Paolo Rovero

Competitive antagonists discriminate between NK2 tachykinin receptor subtypes

1 We have compared the ability of various tachykinins and selective tachykinin receptor agonists to induce contraction of the endothelium‐denuded rabbit pulmonary artery (RPA) and hamster trachea (HT) and have estimated the affinity of some newly developed NK2 selective antagonists in the same tissues. 2 In confirmation of previous findings, experiments with the agonists indicated that NK2 receptors are the main if not the sole mediators of the response to tachykinins in both RPA and HT. No evidence for significant degradation of neurokinin A (NKA) was found in either tissue when experiments were repeated in the presence of a mixture of peptidase inhibitors (thiorphan, captopril and bestatin, 1 μm each). 3 The peptide antagonists tested were: Peptide I = [Tyr5, d‐Trp6,8,9, Arg10]‐NKA(4–10); Peptide II = [Tyr5, d‐Trp6,8,9, Arg10]‐NKA(3–10); Peptide III = Ac‐Leu‐Asp‐Gln‐Trp‐Phe‐Gly‐NH2. The three peptides produced a concentration‐dependent rightward shift of the concentration‐response curve to NKA in both RPA and HT with no significant depression of the maximal response attainable. The slopes of the Schild plots were not significantly different from unity, indicating a competitive antagonism. Peptides I and II were about 100 times more potent in the RPA than in the HT, while Peptide III was about 100 times more potent in the HT than RPA. 4 The pA2 values obtained in these two tissues with the three antagonists were not significantly different when tested in the absence or presence of peptidase inhibitors, or when a selective NK2 receptor agonist, [βAla8]‐NKA(4–10) was used instead of NKA. Similar pA2 values were obtained after 15 or 90 min of incubation with the antagonists. Peptides I, II and III had no inhibitory effect on contractions produced by noradrenaline in the RPA or by carbachol in the HT. 5 Peptides I, II and III showed weak or no antagonistic activity toward the vasodilatator effect of substance P in the dog carotid artery (NK1 receptor‐mediated) or toward the contractile effect of neurokinin B in the rat portal vein (NK3 receptor‐mediated). 6 These results provide pharmacological evidence for heterogeneity of NK2 receptors in the RPA and HT. The NK2 receptors present in these tissues are not discriminated by natural tachykinins or selective agonists, but are recognized with very different affinity by NK2 receptor antagonists.

Synthesis and Conformational Analysis of a Cyclic Peptide Obtained via i to i+4 Intramolecular Side-Chain to Side-Chain Azide-Alkyne 1,3-Dipolar Cycloaddition

Intramolecular side-chain to side-chain cyclization is an established approach to achieve stabilization of specific conformations and a recognized strategy to improve resistance toward proteolytic degradation. To this end, cyclizations, which are bioisosteric to the lactam-type side-chain to side-chain modification and do not require orthogonal protection schemes, are of great interest. Herein, we report the employment of Cu(I)-catalyzed 1,3-dipolar cycloaddition of side chains modified with azido and alkynyl functions and explore alternative synthetic routes to efficiently generate 1,4-disubstituted [1,2,3]triazolyl-containing cyclopeptides. The solid-phase assembly of the linear precursor including epsilon-azido norleucine and the propargylglycine (Pra) in positions i and i+4, respectively, was accomplished by either subjecting the resin-bound peptide to selective on-resin diazo transformation of a Lys into the Nle(epsilon-N3) or the incorporation of Fmoc-Nle(epsilon-N3)-OH during the stepwise build-up of the resin-bound peptide 1b. Solution-phase Cu(I)-catalyzed 1,3-dipolar cycloaddition converts the linear precursor Ac-Lys-Gly-Nle(epsilon-N3)-Ser-Ile-Gln-Pra-Leu-Arg-NH2 (2) into the 1,4-disubstituted [1,2,3]triazolyl-containing cyclopeptide [Ac-Lys-Gly-Xaa(&(1))-Ser-Ile-Gln-Yaa(&(2))-Leu-Arg-NH2][(&(1)(CH2)4-1,4-[1,2,3]triazolyl-CH2&(2))] (3). The conformational preferences of the model cyclopeptide 3 (III), which is derived from the sequence of a highly helical and potent i to i+4 side-chain to side-chain lactam-containing antagonist of parathyroid hormone-related peptide (PTHrP), are compared to the corresponding lactam analogue Ac[Lys(13)(&(1)),Asp(17)(&(2))]hPTHrP(11-19)NH2 (II). CD and NMR studies of 3 and II in water/hexafluoroacetone (HFA) (50:50, v/v) revealed a high prevalence of turn-helical structures involving in particular the cyclic regions of the molecule. Despite a slight difference of the backbone arrangement, the side-chains of Ser, Gln, and Ile located at the i+1 to i+3 of the ring-forming sequences share the same spatial orientation. Both cyclopeptides differ regarding the location of the turn-helical segment, which in II involves noncyclized residues while in 3 it overlaps with residues involved in the cyclic structure. Therefore, the synthetic accessibility and conformational similarity of i to i+4 side-chain to side-chain cyclopeptide containing the 1,4-disubstituted [1,2,3]triazolyl moiety to the lactam-type one may result in similar bioactivities.

The rat isolated portal vein: a preparation sensitive to neurokinins, particularly neurokinin B

The rat isolated portal vein is a pharmacological preparation more sensitive to neurokinin B than to any other neurokinin or tachykinin. The preparation is more sensitive to C-terminal partial sequences of substance P (SP) particularly SP-(6-11) than to the whole undecapeptide. The order of potency of neurokinins is as follows: neurokinin B greater than neurokinin A greater than substance P. The preparation shows high sensitivity also to kassinin and eledoisin. Comparative tests performed with strips of the rat portal vein suspended in a microbath under continuous perfusion (system 1) or in ordinary baths for isolated smooth muscles (system 2) have given similar results and have shown that the myotropic effect of neurokinin B is not modified by a variety of antagonists of endogenous agents as well as by inhibitors of the arachidonic acid cascade. The present results suggest that neurokinin B contracts the rat portal vein by activating specific receptors, presumably located on the smooth muscle membrane, different from those of biologically active amines and peptides which are active stimulants of the vein. Neurokinin B is ten times more active than neurokinin A and at least 100 times more than substance P. Such an order of potency of agonists suggests the existence of a new neurokinin receptor type, particularly sensitive to neurokinin B.

Antibodies from patients with rheumatoid arthritis target citrullinated histone 4 contained in neutrophils extracellular traps

Background Histone deimination regulates gene function and contributes to antimicrobial response, allowing the formation of neutrophil extracellular traps (NETs). Deiminated proteins are target of anti-citrullinated peptides antibodies (ACPA) in rheumatoid arthritis (RA). Objective The objective of this paper is to test the hypothesis that RA sera react with deiminated histones contained in NETs. Methods Neutrophils from peripheral blood were stimulated with A23187 and acid treated; NETosis was induced by phorbol myristate acetate, and NET proteins were isolated. Sera were tested by immunoblot on acid extracted proteins from neutrophils and from NETs, and by ELISA on deiminated histone H4 or H4-derived peptides. Bands reactive with RA sera were excised from gels, digested with trypsin and subjected to matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) analysis, before and after derivatisation to detect citrullinated peptides. Results RA sera reacted with a deiminated antigen of 11 KDa from activated neutrophils, recognised also by anti-H4 and antideiminated H4 antibodies. A similar reactivity was observed with NET proteins. The antigen from neutrophils or NETs was identified as citrullinated H4 by MALDI-TOF analysis. By ELISA, RA sera bound in vitro citrullinated H4. Citrullinated H4 14–34 and 31–50 peptides detected antibodies in 67% and 63% of RA sera and in less than 5% of controls; antibody titre was correlated with anti-CCP2. Conclusions Citrullinated H4 from activated neutrophils and NETs is a target of antibodies in RA, and synthetic citrullinated H4-derived peptides are a new substrate for ACPA detection. As NETosis can generate antigens for ACPA, these data suggest a novel connection between innate and adaptive immunity in RA.

Structure-activity studies of neurokinin A

A structure-activity study on neurokinin A and its C-terminal fragment NKA (4-10) has been performed in order to find selective agonists for the NK-2 receptor and identify chemical modifications suitable for protecting the peptides from degradation, while maintaining activity. Five series of compounds have been prepared and tested: 1. the complete series of the L-Ala monosubstituted analogues of NKA; 2. a series of NKA fragments from the C- or N-terminal; 3. the complete series of NKA (4-10) analogues monosubstituted with beta-Ala; 4. a series of NKA (4-10) analogues with monosubstitutions in pos. 4, 8, 10 or multisubstitutions in two or more of the same positions; and 5. a series of 6 NKA (4-10) analogues monosubstituted with 1-amino,1-cyclohexane carboxylic acid residue. It has been found that the most selective agonists for the NK-2 receptor system are [beta Ala8]NKA (4-10) and [Nle10]NKA (4-10). Protection from aminopeptidase may be obtained by acetylation of the N-terminal amide of NKA (4-10), while partial protection from endopeptidases should be expected from the presence of beta-Ala in position 8. Conformational constraints induced with 1,amino,1-cyclohexane carboxylic acid residue gave weakly active compounds. Multiple substitutions reduce rather than potentiating the favorable effects of the corresponding monosubstituted compounds.

The activity of peptides of the endothelin family in various mammalian smooth muscle preparations

The activity of natural endothelins such as ET-1, ET-2, ET-3 and of sarafotoxin S6b (SRFTX) as compared to that of the C-terminal hexapeptide ET-(16-21) was investigated in several smooth muscle preparations in the presence of indomethacin, captopril, bestatin and thiorphan. In some tissues (rat thoracic aorta, guinea-pig ileum, human urinary bladder, renal pelvis or renal artery), ET-(16-21) had little if any agonist activity. In other preparations (guinea-pig bronchus, rat vas deferens, rabbit pulmonary artery) ET-(16-21) was a full agonist. The amidated form of ET-(16-21) was either inactive or less potent than ET-(16-21). These findings provide evidence that at least two receptors exist for peptides of the ET family; these receptors were termed ETA and ETB. ET-(16-21) is a full agonist at ETB receptors while being inactive or weakly active at ETA receptors. The free acid of tryptophan in position 21 plays a key role in the activity of these peptides at ETB receptors.

A potent and selective agonist for NK-2 tachykinin receptor

Replacement of the glycine in position 8 of the C-terminal heptapeptide NKA(4-10) with beta-alanine give rise to a potent and selective agonist for the NK-2 tachykinin receptor. The affinity of [beta-Ala8]-NKA(4-10) to the NK-2 receptor is enhanced by almost one order of magnitude as compared to NKA(4-10), while affinity decreases at about the same extent at NK-1 and NK-3 receptors, respectively. Synthesis and biological activities of a series of NKA(4-10) analogues systematically replaced in each position with beta-alanine are also reported.

Urantide: an ultrapotent urotensin II antagonist peptide in the rat aorta

In this study we describe the ability of two human urotensin‐II (hU‐II) derivatives [Pen5,Orn8]hU‐II(4–11) and [Pen5,DTrp7,Orn8]hU‐II(4–11) (urantide) to block hU‐II‐induced contractions in the rat isolated thoracic aorta. Both compounds competitively antagonized hU‐II‐ induced effects with pKB=7.4±0.06 (n=12) and pKB=8.3±0.09 (n=12), respectively. In contrast, neither [Pen5,Orn8]hU‐II(4–11) nor urantide (1 μM each) was able to modify noradrenaline‐ or endothelin 1‐induced contractile effects. At micromolar concentrations, [Pen5,Orn8]hU‐II(4–11) produced weak (25% of hU‐II maximum) agonist responses in the rat aorta, whereas urantide was totally uneffective as agonist up to 1 μM. In addition, [Pen5,Orn8]hU‐II(4–11) and urantide displaced [125I]urotensin II from specific binding at hU‐II recombinant receptors (UT receptors) transfected into CHO/K1 cells (pKi=7.7±0.05, n=4 and pKi=8.3±0.04, n=4, respectively). To our knowledge, urantide is the most potent UT receptor antagonist so far described, and might represent a useful tool for exploring the (patho)physiological role of hU‐II in the mammalian cardiovascular system.

The C-terminal hexapeptide, endothelin-(16-21), discriminates between different endothelin receptors.

Short communication. The C-terminal hexapeptide, endothelin (16-21), dicriminates different endothelin receptors which mediate the response to endothelin-1 in the guinea-pig bronchus and rat thoracic aorta

Tachykinin receptors in the guinea-pig isolated bronchi

The aim of the study was to assess which tachykinin receptors mediate the contractile response in the guinea-pig isolated bronchi. Experiments with natural tachykinins and receptor-selective tachykinin agonists were performed in the absence or presence of peptidase inhibitors and in bronchi pretreated with phenoxybenzamine. Both NK-1 (substance P, substance P methylester and septide) and NK-2 (neurokinin A, [beta-Ala8]neurokinin A-(4-10) and MDL 28,564) receptor agonists produced concentration-dependent contraction. NK-3 agonists (senktide and [MePhe7]neurokinin B) were active only at high concentrations. Phenoxybenzamine pretreatment reduced the maximal response to NK-1 agonists and produced a rightward shift of the curve to NK-2 agonists, without depression of the maximum. Five tachykinin antagonists selective for the NK-1 (L 668,169) or the NK-2 (MEN 10,207, MEN 10,376, L 659,877 and R 396) receptor were tested against substance P methylester and [beta-Ala8]neurokinin A-(4-10). The results indicated that these receptor-selective antagonists maintain their characteristic even when tested in a multireceptor assay such as the guinea-pig bronchus. The rank order of potency of NK-2 antagonists against [beta-Ala8]neurokinin A-(4-10) was MEN 10,207 = MEN 10,376 greater than L 659,877 much greater than R 396. This pattern, with the observation of the full agonist activity of MDL 28,564, indicates that in addition to NK-1 receptors, NK-2 receptors also are present in the guinea-pig bronchi and belong to the same subtype (NK-2A) as present in the rabbit pulmonary artery.