Alessandro Sisto

Comparison of tachykinin NK1 and NK2 receptors in the circular muscle of the guinea‐pig ileum and proximal colon

1 The aim of this study was the pharmacological characterization of tachykinin NK1 and NK2 receptors mediating contraction in the circular muscle of the guinea‐pig ileum and proximal colon. The action of substance P (SP), neurokinin A (NKA) and of the synthetic agonists [Sar9]SP sulphone, [Glp6,Pro9]SP(6–11) (septide) and [βAla8]NKA(4–10) was investigated. The affinities of various peptide and nonpeptide antagonists for the NK1 and NK2 receptor was estimated by use of receptor selective agonists. 2 The natural agonists, SP and NKA, produced concentration‐dependent contraction in both preparations. EC50 values were 100 pm and 5 nm for SP, 1.2 nm and 19 nm for NKA in the ileum and colon, respectively. The action of SP and NKA was not significantly modified by peptidase inhibitors (bestatin, captopril and thiorphan, 1 μm each). 3 Synthetic NK1 and NK2 receptor agonists produced concentration‐dependent contraction of the circular muscle of the ileum and proximal colon. EC50 values were 83 pm, 36 pm and 10 nm in the ileum, 8 nm, 0.7 nm and 12 nm in the colon for [Sar9]SP sulphone, septide and [βAla8]NKA(4–10), respectively. The pseudopeptide derivative of NKA(4–10), MDL 28,564 behaved as a full or near‐to‐full agonist in both preparations, its EC50s being 474 nm and 55 nm in the ileum and colon, respectively. 4 Nifedipine (1 μm) abolished the response to septide and [Sar9]SP sulphone in the ileum and produced a rightward shift and large depression of the response in the colon. The response to [βAla8]NKA(4–10) was abolished in the ileum and largely unaffected in the colon. 5 The NK1 receptor antagonists, (±)‐CP 96,34, FK 888 and GR 82,334 competitively antagonized the response to septide and [Sar9]SP sulphone in both preparations without affecting that to [βAla8]NKA(4–10). In general, the NK1 receptor antagonists were significantly more potent toward septide than [Sar9]SP sulphone in both preparations. 6 The NK2 receptor antagonists, GR 94,800 and SR 48,968 selectively antagonized the response to [βAla8]NKA(4–10) without affecting that to [Sar9]SP sulphone or septide in the ileum and colon. SR 48,968 produced noncompetitive antagonism of the response to the NK2 receptor agonist in the ileum and competitive antagonism in the colon. 7 MEN 10,376 and the cyclic pseudopeptide MEN 10,573 antagonized in a competitive manner the response to [βAla8]NKA(4–10) in the ileum and colon. While MEN 10,573 was equipotent in both preparations, MEN 10,376 was significantly more potent in the colon than in the ileum. MEN 10,376 was also effective against septide in both preparations, without affecting the response to [Sar9]SP sulphone. MEN 10,573 antagonized the response to [Sar9]SP sulphone and septide in both preparations, pKB values against septide being intermediate, and significantly different from, those measured against [βAla8]NKA(4–10) and [Sar9]SP sulphone. 8 These findings show that tachykinin NK1 and NK2 receptors mediate contraction of the circular muscle of the guinea‐pig ileum and colon. In both preparations NK1 receptor antagonists display higher apparent affinity when tested against septide than [Sar9]SP sulphone. These findings are compatible with the proposed existence of NK1 receptor subtypes in guinea‐pig, although alternative explanations (e.g. agonist binding to different epitopes of the same receptor protein) cannot be excluded at present. Furthermore, an intraspecies heterogeneity of the NK2 receptor in the circular muscle of the guinea‐pig ileum and colon is suggested.

α,α-Cyclopentaneglycine Dipeptides Capped with Biaryls as Tachykinin NK2 Receptor Antagonists

The NK2 receptor belongs to the family of tachykinin neurotransmitters. It has been reported to be involved in several pathological conditions, and selective antagonists are potentially useful drugs for the treatment of asthma, irritable bowel syndrome, cystitis, and depression. Starting from in‐house capped dipeptide libraries, we were able to identify a number of molecules with sub‐nanomolar binding affinity for the hNK2 receptor. All were characterized by a rigid core structure with a strong constraint induced by an α,α‐cyclopentaneglycine fragment. Herein we report the further elaboration of three initial basic structures. The planar benzothiophene group was substituted with a series of biphenyl and heterobiphenyl moieties that are well tolerated in terms of receptor affinity. The new compounds also maintained good antagonist potency in an in vitro functional assay, and a number of them showed significant in vivo activity after intravenous administration in our guinea pig model.

Absorption, emission, and chiroptical spectra of neurokinin 1 tachykinin receptor antagonists: the role of charge-transfer states on the biological activity.

A spectroscopic investigation, based on both electronic absorption and emission spectra as well as on chiroptical data, was performed on novel neurokinin 1 (NK1) tachykinin receptor antagonists, exhibiting interesting biological activity. These pseudopeptides have two fluorophores, i.e., indole (I) and naphthalene (N), and a central scaffold with different conformational mobility. Absorption spectra in methanol show the presence of a new band with respect to the sum spectrum of the isolated chromophores at around 285 nm, the intensity of which linearly increases as the bioactivity increases. This absorption disappears by using dioxane as solvent. It is ascribed to an intramolecular I‐N charge‐transfer (CT) complex that forms to different extent, depending on the flexibility of the scaffold. Under this condition, the molecules fold and apparently attain the correct conformation for competing substance P binding to the NK1 receptor, lending plausibility to the role of dipolar charged, spatially close aromatic moieties as topochemical elements in the mechanism of action of substance P antagonists. The excited‐state behavior parallels that in the ground state, as the quenching of the singlet state at 340 nm is found to be linearly dependent on the biological activity, too. Upon decreasing solvent polarity (methanol vs dioxane) the emission of the dipolar state at around 370 nm disappears, while exciplex emission in the range of 400–500 nm occurs. This transition from charge‐separated to exciplex‐like states by lowering the dielectric constant of the medium very likely reflects a change in the structural features of the intramolecular I‐N stacked complex, from a twisted or an asymmetrically overlapped conformation of the indolyl and naphthyl rings to a face‐to‐face geometry. Implications of the rigidity of the molecules, arising from the formation of the intramolecular CT complex, on the ellipticity are briefly discussed.