1H-NMR studies of receptor-selective substance P analogues reveal distinct predominant conformations in DMSO-d6

Proton nmr parameters are reported for DMSO-d6 solutions of two receptor-selective substance P analogues: Ac[Arg6,Pro9]SP6-11, which is selective for the NK-1 (SP-P) receptor and [pGlu6,N-MePhe8]SP6-11, which selectively activates the NK-3 (SP-N) receptor. Full peak assignments of both analogues were obtained by COSY experiments. The chemical shifts, coupling constants, and temperature coefficients of amide proton chemical shifts as well as NOESY effects and calculated side-chain rotamer populations of Phe side chains are reported for both peptides. Analysis of coupling constants and temperature coefficients together with the nuclear Overhauser enhancement spectroscopy effects suggest that Ac[Arg6,Pro9]SP6-11 has a trans configuration about the Phe8-Pro9 amide bond and the preferred conformation of this analogue has a type I beta-turn. The nmr data for [pGlu6,N-MePhe8]SP6-11 suggest that this peptide exists as a mixture of cis-trans isomers in which the cis isomer can preferably adopt a type VI beta-turn conformation, and the trans isomer can adopt a gamma-turn conformation. There are indications that the two last turns are stabilized by a hydrogen bond between the syn carboxamide proton and the pGlu ring carbonyl.     

The Conformational Analysis of Substance P Analogs Using High-Field NMR Techniques

Abstract

High-field nuclear magnetic resonance measurements were carried out on substance P fragments SP_4–11 [pGlu⁵]-SP_5–11 and [pGlu⁶]SP_6–11 both at 400 and at 500 MHz. A spectral simulation was carried out on two of these peptides and the coupling constants were interpreted in terms of the conformations. The J_NH-CHa coupling constants are all ～8 Hz, with the exception of glycine, indicating no preferred conformation for the backbone. For the amino acids other than p-Glu, a comparison of the coupling constant data suggests the same relative rotamer populations for the side chains. Proton longitudinal relaxation time data were measured for all three peptides and support the above conclusions.     

Prostacyclin release induced by neurokinins in cultured human endothelial cells

The effects of neurokinins (NK) and related peptides on the secretion of 6-keto-prostaglandin F1 alpha, a stable metabolite of prostacyclin, were measured. These peptides enhanced three- to five-fold the basal secretion rate with the following rank order of potency (based on threshold concentrations for a significant output): substance P (SP) greater than or equal to NKA greater than SP 4-11 greater than or equal to [pGlu6]SP 6-11 = SP 7-11.NKB and SP 1-9 were inactive. Ac[Arg6, Sar9, Met(O2)11]SP, a NK1 receptor selective agonist, was more potent than other selective agonists for the NK2 and NK3 receptor subtypes. These results suggest that the NK receptors, which mediate the release of prostacyclin from human endothelial cells, belong to the NK1 subtype.     

Interaction of a substance P agonist and of substance P antagonists with lipid membranes. A thermodynamic analysis.

The molecular characteristics of the neuropeptide substance P (SP), its agonist [Sar9,Met-(O2)11]SP, and three of its antagonists [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP, [D-Arg1,D-Trp7,9,Leu11]SP, and [D-Pro2,D-Trp7,9]SP were investigated at the air/water interface and when bound to lipid monolayers and bilayers. Measurement of the Gibbs adsorption isotherm showed that the surface areas of SP and its agonist (240 +/- 5 A2 at biologically relevant concentrations) were distinctly larger than those of the antagonists (138 +/- 5 A2) [Seelig, A. (1990) Biochim. Biophys. Acta 1030, 111-118]. The surface activity of the peptides increased in the order [Sar9,Met(O2)11]SP less than SP less than [D-Pro2,D-Trp7,9]SP less than [D-Arg1,D-Trp7,9,Leu11]SP = [D-Arg1,D- Pro2,D-Trp7,9,Leu11]SP and correlated with the respective binding affinities to lipid membranes. The agonist did not insert into neutral and negatively charged bilayers or into densely packed lipid monolayers (at surface pressures greater than 31 mN/m). In contrast, the three antagonists gave rise to a strong binding both to neutral and to charged lipid monolayers and bilayers. The degree of binding was evaluated from the area increase of lipid monolayers upon peptide insertion, and the binding isotherms were analyzed in terms of the Gouy-Chapman theory. At the monolayer-bilayer equivalence pressure of approximately 32 mN/m, the binding can be described by a surface partition equilibrium with binding constants of (4.5 +/- 0.1) x 10(3) M-1 for [D-Pro2,D-Trp7,9]SP and (1.3 +/- 0.1) x 10(4) M-1 for both [D-Arg1,D-Trp7,9,Leu11]SP and [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP for pure palmitoyloleoylphosphatidylcholine (POPC) membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substance P enhancement of inhibitory avoidance learning: mediation by the N-terminal sequence.

Experiments were performed to investigate the effects of intraperitoneally administered undecapeptide substance P (SP), its N-terminal fragment SP(1-7) (SPN) and the C-terminal analog [pGlu6]-SP(6-11) (SPC) on inhibitory avoidance learning, using a one-trial up-hill avoidance task. In Experiment 1 rats were injected with either SP (50 micrograms/kg), SPN (3.3, 33, 167, 333 micrograms/kg) or SPC (2.7, 27, 134, 268 micrograms/kg) immediately after the training trial. Controls received the diluent vehicles. When tested 24 hr later, rats injected with 50 micrograms/kg SP (37 nmol/kg) and 167 micrograms/kg SPN (185 nmol/kg) exhibited longer step-up latencies than vehicle-treated controls. None of the other doses of SPN nor of the C-terminal fragment influenced performance. In Experiment 2, 167 micrograms/kg SPN or vehicle was injected posttrial either immediately or 5 hr after the training trial. Retention latencies 24 hr later were longer for rats treated with 167 micrograms/kg SPN immediately after the training trial. Performance of the SPN 5-hr delay group did not differ from that of the vehicle-injected controls, ruling out proactive effects of SPN on recall.     

Long-term facilitation of social recognition in rats by vasopressin related peptides: a structure-activity study.

The effect of several vasopressin-related peptides was investigated in the social recognition paradigm, that consists of two successive encounters of a resident and a juvenile rat. The decrease of social investigation time of the resident rat during the second encounter served as a measure for social recognition. Single administration (3.0 micrograms, s.c.) of the vasopressin (AVP)-related peptides AVP-(1-8), AVP-(1-7) or AVP-(1-6), injected just after the first encounter, resulted in social recognition after 24 hours. Such an effect was not observed after placebo treatment or an injection with AVP-(1-5), [pGlu4, Cyt6]AVP-(4-8), [pGlu4, Cyt6]-AVP-(4-9), AVP-(7-9) or oxytocin-(1-6)-NH2. The peptide AVP-(1-6) was also active when administered in a dose of 0.3 micrograms in contrast to other peptides. Thus, vasopressin related peptides induce long term facilitation of social recognition and this action resides in the covalent ring structure of vasopressin. This effect resembles the vasopressin-induced facilitation of particular memory processes, as revealed with other behavioral paradigms.     

Reduced peptide bond pseudopeptide analogues of substance P. A new class of substance P receptor antagonists with enhanced specificity

Each of the last 6 peptide bonds in the COOH terminus of [Leu11]substance P [( Leu11]SP) and [Nle11]spantide were replaced with [CH2NH], and each analogue was tested for SP agonist or antagonist activity by determining its ability to interact with SP receptors on dispersed acini from guinea pig pancreas. Each of the 6 spantide and 5 of the 6 SP analogues had no agonist activity, whereas [psi 9-10]SP was an agonist. For the spantide pseudopeptides, the psi 10-11 analogue (Ki,2.8 microM) was equipotent as an antagonist to spantide itself, whereas the psi 9-10, psi 8-9, psi 7-8, and psi 6-7 analogues were 2.5, 7, 5, and 3 times less potent. For the SP pseudopeptides, the psi 10-11 analogue was the most potent antagonist (Ki, 6.2 microM), whereas the psi 8-9, psi 7-8, and psi 6-7 analogues were 7-, 36-, and 39-fold less potent. There was a close correlation between the ability of each pseudopeptide to inhibit binding of 125I-Bolton-Hunter-SP and to affect amylase secretion. [psi 10-11]SP inhibited SP-stimulated amylase release in a competitive manner, and its inhibitory ability was specific for the SP receptor. Despite [psi 10-11]SP, spantide, and [psi 10-11]spantide having similar affinities for the SP receptor (Ki, 2-6 microM), for inhibition of binding of 125I-[Tyr4]bombesin, the analogues differed with [psi 10-11]SP having a 50-fold lower affinity than for the SP receptor, whereas [psi 10-11]spantide had a 4-fold lower affinity and spantide a 1.5-fold lower affinity for the SP receptor. These results demonstrate that SP pseudopeptides represent a new class of SP receptor antagonists and, in contrast to the currently described SP receptor antagonists, are more specific for SP receptors.     

Naloxone blocks conditioned place preference induced by substance P and [pGlu6]-SP(6-11).

The effect of prior treatment with the opioid receptor (opioceptor) antagonist naloxone on conditioned place preference produced by the neurotachykinin substance P (SP) and its C-terminal hexapeptide analog [pGlu6]-SP(6-11) (SPC) was investigated in rats. Place conditioning was assessed using a circular open field partitioned into four quadrants that were equally preferred by the rats prior to drug treatment. On three successive days, rats received an intraperitoneal (i.p.) injection of naloxone-HCl (1 mg/kg) or vehicle 15 min before an i.p. injection of either 37 nmol/kg SP, equimolar dosed SPC or corresponding diluent vehicle. After injection the rats were placed into their assigned treatment corral for 15 min. During the test for conditioned corral preference (CCP), when provided a choice between the four quadrants, rats injected with SP or SPC spent more time in the treatment corral compared to vehicle controls, indicative of a positive reinforcing action of these peptides. The pre-treatment with naloxone blocked the positive reinforcing effects of both SP and SPC; when injected alone, naloxone did not influence the preference behavior. Gross locomotor activity was affected by neither treatment. Thus, the positive reinforcing effects of SP and SPC may be mediated via interactions with the endogenous opioid system(s).     

The dog common carotid artery: a sensitive bioassay for studying vasodilator effects of substance P and of kinins

In order to develop a sensitive pharmacological preparation which would allow the measurement of the inhibitory effects of kinins and substance P (SP) in vascular smooth muscles, several large arteries of the dog were studied in vitro. The common carotid artery was found to be one of the most sensitive preparations to SP and kinins. When contracted with low concentrations of noradrenaline (between 3.0 x 10(-8) and 3.0 x 10(-7) M), this artery responds to SP (6.5 x 10(-11)-6.5 x 10(-9) M) and bradykinin (BK) (8.1 x 10(-11)-9.1 x 10(-8) M) with relaxations that are proportional to the concentrations of the two peptides. SP and BK appear to exert their relaxant effects through the activation of specific receptors as the exposure of the common carotid artery to concentrations of [Leu8]-angiotensin II, propranolol, methysergide, cimetidine, or atropine sufficient to inhibit the effects of the corresponding agonists do not affect the relaxing effect of SP and BK. [Leu8]-des-Arg9-BK (1.0 x 10(-6) M), indomethacin (2.8 x 10(-5) M), and lioresal (4.7 x 10(-5) M) are also inactive. When the dog common carotid artery is desensitized with high concentrations of SP, BK, eledoisin, and physalaemin a cross-desensitization is observed only between SP and physalaemin. These results support the conclusion that SP and kinins act on different receptors. The order of potency of kinins is the following: BK = [Tyr(Me)8]-BK greater than des-Arg9-BK, suggesting that the receptor for kinins is of the B2 type. The order of potency of peptides related to SP is SP greater than C-terminal 4-11 greater than C-terminal hexapeptide 6-11, similar to that observed in other vascular preparations. The results summarized in this paper indicate that the dog common carotid artery is a preparation sensitive to SP and BK and useful for studying the relaxant effect of these two peptides on vascular smooth muscles.     

Internalization of [3H]substance P analogues in NK-1 receptor transfected CHO cells

The internalization of [3H]propionyl[Met(O2)11]SP(7-11) which binds one binding site and of [3H][Pro9]SP which binds the two binding sites associated with the NK-1 receptor has been examined in CHO cells. The quantity of [3H][Pro9]SP measured inside the cytoplasm in kinetic experiments is fully temperature-dependent. In contrast, [3H]propionyl[Met(O2)11]SP(7-11) internalization reaches the same extent whatever the temperature, although the rate slowed down with lower temperature. The extent of internalization of [3H][Pro(9)]SP relative to the total specific bound is biphasic, when the extent of internalization of [3H]propionyl[Met(O2)11]SP(7-11) remains constant. For [3H][Pro9]SP, a high-affinity high-yield component inhibited in the presence of propionyl[Met(O2)11]SP(7-11) and a low-affinity low-yield component in the internalization process could be determined. Saturation studies show that [3H][Pro9]SP-binding parameters are insensitive to both phenylarsine oxide and monensin treatment, whereas [3H]propionyl[Met(O2)11]SP(7-11) maximal binding is decreased in both cases. Altogether, these data suggest that the two radiolabeled peptides should not follow the same internalization pathway.     

Specificity of antisera obtained to substance P and its C-terminal hexapeptide

Synthetic fragments and analogs were used to characterize specificity of antisera to SP and SP6-11. [Tyr8] SP and [Lys6] SP6-11 were both used as radioiodinated ligands. The latter was conjugated with Bolton-Hunter reagents before labelling. In both systems, the C-terminal pentapeptide SP7-11 was the shortest fragment showing antigenic identity with Substance P molecule. Substitution of different amino acid residues in SP6-11 by His or Gly showed that all but Glu6 take part in the structure of the antigenic determinant.     

NK1 receptor activation in rat rostral ventrolateral medulla selectively attenuates somato-sympathetic reflex while antagonism attenuates sympathetic chemoreflex

The effects of activation and blockade of the neurokinin 1 (NK1) receptor in the rostral ventrolateral medulla (RVLM) on arterial blood pressure (ABP), splanchnic sympathetic nerve activity (sSNA), phrenic nerve activity, the somato-sympathetic reflex, baroreflex, and chemoreflex were studied in urethane-anesthetized and artificially ventilated Sprague-Dawley rats. Bilateral microinjection of either the stable substance P analog (pGlu5, MePhe8, Sar9)SP(5-11) (DiMe-SP) or the highly selective NK1 agonist [Sar9, Met (O(2))11]SP into the RVLM resulted in an increase in ABP, sSNA, and heart rate and an abolition of phrenic nerve activity. The effects of [Sar9, Met (O(2))11]SP were blocked by the selective nonpeptide NK1 receptor antagonist WIN 51708. NK1 receptor activation also dramatically attenuated the somato-sympathetic reflex elicited by tibial nerve stimulation, while leaving the baroreflex and chemoreflex unaffected. This effect was again blocked by WIN 51708. NK1 receptor antagonism in the RVLM, with WIN 51708 significantly attenuated the sympathoexcitatory response to hypoxia but had no effect on baseline respiratory function. Our findings suggest that substance P and the NK1 receptor play a significant role in the cardiorespiratory reflexes integrated within the RVLM.     

Synthesis and biological activity of substance P analogues

The synthesis of the hexapeptide [Glu6]SP6-11 and its glycosylated analogue at the Glu6 gamma-carboxyl position by solution procedures according to several strategies is discussed. The biological activity of SP, [Glu6]SP6-11 (VI) and [Glu(beta-D-Glcp)6]SP6-11 (VIII) have been determined and compared to SP by the GPI and RVD assays. The introduction of a beta-D-glucopyranosyl moiety at the sixth position of the [Glu6]SP6-11 did not affect to a great extent the in vitro activity pattern of the parent hexapeptide.     

Comparative study of SP[6-11] and its analogs using simulated annealing

In this study we compared the steric structures of the bioactive part of substance P (SP[6-11]) and its analogs (NY3460 and pHOPA-SP5). The molecular dynamics-simulated annealing method was used to explore the conformational space, and the structural differences and similarities of these molecules were identified. For the three peptides, the conformational distributions were represented in Ramachandran density plots. The occurring secondary structural elements of the investigated molecules were identified, namely alpha-Helix, type III beta-Turn, gamma-Turn, and inverse gamma-Turn. For SP[6-11] and its two analogs, different intramolecular interactions (H-bonds between the main-chain atoms, aromatic-aromatic interactions, and amino-aromatic interactions) that can stabilize the various conformations of the three peptides were investigated. Detailed examination of these intramolecular interactions revealed that H-bonds between the main-chain atoms are relevant in the determination and stabilization of the conformer structures of the peptides, while the aromatic-aromatic interactions do not play an important stabilizing role. Furthermore, in the conformers of NY3460 and pHOPA-SP5, different types of amino-aromatic interactions were identified that contribute to the formation of the various structures of these peptides. For all three molecules, the orientations of the side chains were investigated and the rotamer populations were determined.     

Influence of proline and β-turn mimetics on the cyclization of penta- and hexapeptides

Summary Proline and Pro-derived peptidomimetics, such as meoxPro-Oic (4-methoxy-proline-octahydro indolic acid), and DBF (2-aminoethyl-6-dibenzofuran propionic acid) were introduced into thymopentin-derived penta-[SP5-] and hexa-[SP6-] peptides and penta-, hexa- and hepta-alanine. Surprisingly, we found that cyclomonomer formation in the investigated penta- and hexapeptides was drastically hindered by the presence of proline regardless of position.     

Antagonism by GRP(18-27) and substance P analogues on insulin release stimulated by GRP(18-27).

The antagonistic effects of [D-Phe25]gastrin-releasing peptide (GRP)(18-27) and [D-Arg1,D-Pro2,D-Trp7,9,Leu11]substance P (SP) on the stimulation of insulin release by GRP(18-27) from isolated canine pancreas were compared with that of [Ala23]GRP(18-27). The stimulation of insulin release by 1 nM GRP(18-27) was reduced to 24.1% and 15.4% by the prior infusion of 1 microM of [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP and 10 microM of [D-Phe25]GRP(18-27), respectively. Glucagon release by GRP(18-27) was not affected by these peptides using the above concentrations. The results indicate that these peptides are antagonists of bombesin-like peptide receptors on pancreatic B-cells, although the inhibitory activities are lower than that of [Ala23]GRP(18-27).     

Effect of substance P/bombesin antagonists on the release of growth hormone by GHRP and GHRH.

The substance P(SP)/bombesin (Bn) antagonists [DArg1DTrp7,9Leu11] SP(P-7482), [DArg1-DPro2DTrp7,9Leu11]SP (P-7483), [DArg1DPhe5DTrp7,9Leu11]SP(P-7492), and the growth hormone releasing hormone (GHRH) antagonist [DArg2Ala8,9,15]GHRH(1-29)(DC21-366) were tested for their in vitro effects on the release of growth hormone (GH) in the presence of GHRH and growth hormone releasing peptide, HisDTrpAlaTrpDPheLysNH2(GHRP). P-7492, P-7483, and P-7482 decreased, dose-dependently, the release of GH by GHRP (IC50 = 0.2 microM, 0.85 microM, and 6 microM, respectively). These antagonists had only a 10-15% inhibitory effect on the stimulated GH release of GHRH even at high dosage. DC21-366 decreased the stimulated release of GH by GHRH (IC50 = 0.16 microM) but not by GHRP. Neither SP nor Bn had GH releasing or inhibitory effects in this system.     

Spinal actions of substance P analogues on cardiovascular responses in the rat: a structure-activity analysis

Ten substance P (SP) analogues were tested for their effects on mean arterial pressure and heart rate following intrathecal administration in the pentobarbital anaesthetized rat. The 10 analogues are [D-Pro4,D-alpha Npa7,9,10]SP(4-11) (A-I), (D-alpha Npa7,9,10]SP (A-II), [D-Trp7,9,10]SP (A-III), [D-Pro4,D-Npa7,9,Phe11]SP(4-11) (A-IV), [D-Pro4,D-beta Npa7,D-alpha Npa9,D-Phe11]SP(4-11) (A-V), [D-Pro4,Lys6,D-Trp7,9,10,Phe11]SP(4-11) (A-VI), [D-Pro4,D-Trp7,9,10,Phe11]SP(4-11) (A-VII), [D-Pro4,D-Trp7,9,10,Trp11]SP(4-11) (A-VIII), [D-Trp7,9,10,Trp11]SP (A-IX), and [D-Pro4,D-Phe7,9,10,Phe11]SP(4-11) (A-X). At 6.5 nmol, the analogues containing the amino acid D-Npa (A-I, A-II, A-IV, and A-V) or D-Phe (A-X) in positions 7, 9, or 10 of SP or its C-terminal octapeptide are devoid of the long-lasting cardio- and vaso-depressor effects, which are otherwise seen with analogues containing the amino acid D-Trp (A-III, A-VI, A-VII, A-VIII, and A-IX) in the same positions. Some of the analogues containing D-Npa maintain the initial hypotensive effect seen with SP while the analogue containing D-Phe produces only a small hypertensive response. The 10 analogues when tested at a dose that failed to alter basal mean arterial pressure and heart rate did not block the cardiovascular responses elicited by SP and no cross desensitization was observed between SP and these analogues. It appears that these SP analogues exert cardiovascular effects in the rat spinal cord probably without interacting with SP receptors.     

500 MHz NMR characterization of synthetic bombesin and related peptides in DMSO-d6 by two-dimensional techniques

The proton NMR characterization of bombesin has been carried out at 500 MHz in DMSO-d6 using two-dimensional homo- and 1H-13C hetero-correlated techniques. All resonances in the NMR spectra have been assigned and several coupling constants have been measured. The backbone J alpha CH-NH coupling constants have constant values that vary between 7.8 and 8.2 Hz and indicate an unfolded structure in DMSO-d6. Discrepancies with data recently obtained at 300 MHz [(1987) Eur. J. Biochem. 168, 193-199] are discussed.