Design and synthesis of side-chain conformationally restricted phenylalanines and their use for structure-activity studies on tachykinin NK-1 receptor.
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.
Josien
H
H
Laboratoire de Chimie Organique Biologique, CNRS URA 493, Université P. et M. Curie, Paris, France.
Lavielle
S
S
Brunissen
A
A
Saffroy
M
M
Torrens
Y
Y
Beaujouan
J C
JC
Glowinski
J
J
Chassaing
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D005449:Fluorenes / Q000737:chemistry; D005998:Glycine / Q000031:analogs & derivatives / Q000737:chemistry; D006168:Guinea Pigs; D007082:Ileum / Q000378:metabolism; D007189:Indans / Q000737:chemistry; D008297:Male; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry; D011487:Protein Conformation; D051381:Rats; D018040:Receptors, Neurokinin-1 / Q000378:metabolism*; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013373:Substance P / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism; D013816:Thermodynamics
D005449:Fluorenes; D007189:Indans; D018040:Receptors, Neurokinin-1; C087557:2-(9-fluorenyl)glycine; C087558:2-(indanyl)glycine; D013373:Substance P; D010649:Phenylalanine; C087556:3,3-diphenylalanine; D005998:Glycine