
authors.initials: B
authors.lastname: Marron
authors.forename: T
authors.initials: T
authors.lastname: Rimele
authors.forename: J
authors.initials: J
authors.lastname: Shaffer
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D008547:Melanophores; D019151:Peptide Library; D011979:Receptors, Pituitary Hormone; D020539:Sequence Analysis, Protein / Q000379:methods*; D014981:Xenopus
chemical_list: D019151:Peptide Library; D011979:Receptors, Pituitary Hormone; C029446:MSH receptor