Characterization of the bioactive conformation of the C‐terminal tripeptide Gly‐Leu‐Met‐NH₂ of substance P using [3‐prolinoleucine10]SP analogues

Abstract

Residue Leu10 of substance P (SP) is critical for NK-1 receptor recognition and agonist activity. In order to probe the bioactive conformation of this residue, cis-and trans-3-substituted prolinoleucines were introduced in position 10 of SP. The substituted SP analogues were tested for their affinity to human NK-1 receptor specific binding sites (NK-1M and NK-1m) and their potency to stimulate adenylate cyclase and phospholipase C in CHO cells transfected with the human NK-1 receptor. [trans-3-prolinoleucine10]SP retained affinity and potency similar to SP whereas [cis-3-prolinoleucine10]SP shows dramatic loss of affinity and potency. To analyze the structural implications of these biological results, the conformational preferences of the SP analogues were analyzed by NMR spectroscopy and minimum-energy conformers of Ac-cis-3-prolinoleucineNHMe, Ac-trans-3-prolinoleucine-NHMe and model dipeptides were generated by molecular mechanics calculations. From NMR and modeling studies it can be proposed that residue Leu10 of SP adopts a gauche(+) conformation around the v 1 angle and a trans conformation around the v 2 angle in the bioactive conformation. Together with previously published results, our data indicate that the C-terminal SP tripeptide should preferentially adopt an extended conformation or a PPII helical structure when bound to the receptor.

Original article In vitro pharmacological evaluation of the radiolabeled C-terminal substance P analogue Lys-Phe-Phe-Gly-Leu-Met-NH2: Does a specific binding site exist?

“…Numerous structureactivity relationship stud ies have shown that the Cterminal component of neurokinin ligands (especially the Cterminal penta peptide PheXGlyLeuMetNH 2 motif, where X is a hydrophobic amino acid residue) is critical for spe cific binding of highaffinity agonists to receptors in the tachykinin family [4,20,22,26,27,33].…”

Incorporation of vinylogous scaffolds in the C‐terminal tripeptide of substance P

“…The previous use of β ‐amino acid substituted‐SP analogs [HGly 9 ]SP, [(R)‐ β 2 HAla 10 ]SP and [(S)‐ β 3 HAla 9 ]SP had shown that an ‘extra’ atom in the peptidic backbone introduced in position 9 in the sequence of SP can be tolerated if a destabilizing interaction associated with this ‘extra’ methylene can be compensated by a positive interaction with a methyl group (18). The isobutyl side chain of leucine‐10 in the sequence of SP establishes a crucial interaction, mainly within the NK‐1M‐specific binding site, its specific role had been ascertained according to the behaviors of two series of peptides (12, 18, 19). To summarize, positions 9, 10 or 11 in the sequence of SP can be replaced by a proline (position 9) or a C β ‐substituted proline (position 10 or 11), assuming that in position 10 the orientation of the isobutyl side chain is fixed by the trans relationship between the peptidic backbone and the C β ‐substitution.…”

“…For all the steps performed manually, completion of the coupling and deprotection steps was monitored with the Kaiser's test. Final steps performed on the ABI Synthesizer, HF cleavage and subsequent high‐performance liquid chromatography (HPLC) purifications were performed as previously described (12), except that all HPLC columns were heated at 50°C.…”

“…Nuclear magnetic resonance (NMR) experiments were recorded with peptides VI or VII (3 mg) dissolved in 2 H 2 O (550 μ L) at 303 K and 318 K, respectively, on a Bruker Avance spectrometer (Bruker, Lisses, France). Proton assignments were obtained as previously described (12).…”

“…Glycine can be favorably replaced by sarcosine or proline (9, 10) without significant decrease in binding affinity and biological activity associated with the (NK)‐1 receptor, whatever the binding sites considered are (NK‐1M and NK‐1m) and the associated second messenger pathways (adenylate cyclase and phospholipase C, respectively) (10). The tripeptide Gly‐Leu‐Met‐NH 2 preferentially adopts a more or less extended conformation or a PPII helical structure, when bound to the NK‐1 receptor (11, 12). Conversely, a type II′ β ‐turn constraint introduced around residues 9 and 10 confers antagonistic properties to the corresponding substituted SP analogs (13, 14).…”