A comparison of the effects of substance P and shorter analogues on the synaptosomal ATPases activities in the rat brain

The influence in vitro of SP and C-terminal fragments of analogues SP(5-11) (pyroGlu5, Tyr8); SP(6-11) (pyroGlu6, Tyr8); SP(6-11) (pyroGlu6, D-Phe7); SP(6-11) (pyroGlu6, D-Phe8) on the (Ca, Mg) and (Na, K) ATPases activities from synaptosomal membranes of cerebral cortex and hippocampus of rat brain were compared. The data obtained in this study indicate the following: 1. Substance P stimulates the activities of (Na, K) and (Ca, Mg) ATPases more effectively in synaptosomal membranes from hippocampus than cerebral cortex. 2. Heptapeptide SP(5-11) (pyroGlu5, Tyr8) causes a more distinct increase of (Ca, Mg) ATPase activity in cortical synaptosomal membranes than SP does. 3. The change of L-Phe conformation to D in position 7 in hexapeptide induces reduction of enzymes activities in hippocampus. 4. Especially important for the maintenance of biological activity of drugs is the replacement of Gln5 with pyroGlu6 and conformation of Phe residues. 5. SP and shorter analogues of fragments SP C-terminal SP regulate the active cation transport in synaptosomal membranes of cerebral cortex and hippocampus.     

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.     

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.     

Immunochemical analysis of substance P using its synthetic fragments and analogs

Synthetic fragments and analogs were used to characterize specificity of antisera to substance P. Both, the C-terminal hexapeptide and the pentapeptide completely inhibited binding of 125I-[Tyr8]substance P by these antisera, showing the antigenic identity with substance P. Synthetic fragments shorter than peptide (7-11) did not react with anti-substance P antisera in this system. Substitution of amino acids in different positions in the fragments (6-11) or (7-11) by histidine or glycine revealed that all five amino-acid residues take part in a structure of the antigenic determinant.     

Structural and biological effects of a beta2- or beta3-amino acid insertion in a peptide.

Molecular mechanics calculations on conformers of Ac-HGly-NHMe, Ac-beta2-HAla-NHMe and Ac-beta3-HAla-NHMe indicate that low-energy conformations of the beta-amino acids backbone, corresponding to gauche rotamers around the Calpha-Cbeta bond, may overlap canonical backbone conformers observed for alpha-amino acids. Therefore, Substance P (SP) was used as a model peptide to analyse the structural and biological consequences of the substitution of Phe7 and Phe8 by (R)-beta2-HPhe and of Gly9 by HGly (R)-beta2-HAla or (S)-beta3-HAla. [(R)-beta2-HAla9]SP has pharmacological potency similar to that of SP while [HGly9]SP and [(S)-beta3-HAla9]SP show a 30- to 50-fold decrease in biological activities. The three analogues modified at position 9 are more resistant to degradation by angiotensin converting enzyme than SP and [Ala9]SP. NMR analysis of these SP analogues suggest that a beta-amino acid insertion in position 9 does not affect the overall backbone conformation. Altogether these data suggest that [HGly9]SP, [(S)-beta3-HAla9]SP and [(R)-beta2-HAla9]SP could adopt backbone conformations similar to that of SP, [Ala9]SP and [Pro9]SP. In contrast, incorporation of beta2-HPhe in position 7 and 8 of SP led to peptides that are almost devoid of biological activity. Thus, a beta-amino acid could replace an alpha-amino acid within the sequence of a bioactive peptide provided that the additional methylene group does not cause steric hindrance and does not confine orientations of the side chain to regions of space different from those permitted in the alpha-amino acid.     

Hydrolysis of the synthetic chromophoric hexapeptide Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe catalyzed by bovine pepsin A. Dependence on pH and effect of enzyme phosphorylation level

Inactivation of substance P and its C-terminal hexapeptide analog [p-Glu6]substance P6-11 was studied in rat parotid and hypothalamic slices. It was found that in the parotid slice system the decay of substance P induced K+ release occurs concurrently with a decrease in the biologically active concentration of the peptide in the medium. The inactivation was further studied using [p-Glu6]substance P6-11 as substrate in the parotid and in the hypothalamic slice systems. In both tissue preparations the hexapeptide is degraded to small peptide fragments by metalloendopeptidase. Separation of the peptide fragments by high performance liquid chromatography and determination of their amino acid composition showed that in the hypothalamic slice system the major cleavage of the hexapeptide analog occurs between Phe8-Gly9 with minor cleavage sites between Phe7-Phe8 and Gly9-Leu10. In the rat parotid slice system the major cleavage occurs between Gly9-Leu10 with a minor cleavage site between Phe7-Phe8. The degradation of the hexapeptide analog in the hypothalamic system was inhibited 77% and 67% by treatment with 1 mM p-chloromercuriphenylsulfonate and p-chloromercuribenzoate, respectively, whereas in the parotid system these reagents inhibited the degradation of the hexapeptide only by 15% and 8%. These results may indicate that different proteases in the parotid and hypothalamus are involved in degradation of substance P. Kinetic studies, including the use of various inhibitors as well as competition by the peptide hormones somatostatin, LHRH, TRH and Leu-enkephalin-NH2, revealed that in both tissues the hexapeptide analog is a preferred substrate for degradation by protease of considerable specificity towards the C-terminal sequence of substance P. It is suggested that this metalloendopeptidase may be important in the termination of the substance P response.     

Monoclonal antibodies against different epitopes of peptide hormones. Use of photoreactive analogues in studies on vasopressin.

In order to produce monoclonal antibodies directed against different epitopes of the neurohypophyseal hormone vasopressin, the hormone was coupled to carrier proteins via photoreactive groups at different positions in the vasopressin sequence: [2-(4-azidophenylalanine), 8-arginine]vasopressin (peptide P1, photoreactive group at position 2) and desamino-[8-N6-(4-azidophenylamidino)lysine]vasopressin (peptide P2, photoreactive group at position 8) were conjugated to thyroglobulin by flash photolysis. Monoclonal antibodies against these conjugates bound ([3H]8-arginine]vasopressin with dissociation constants ranging over 40-400 nM. Epitope analysis by means of competitive ELISA showed that the monoclonal antibody obtained with peptide P1 as hapten was directed against the C-terminal acyclic tripeptide when its conformation was stabilized by interaction with the disulphide-linked cyclic hexapeptide. In contrast, the epitope analysis of three monoclonal anti-(peptide P2) antibodies demonstrated that they recognized antigenic determinants in the cyclic hexapeptide ring, mainly the hydrophobic surface formed by Tyr2 and Phe3. Our results suggest that monoclonal antibodies against different epitopes in small peptide hormones can be generated selectively by using photoreactive peptides in such a way that different antigenic sites are exposed in the hapten-carrier conjugate.     

Involvement of the second extracellular loop (E2) of the neurokinin-1 receptor in the binding of substance P. Photoaffinity labeling and modeling studies

Substance P (SP) interacts with the neurokinin-1 (NK-1) G-protein-coupled receptor, which has been cloned in several species. In the present study, the domains of the NK-1 receptor involved in the binding of SP and SP-(7-11) C-terminal fragment have been analyzed using two peptide analogs containing the photoreactive amino acid para-benzoylphenylalanine ((p-Bz)Phe) in position 8 of their sequence. This study was carried out with [BAPA-Lys(6),(p-Bz)Phe(8),Pro(9),Met(O(2))(11)]SP-(7-11) and [BAPA(0),(p-Bz)Phe(8)]SP on both rat and human NK-1 receptors expressed in CHO cells. Combined trypsin and endo-GluC enzymatic complete digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis led to the identification of the same domain of covalent interaction, (173)TMPSR(177), for the two photoactivatable peptides. Further digestion of this fragment with carboxypeptidase Y led to the identification of (173)TMP(175) in the second extracellular loop (E2) of the NK-1 receptor as the site of covalent attachment. Models of the conformation of this E2 loop in the human NK-1 receptor were generated using two different strategies, one based on homology with bovine rhodopsin and the other based on the solution conformation preferences of a synthetic peptide corresponding to the E2 loop.     

Tachykinin-induced vasodilatation in rat skin measured with a laser-Doppler flowmeter: evidence for receptor-mediated effects

Vasodilatation was induced by perfusion of the tachykinins substance P (SP), neurokinin A and neurokinin B and the analogues [Glp6, D-Pro9]SP-(6-11) and [Glp6, L-Pro9]SP-(6-11) over the base of vacuum-induced blisters on the rat footpad. Vasodilatation was measured as change in blood flow using a laser-Doppler flowmeter. The tachykinins induced vasodilatation in a dose-response manner with a threshold of approximately 3 pmol and pD2's of 6.48, 6.13 and 6.21 for SP, neurokinin A and neurokinin B respectively. The D- and L-Pro analogues of [Glp6, Pro9]SP-(6-11) also induced vasodilatation in a dose-dependent manner. The L-Pro analogue was more potent than the D-Pro analogue (D/L ratio of the EC50's = 21) which suggests the involvement of an NK-1 type receptor in the mediation of small vessel vasodilatation. The vasodilatation to SP was reduced by 64% and 59% in capsaicin- and antihistamine-pretreated animals respectively, demonstrating the involvement of capsaicin-sensitive primary afferent nerves and mast cells in the vasodilatation component of the neurogenic inflammatory response.     

Analogs of Substance P modified at the C-terminus which are both agonist and antagonist of the NK-1 receptor depending on the second messenger pathway.

The initial goal of this study was to analyze, using photolabeling, the interactions between Substance P and its tachykinin NK-1 receptor. Therefore, the photoreactive amino acid para-benzoyl-phenylalanine (pBzl)Phe was incorporated into the Substance P sequence from position 4 to 11 leading to Bapa0[(pBzl)Phex]SP analogs. Biotinyl sulfone-5-aminopentanoic acid (Bapa) was introduced in order to purify the covalent complex. These photoreactive SP analogs were first assayed for their affinity for the two binding sites associated with the NK-1 receptor, as well as for their potency in activating the phospholipase C and adenylate cyclase pathways. All analogs photoreactive from position 4 to 11 have moderate to high affinity for the two NK-1 receptor-binding sites, except for the analog modified at position 7. This affinity could be correlated to their potency to activate the phospholipase C and adenylate cyclase pathways, except for the analog photoreactive at position 11. Bapa0[(pBzl)Phe11]SP was found to be an agonist in the phospholipase C pathway and an antagonist in the adenylate cyclase pathway, other analogs modified at position 11 were therefore analyzed. Among these, Bapa0[Pro9, (pBzl)Hcy(O2)11]SP is a partial agonist, whereas Bapa0[Hcy(ethylaminodansyl)11]SP is a full agonist in the phospholipase C pathway, the two analogs being antagonist in the adenylate cyclase pathway. These results show that analogs of SP can be simultaneously agonist at one binding site and antagonist at the other binding site associated with the NK-1 receptor.     

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.     

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.     

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.     

Peptide analogues of angiotensinogen. Effect of peptide chain length on renin inhibition

Renin inhibition was evaluated for a series of peptide analogues of angiotensinogen with different chain lengths. Systematic deletion of amino acid residues from the hexapeptide BocPheHisLeuR-ValIleHisOCH3 showed that the presence of residues at the N-terminal Phe and His positions was essential for efficient enzyme-inhibitor binding whereas the C-terminal Ile and His residues were much less important. Synthesis of a tetrapeptide analogue shortened at the C-terminus and containing modified side chains produced a potent inhibitor of renin which demonstrated hypotensive activity in a salt depleted monkey.     

NMR and molecular dynamics studies of tachykinins: conformation of substance P fragment 4-11

The conformation of the C-terminal octapeptide fragment of Substance P (SP4-11, Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) has been investigated by 2D-NMR and MD methods. The octapeptide exists in a blend of conformations. The molecule seems to shuttle between conformations with gamma-bends either at Phe5 or Gly6 or Gln3 or Leu7 and between a nearly extended structure.     

Structure-activity studies with somatostatin: role of lysine in positions 4 and 9 for gastric activity

The gastric inhibitory activity of somatostatin analogues modified in position 4 or 9, was investigated in conscious cats prepared with gastric fistulae. Gastric secretion was stimulated with pentagastrin. Deletion of Lys4, or substitution with an alcoholic (Thr) or acidic (Glu) residue yielded analogues with reduced (10% or less) potency compared with somatostatin. In comparison, [Phe4]somatostatin and modified Phe4 analogues [( p-NH2-Phe4]-, [F5Phe4]- and [Phe4,D-Trp8]somatostatin) were approximately equipotent with somatostatin. The high potency of the Phe4 analogues illustrates that a basic side-chain in position 4 is not essential for gastric activity. In contrast, [Thr9]-, [Glu9]-, [Phe9]- and [p-NH2-Phe9]somatostatin were all inactive (less than 5% potency of somatostatin) in the stomach. Thus the lysyl residue in position 9 is more critical than that in position 4 for somatostatin's gastric activity.     

Antibiotic activity and structural analysis of the scorpion-derived antimicrobial peptide IsCT and its analogs

IsCT is a non-cell-selective antimicrobial peptide isolated from the scorpion Opisthacanthus madagascariensis that has potent cytolytic activity against both mammalian and bacterial cells. To investigate the structure-activity relationships of IsCT and to design novel peptide antibiotics with bacterial cell selectivity, we synthesized several analogs of IsCT and determined their three-dimensional structures in solution by 2D-NMR spectroscopy. IsCT has a linear alpha-helical structure from Gly3 to Phe13, and [K7]-IsCT has a linear alpha-helical structure from Leu2 to Phe13. [K7, P8, K11]-IsCT, which has a bend in its middle region, exhibited the highest antibacterial activity without hemolytic activity, suggesting that its proline-induced bend is an important determinant of this selectivity. Tryptophan fluorescence showed that the high selectivity of [K7, P8, K11]-IsCT toward bacterial cells is closely correlated with its highly selective interaction with negatively charged phospholipids. Its potent activity against antibiotic-resistant bacteria suggests that [K7, P8, K11]-IsCT may serve as a promising lead candidate in the development of new peptide antibiotics.     

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.     

Dehydro-dermorphins. II. Synthesis and biological activity of unsaturated dermorphin hexa and heptapeptides

The Phe3 and/or Tyr5 residues in dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2) and its N-terminal hexapeptide-amide were replaced by delta-Phe or by Phe5 in order to examine the effect on opioid activity. On GPI preparation, the substitution of Phe5 for Tyr5 was well tolerated, whereas the hexa and heptapeptides containing delta Phe in position 3 and/or 5 displayed low potency. The unsaturation at position 3 alone or at positions 3 and 5 was particularly detrimental to mu activity. In the tail flick test, the influence of unsaturation or substitution at positions 3 and 5 generally matched the results of the in vitro assay. Dehydropeptides showed comparatively low antinociceptive effects and [Phe5] analogues displayed about 50% of the analgesic potency of the original peptides.     

Synthesis and purification of two SP (6-11) analogues with enhanced selectivity for the NK-1 receptor.

Two hexapeptide analogues of Substance P (6-11) have been synthesized. Replacement of Gly9 by proline provides a peptide with tenfold enhanced selectivity for the NK-1 receptor. The corresponding proline-containing glycopeptide incorporating a beta-D-glucopyranosyl residue linked to the side-chain of Glu6 was 100 times more selective than Substance P for the same receptor.