Design and Synthesis of Side-Chain Conformationally Restricted Phenylalanines and Their Use for Structure-Activity Studies on Tachykinin NK-1 Receptor

Article abstract of DOI:10.1021/jm00037a009

Constrained analogues of phenylalanine have been conceptually designed for analyzing the binding pockets of Phe⁷ (S₇) and Phe⁸ (S₈), two aromatic residues important for the pharmacological properties of SP, i.e., L-tetrahydroisoquinoleic acid, L-diphenylalanine, L-9-fluorenylglycine (Flg), 2-indanylglycine, the diastreomers of L-1-indanylglycine (Ing) and L-1-benzindanylglycine (Bfi), and the Z and E isomers of dehydrophenylalanine (Δ^ZPhe, Δ^EPhe).Binding studies were performed with appropriate ligands and tissue preparations allowing the discrimination of the three tachykinin binding sites, NK-1, NK-2, and NK-3.The potencies of these agonists were evaluated in the guinea pig ileum bioassay.According to the binding data, we can conclude that the S₇ subsite is small, only the gauche(-) probe <(2S,3S)-Ing⁷>SP presents a high affinity for specific NK-1 binding sites.Surprisingly, the <Δ^EPhe⁷>SP analogue, which projects the aromatic ring toward the trans orientation, is over 40-fold more potent than the Z isomer, <Δ^ZPhe⁷>SP.A plausible explanation of these conflictual results is that either the binding protein quenches the minor trans rotamer of <(2S,3S)-Ing⁷>SP in solution or this constrained amino acid side chain rotates when inserted in the protein.In position 8, the high binding affinities of 8>SP and <(2S,3S)-Bfi⁸>SP suggest that the S₈ subsite is large enough to accept two aromatic rings in the gauche(-) and one aromatic ring in the trans direction.Peptides bearing two conformational probes in positions 7, 8 or 9 led to postulate that S₇, S₈, and S₉ subsites are independent from each other.The volumes available for side chains 7 and 8 can be estimated to be close to 110 and 240 Angstroem³, respectively.The large volume of the S₈ subsite raises question on the localization of the SP-binding site in the NK-1 receptor.If SP were to bind in the transmembrane domains, the cleft defined by the seven transmembrane segments must rearrange during the binding process in order to bind a peptide in an α-helical structure and at least one large binding subsite in position 8.Thus, indirect topographical analysis with constrained amino acids might contribute to the analysis of the receptor/ligand dynamics.Finally, this study demonstrates that a good knowledge of the peptidic backbone structure and a combination of constrained amino acids are prerequisites to confidently attribute the preferred orientation(s) of an amino acid side chain.