The importance of the C-terminal Phe of gastrin and structural requirements at position 17 for binding to the human CCK2 receptor were assessed using analogs of [Leu15]G(11-17). The following peptides were synthesized, Ac[Leu15]G(11-17), Ac[Leu15]G(11-16)NH2, [Leu15]G(11-17), [Leu15, Ala17]G(11-17), [Leu15, Abu17]G(11-17), [Leu15Val17] G(11-17), [Leu15, Leu17]G(11-17), [Leu15, Cha17]G(11-17), [Leu15, Trp17]G(11-17), [Leu15, Tic17]G(11-17), [Leu15, D-Phe17] G(11-17) and [Leu15, p-X-Phe17]G(11-17), where X=F, Cl, Bt, I, OH, CH3, NH2 and NO2. Competition binding experiments with [3H]CCK-8 were performed using human CCK2 receptors stably expressed in CHO cells. Phe17 was shown to be important for binding. A hydrophobic side-chain larger than Leu is required at position 17 but aromaticity does not appear to be essential. Constraint of the aromatic side-chain either in the g(+) or g(-) conformation, as in the case of Tic, results in a significant decrease in affinity. In addition, the peptide conformation induced by incorporation of D-Phe decreases binding. The size and electron withdrawing/donating properties of the para substituent are not important for interaction with the receptor. The current study shows that the use of des-Phe analogs of gastrin is not a viable strategy for development of antagonists for the human CCK2 receptor.
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