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

Abstract

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 diastereomers of L-1-indanylglycine (Ing) and L-1-benz[f]indanylglycine (Bfi), and the Z and E isomers of dehydrophenylalanine (delta ZPhe, delta 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 [delta EPhe7]SP analogue, which projects the aromatic ring toward the trans orientation, is over 40-fold more potent than the Z isomer, [delta 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 [Flg8]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 A3, 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 alpha-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.

Solution Structures in SDS Micelles and Functional Activity at the Bullfrog Substance P Receptor of Ranatachykinin Peptides

“…It has been shown that the binding pocket for Phe 8 of SP is much larger than the binding pocket for Phe 7 of SP on the NK 1 receptor (estimated volumes of 110 and 240 Å 3 , respectively). 37 Thus, it could be either the difference in hydrophobicity between Ile and Tyr or it could be the difference in size of the side chains. Due to the small size of the side chain of Ile (relative to the side chain of Tyr) fewer stabilizing intermolecular noncovalent interactions may be formed between the receptor and the Ile side chain.…”

Further Delineation of the Two Binding Sites (R*n) Associated with Tachykinin Neurokinin-1 Receptors Using [3-Prolinomethionine11]SP Analogues

“…: 33 1 44 27 55 09; Fax: 33 1 44 27 71 50; E-mail: sagan@ccr.jussieu.fr. 1 The abbreviations used are: SP, substance P (Arg-Pro-Lys-Pro-GlnGln-Phe-Phe-Gly-Leu-Met-NH 2 ); CHO, Chinese hamster ovary; Fmoc, 9-fluorenylmethyloxycarbonyl; HPLC, high pressure liquid chromatography; MBHA, ␣-p-methylbenzhydrylamine; h, human; [P (12) and also for the binding and stimulation of second messengers (13)(14)(15). Therefore, to further delineate the molecular interactions of this C-terminal amino acid with both binding sites of the human NK-1 tachykinin receptor, we have designed constrained analogues of methionine, i.e.…”

An Efficient Synthesis of Novel Adamantane β-Amino Acid: 2-(Adamant-2-yl)-3-aminopropanoic Acid

“…The resulting chemical transformation was confirmed by spectral data (MS, NMR and IR). The efficacy of reduction was clearly confirmed by the absence of the characteristic signals in IR and 13 C NMR spectra belonging to the CN group and C=C bonds.…”