Creation and functional screening of a multi-usepeptide library.

Abstract

One of the most striking aspects of the GCR family is the sheer number of structures which serve as ligands. At a minimum the range includes photons (with a retinal cofactor), odorants, carbohydrates, lipids, and peptides. The peptides are of particular interest to neurobiology and endocrinology because they encompass many of the neurotransmitters, neuromodulators, and hormones. As a result, we have been interested in applying the recently described synthetic peptide libraries (2-5), which offer diverse peptides to investigations of GCR function.

Down-regulation of Melanocortin Receptor Signaling Mediated by the Amino Terminus of Agouti Protein in XenopusMelanophores

“…Our results are based on the endogenous melanocortin receptor of Xenopus melanophores, which exhibits agonist and antagonist profiles in between those of the mammalian Mc1r and Mc4r (48,49), but whose sequence is not known. Compared with most assays for G s -coupled receptors in mammalian cells, a principle advantage of the melanophore assay is the ability to examine multiple time points to assure that our analyses are performed at equilibrium.…”

Putting Thought to Paper: A μARCS Protease Screen

“…Later, application of the matrix diffusion technique expanded into highthroughput screening (HTS) with gel permeation methods for screening combinatorial peptide libraries. 4,5 Currently, combinatorial compounds synthesized on polymeric beads are resuspended in an agarose gel containing the target of interest. Cleavage of the chemical compounds from the beads, and subsequent diffusion of the compounds into the gel, permit interactions with the target.…”

Combinatorial diffusion assay used to identify topically active melanocyte-stimulating hormone receptor antagonists.

“…Another method has relied on random screening of thousands of compounds. In this report we describe the use of a combinatorial diffusion assay, an assay that separates molecules in time and space (11), to randomly search for tripeptide and tripeptide-like molecules that antagonize the ca-MSH receptor. The small and uncomplicated structure of these peptide antagonists provides useful structural information for determining receptor interactions.…”

“…It is well known that injection of ca-MSH into a variety of animals including humans causes an increase in skin tone beyond basal levels (1,2), and there is evidence in some species that plasma a-MSH may correlate with changes in background and animal color (12)(13)(14), but these do not provide conclusive information with regard to the extent of MSH involvement in the setting or maintenance of basal tone. Support for the possible involvement of a-MSH in basal skin tone comes primarily from experiments in which removal of the pituitary gland induces pallor in fish, amphibians, and lower mammals (15)(16)(17)(18) and from anecdotal clinical reports, given without explanation or speculation, that humans become abnormally pale following the loss of pituitary function due to disease or following hypothesectomy for treatment of metastatic breast cancer (19 (11) by using fluorenylmethoxycarbonyl (Fmoc) chemistry on 4-methylbenzhydrylamine (MBHA)-linked polystyrene resin in combination with a standard simultaneous multiple peptide synthesis protocol (21,22). Briefly, 0.5 mmol of derivatized amino acid was dissolved in 1.25 ml of N-methylpyrrolidone (NMP)/1.0 ml of N,N-dimethylformamide containing 0.45 M O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and 0.45 M 1-hydroxybenzotriazole (HOBT).…”

“…The kinetics of bond cleavage from MBHA-linked resin has been described elsewhere (see ref. 11). The 10 pmol (or 4%) release level per bead was used so that responses from molecules with potencies as low as 100 ,uM could be observed.…”