In vitro selection of a peptide antagonist of growth hormone secretagogue receptor using cDNA display

Abstract

G protein-coupled receptors (GPCRs) are major drug targets, and their ligands are currently being explored and developed by many pharmaceutical companies and independent researchers. Class A (rhodopsin-like) GPCRs compose a predominant GPCR family; therefore, class A GPCR ligands are in demand. Growth hormone secretagogue receptor (GHS-R) is a class A GPCR that stimulates food intake by binding to its peptide ligand, ghrelin. Therefore, antagonists of GHS-R are expected to exert antiobesity function. In this article, we describe the use of cDNA display to screen for successfully and identify an antagonistic peptide of GHS-R. The antagonistic peptide inhibited the ghrelin-induced increase in intracellular Ca 2þ in vitro (IC 50 ¼ approximately 10 μM) and repressed the contraction of isolated animal stomach in response to ghrelin. Furthermore, peripheral administration of the peptide inhibited the food intake of mice. This work provides new insight into the development of antiobesity drugs and describes a method for the discovery of unique peptide ligands for class A GPCRs.aptamer | in vitro display | peptide drug | ligand screening | cell-based selection

Peptide mimetic of N‐terminal ghrelin enhances ghrelin‐induced growth hormone secretion and c‐Fos expression in mice

“…The peptide inhibited intracellular calcium release even better then peptidomimetic antagonist [ d ‐Lys3]‐GHRP‐6 (IC 50 = 25 nmol L ‐1 ). Therefore, similar in vivo effects regarding food intake were expected . Moreover, this peptide shares some sequence similarity with the first eight amino acids of peptides identified by screening the cDNA combinatorial library of 28 residue‐long ghrelin peptide analogues .…”

Increasing the library size in cDNA display by optimizing purification procedures

“…This technology allows researchers to screen large combinatorial libraries against molecules on a cell surface (e.g. receptors) [9], and to use peptide libraries containing two or more disulfide bonds [10,11]. Although the cDNA display method was useful for in vitro peptide and protein selection, its productivity was hindered by the generation of mRNA/cDNA-protein fusion molecules; only around 0.1% of the initial mRNA was ligated to proteins with a puromycin-linker [8].…”

Creation of Functional Peptides by Evolutionary Engineering with Bioorthogonal Incorporation of Artificial Components

“…Threfore, direct in vitro selection of peptides with inhibitory activity has been reported (30)(31)(32). Especially, Li and Roberts introduced penicillin molecule into a mRNA-display peptide library by post-translational modification and isolated peptide-drug conjugates with at least 100-fold higher activity against Staphylococcus aureus penicillin-binding protein 2a (32).…”