D-amino acid and alanine scans of the bioactive portion of porcine motilin

Abstract A recent systematic study of porcine motilin fragments has clearly shown that biological activity resides in the amino-terminal end. The amino-terminal tetradecapeptide retains more than 90% of the potency of the full molecule. We now examined the effect of replacement of residues 1 through 11 by either their d -isomer or by alanine in [Leu 13 ]pMOT(1–14). Peptides were synthesized using Fmoc solid phase methodology, purified by HPLC, and assayed for their ability to displace bound motilin (rabbit antral smooth muscle homogenate) and to induce contractions (isolated rabbit duodenal segments). The negative logarithm of the concentration displacing 50% of the tracer (pIC 50 ), or producing 50% of the maximal contractile response (pEC 50 ), was determined. All compounds were still full agonists. A reduction in potency of more than two log units was seen for the compounds in which residues 1 (Phe), 4 (Ile), and 7 (Tyr) were replaced by Ala and residues 3 (Pro), 4 (Ile), and 6 (Thr) by their d -isomer. The largest drop was noted for the analogs substituted at position 4. For all compounds there was an almost perfect correlation between the pIC 50 and the pEC 50 values ( r = 0.96), although the pEC 50 was consistently smaller. These results show that the biological activity of motilin is mainly determined by the first seven residues. The pharmacophore consists of the aromatic rings from Phe 1 and Tyr 7 and the aliphatic side chains from Val 2 and Ile 4 . Pro 3 , Phe 5 , and Thr 6 may stabilize the bioactive conformation.