Synthesis and biological activity of substance P C-terminal hexapeptide analogues: structure-activity studies

A series of analogues of the C-terminal hexapeptide of substance P, modified at the glutaminyl residue, was synthesized and their relative activities as spasmogens were determined in the guinea pig ileum and rat colon muscularis mucosae preparations in vitro. In general, when compared to SP6-11, the loss of the carboxamide group has little effect on activity in the colon and reduces activity on the ileum. The exception to this is the Orn6 analogue which retains activity on both preparations and is proposed as a useful tool for structure-activity studies. It is concluded that the hydrogen-bonding potential of the position 6 substituent may be an important determinant of biological activity.     

Synthesis and some biological activities of the tyrosine-8 analog of substance P.

[Tyr8]-substance P, an undecapeptide having the structure Arg-Pro-Lys-Pro-Gln-Gln-Phe-Tyr-Gly-Leu-Met-NH2, has been synthesized by the solid-phase technique on a Beckman automatic peptide synthesizer, appropriately purified and biologically characterized. At twice the dosage, [Tyr8]-substance P showed the same biological activity response as synthetic substance P for stimulation of contraction of the isolated guinea pig ileum and for decrease in the systemic blood pressure of dogs. On the dog's blood pressure, no qualitative differences were observed, but on the isolated gut, the Tyr8 analog gave a more gradual increase in the muscle tone than synthetic substance P. [Tyr8]-substance P released, in vitro, the luteinizing and follicle-stimulating hormones at a very high dosage but did not release growth hormone, prolactin, or thyrotropin.     

Synthesis and biological activity of analogues of the C-terminal hexapeptide of substance P with modifications at glutaminyl and methioninyl residues

Analogues of [Orn⁶]-SP_6-11 have been synthesized in which the methionyl residue is replaced by glutamine γ-carboxamide substituted derivatives. These analogues where tested in three in vitro preparations representative of NK-1, NK-2 and NK-3 receptor types. Substitution of the SCH₃ group of the Met¹¹ side chain by CONHCH₃, CON(CH₃)₂, CONHPh and CONCH₃Ph groups results in analogues which are full agonists in NK-1 and NK-2 preparations with the exception of the Glu[N(CH₃)₂]¹¹ and the Glu(NHCH₃)¹¹ analogues, which are partial agonists at NK-1 and NK-2 receptors respectively. The Glu(NHCH₃)¹¹ analogue shows selectivity for the NK-1 receptor type and is equipotent to the Glu(NCH₃Ph)¹¹ analogue in the same receptor type. The latter analogue is 2.84 times more potent than the parent hexapeptide in the NK-2 preparation. The Glu(NHPh)¹¹ analogue is a full agonist in the NK-3 preparation and equipotent to the parent hexapeptide, in contrast to the other analogues in which Met has been replaced by glutamine γ-carboxamide substituted residues. It is concluded that for NK-1 receptor type the lipophilic character of Met¹¹ side chain is not a determining factor for activity but it is an important factor for activity in the NK-2 receptor type and has a stronger effect when a phenyl group is present, thus leading to analogues which are full agonists and more potent than the parent hexapeptide.     

Synthesis and biological activity of Substance P C-terminal hexapeptide and heptapeptide analogues

The analogues [Glu(OBzl)¹¹]SP_6–11 and [Glu(OBzl)¹¹]SP_5–11 of the C-terminal hexapeptide and heptapeptide of Substance P have been synthesized by conventional solution methods. In each analogue the SCH₃ group of Met¹¹ is replaced by the COOCH₂C₆H₅ group. The in vitro activity of both analogues has been determined on three biological preparations: guinea pig ileum (GPI), rat vas deferens (RVD), and rat portal vein (RPV). The selectivity for the different receptors has been studied by utilizing atropine-treated guinea pig ileum (GPI + At). The results showed that both analogues are mainly active on GPI through the NK-1 receptor and that both analogues are equipotent to Substance P.     

Synthesis of a potent agonist of substance P by modifying the methionyl and glutaminyl residues of the C-terminal hexapeptide of substance P

Analogues of [Orn⁶]-SP_6-11 have been synthesized in which the Met¹¹ residue is replaced by glutamate γ-alkylesters. These analogues were tested in three in vitro preparations representative of NK-1, NK-2, and NK-3 receptor types. Substitution of the SCH₃ group of the Met¹¹ side chain by a COOR (R = methyl, ethyl, n-propyl, n-butyl, cyclohexyl) group results in analogues which are full agonsists in NK-1 and NK-2 preparations but show little agonist activity in the NK-3 preparation. When the SCH₃ group is replaced by a t-butyl ester group and the resulting analogue is a full agonist in all the above preparations and more active than the parent hexapeptide and SP-OCH₃ at NK-1 receptors. It is concluded that for activity at NK-1 receptors methionine can be replaced by γ-t-butyl glutamate without loss of activity, whilst at NK-2 and NK-3 receptors the above substitution increases the activity of [Orn₆]-SP_6-11. Other γ-alkyl esters of the glutamic acid reduce its biological activity.     

Synthesis and biological activity of glycosylated analogs of the C-terminal hexapeptide and heptapeptide of Substance P

The synthesis of two glycosylated analogs of Substance P is described. The activity of the peptides was assayed on the isolated guinea-pig ileum and their degradation was studied using rat hypothalamus slices. While glycosylation noticeably enhances the solubility of the corresponding compounds, the β-glucopyranosyl moiety only slightly modifies the biological half-life and the bioactivity of the glycopeptides.     

Structure-activity studies on the C-terminal hexapeptide of substance P with modifications at the glutaminyl and methioninyl residues

Analogues of [Orn6]-SP6-11 have been synthesized in which the SCH3 group of the Met11 side chain is replaced by other functional groups, such as (CH2)2NH2, COOH, CONH2, and COOR, which have basic, acid, or neutral character and which may act as either H-bonding donors or H-bonding acceptors. These analogues were tested in guinea pig ileum and rat colon muscularis mucosae, in vitro. Substitution of Lys, Gln, or Glu at position 11 caused a marked reduction in biological activity in both tissues. In contrast, the glutamate benzyl ester analogue had only slightly reduced activity in the guinea pig ileum and an increased (4.7 times) activity in the rat colon. It is concluded that charged groups in the side chain at position 11 of SP6-11 reduce the biological activity of SP hexapeptide.     

Sialogogic effects on rat submandibular gland of analogs of the C-terminal hexapeptide of substance P.

The sialogogic response of submandibular glands to analogs of the C-terminal hexapeptide of substance P with various amino acids at the N-terminus was investigated in urethane-anesthetized rats. The rank order of potencies was as follows: SP greater than (pGlu6)SP6-11 much greater than (Dab6)SP6-11 greater than (Orn6)SP6-11 greater than (Gln6)SP6-11 greater than (Lys6)SP6-11 much greater than (Ala6)SP6-11. These results suggest that the sialogogic activity of the analogs of the C-terminal hexapeptide is influenced by the steric effects of the N-terminal amino acid, and the nature of its side chain is of particular importance.     

An approach to the elucidation of self-regulatory mechanism of substance P action. I. Synthesis and biological properties of pentapeptides related both to the substance P C-terminal fragment and enkephalins

Seven pentapeptides, chemically related both to C-terminal fragment of substance P and Met-enkephalin were synthetized and their pharmacological properties were investigated. Peptides I-VI (L-amino acid residue in position 2) antagonized the inhibitory action of D-Ala2-Met-EK-NH2 on isolated vas deferens in vitro but were devoid of opiate-receptor binding activity in radioreceptor studies. Peptide VII (D-Phe2-Met-EK-NH2) exerted a weak inhibitory effect on contractions of transmurally stimulated vas deferens of rat which was abolished by naloxone (10(-8) M) and showed relatively strong but short-lasting analgesic activity in vivo. This peptide at concentration above 10(-5) M partially displaced the 3H-naloxone from its binding sites in striatal membranes. The possible existence of the neuronal substance P-enkephalin self-regulatory mechanism is discussed.     

Structure-activity relationship of the ring portion in backbone-cyclic C-terminal hexapeptide analogs of substance P. NMR and molecular dynamics

The conformations of two backbone-cyclized substance P analogs were derived from homo- and heteronuclear NMR measurements and molecular dynamics simulations carried out in DMSO. The analogs contain subtle variations in the ring chemistry and are compared with biologically active analogs previously examined. The correlation between conformation and activity is used to gain insight into the conformational requirements from the pharmacophore.     

Conformationally restricted C-terminal peptides of substance P. Synthesis, mass spectral analysis and pharmacological properties

Four cyclic analogues of the C-terminal hepta- or hexapeptide of substance P were prepared by the solution method. The cyclizations were obtained by substituting with cysteine the residues normally present in positions 5 or 6 or 11 of substance P and by subsequent disulfide bond formation. The final products were identified by ordinary analytical procedures and advanced mass spectroscopy. The biological activities were determined on three bioassays: the guinea pig ileum, the guinea pig trachea and the rabbit mesenteric vein. Results obtained with these assays indicate that all peptides with a disulfide bridgehead in position 11 are inactive and that a cycle between positions 5 and 6 already strongly reduces the biological activity. The acyclic precursors containing thiol protection groups display weak biological activities. These results further underline the importance of the side chain in position 11 of substance P and suggest that optimal biological activities may require a linear peptide sequence.     

Synthesis of potent antagonists of substance P by modifying the methionyl and glutaminyl residues of its C-terminal hexapeptide and without using d-amino acids

Analogues of [Orn⁶]-SP_6–11 have been synthesized in which the Met¹¹-NH₂ residue is replaced by the α,γ-dimethyl, α,γ-dibenzyl and α,γ-di-tert-butyl esters of glutamic acid. These analogues were tested in three in vitro preparations representative of NK-1, NK-2 and NK-3 receptor types for agonist and antagonist activity. The dimethyl analogue is a selective full agonist in the NK-1 receptor type and a weak antagonist in the other two receptor types, while the dibenzyl and the di-tert-butyl analogues are potent antagonists in the NK-1 receptor type and weak antagonists in the other two receptor types. It is concluded that appropriate modification at the α-carboxamide and the side chain of the methionine residue of substance P may induce antagonism without using d -amino acids.     

Synthesis and biological activity of substance P analogues

The synthesis of the hexapeptide [Glu⁶]SP_6-11 and its glycosylated analogue at the Glu⁶γ-carboxyl position by solution procedures according to several strategies is discussed. The biological activity of SP, [GIu⁶]SP_6-11 (VI) and [Glu(β-d -Glcp)⁶]SP_6-11 (VIII) have been determined and compared to SP by the GPI and RVD assays. The introduction of a β-d -glucopyranosyl moiety at the sixth position of the [Glu⁶]SP_6-11 did not affect to a great extent the in vitro activity pattern of the parent hexapeptide.     

Synthesis and biological activities of substance P antagonists

Several substance P analogues containing various D-amino acid modifications have been synthesized by the solid-phase procedure, detached from the solid support by ammonolysis, and purified by gel filtration combined with reversed-phase chromatography. Three compounds were fair to very potent competitive antagonists of substance P on three bioassays, i.e., guinea pig ileum, rabbit mesenteric vein, and guinea pig trachea. [Arg6,D-Trp10]SP(6-11) is a reasonable antagonist in all three bioassays and [D-Pro4,D-Trp7,9]SP(4-11) is a very potent competitive antagonist with pA2 values ranging around 6.0.     

Synthesis of cyclic and non-cyclic tachykinin partial sequences. 2. Synthesis of the homocyclic substance P(6-11) hexapeptide

The authors describe the synthesis of Gln-Phe-Phe-Gly-Leu-Met by cyclization of H-Leu-Met-Gln-Phe-Phe-Gly using three different methods. The linear sequence was obtained by a (2+4)-segment condensation. The resulting cyclopeptide showed only a small kinin activity on isolated guinea pig ileum compared to substance P, but it is a full agonist.     

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

Synthetic fragments and analogs were used to characterize specificity of antisera toSP and SP_6–11. [Tyr⁸]SP and [Lys⁶]SP_6–11 were both used as radioiodinated ligands. The latter was conjugated with Bolton-Hunter reagents before labelling. In both systems, the C-terminal pentapeptide SP_7–11 was the shortest fragment showing antigenic identity with Substance P molecule. Substitution of different amino acid residues in SP_6–11 by His or Gly showed that all but Glu⁶ take part in the structure of the antigenic determinant.     

Conformation of a biologically active C-terminal hexapeptide analog of the pheromone biosynthesis activating neuropeptide by NMR spectroscopy

The solution conformation of a biologically active C-terminal hexapeptide analog of the pheromone biosynthesis activating neuropeptide Tyr-d -Phe-Ser-Pro-Arg-Leu-NH₂ has been studied by NMR spectroscopy. A β-turn conformation was identified from the NOE connectivities observed for the peptide in a mixed solvent of water and DMSO, indicating that this is the biologically active conformation of the peptide. This study also suggests that the use of such an aqueous-like solvent mixture allows the observation of a preferred conformation for small linear peptides in the presence of conformational averaging.     

Synthesis and conformational study of a cyclic hexapeptide analog of somatostatin containing dehydrophenylalanine

A cyclic hexapeptide analog of somatostatin, cyclo-(Pro-Δ^z-Phe-d -Trp-Lys-Thr-Phe) ( II )? has been synthesized by a combination of solid phase and solution methodology. It shows a potency for inhibition of growth hormone release in vitro about one-tenth that of the corresponding saturated analog, cyclo-(Pro-Phe-d -Trp-Lys-Thr-Phe) ( I ). N.m.r. studies indicate comparable backbone conformations for analogs I and II . However, the sum of our findings from biological evaluation and solution physical data suggest that on the receptor the position-7 phenyl ring of I is adopting a conformation which differs from that of one of the major solution conformers defined previously by n.m.r. studies.