Biotinylated [Leu13]-porcine motilin for use as a receptor probe

202 BIOTINYLATED [LEUI3]-PORCINE MOTILIN FOR USE AS A RECEPTOR PROBE. M. J. Macielag, I. Depoortere*, T. L. Peeters*. BOC Group Technical Center, Health Care Research, New Providence, NJ, USA and (*) Gut Hormone Lab, Gasthuisberg O&N, Leuven, Belgium. Biotinylated peptides are interesting tools for the study of the corresponding receptors, but in small peptides the attachment of the biotin group may easily affect binding. Previous structure-activity studies from our laboratories have demonstrated that the C-terminal octapeptide of porcine motilin plays only a minor role in receptor binding. In contrast, the N-terminal phenylalanine residue is very important. Therefore, a [Leul3]-porcine motilin derivative in which lysine20 was biotinylated was prepared using a novel solid phase synthetic procedure. The derivatized peptide was purified by HPLC and characterized by mass spectral and amino acid analysis. This analogue ([Leu 13, Lys(Bio)20]-motilin) was nearly equipotent to motilin (IC50 = 1.3 nM versus 0.42 nM) in displacing 125I-labeled [Nlel3]-porcine motilin from rabbit antral smooth muscle homogenate. Contractility studies with rabbit duodenal smooth muscle strips afforded a dose-response curve for the biotinylated probe that was nearly identical to that of native porcine motilin (EDs0 = 4.5 nM versus 2.8 nM). In contrast, a commercially available derivative in which the N-terminal amino group was selectively biotinylated (Peninsula Laboratories, Inc.) was markedly less potent (IC50 = 30 nM; EDso = 190 nM). The motilin receptor could be solubilized with 1% cholic acid from antral membranes labeled with the 125I-Leu13, Lys(Bio)20-motilin probe. This solubilized preparation bound to immobilized avidin. The novel biotinylated derivative may prove useful for the localization and isolation of the motilin receptor.

STRUCTURE ANTIGASTRIN (R)-4-BENZAMIDO-5-OXOPENTANE

ACTIVITY BASES

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Makovec F, Peris W, Revel L, Giovanetti R, Mennuni L and Rovati LC. Rotta Research Laboratorium, 20052 Monza, Milan, Italy New (R)-4-benzamido-5-oxopentane bases were synthesized and evaluated in vitro for their capacity to inhibit the binding of [125I](BH)-CCK-8 to either rat peripheral (CCK-A) or central (CCK-B) CCK receptors. In vivo these compounds were studied for the ability to inhibit the acid secretion induced by pentagastrin infusion in the perfused rat stomach. Structure-activity relationships are discussed. The antigastrin activity of the most potent compound of the series, i.e.(R)-l-[4-methyl-l-piperazinyl]-l-oxo-4-[3,5-dichlorophenyl]-5-(8-azaspiro-[4.5]decan--8-yl)5-oxo-pentane (CR 2345) was further evaluated in vivo in both cat and dog (in the cat with gastric fistula and in the dog with Heidenhain pouch ) and compared with that exhibited by the acidic parent compound CR 2194. The characteristic of both CR 2194 and CR 2345, that is, the selectivity for the gastrin receptor, the simple nonpeptidic molecular structure and the activity after oral administration, indicate that both these compounds are a useful tool in the study of the biological effects of gastrin and potential agents for diagnostic or therapeutic use.