The ring size of monocyclic ET-1 controls selectivity and signaling efficiency at both endothelin receptor subtypes.

Cardiovascular diseases (CVDs) like hypertension are a major cause for death worldwide. In the cardiovascular tissue, the endothelin system-consisting of the receptor subtypes A (ETA R) and B (ETB R) and the mixed agonist endothelin 1 (ET-1)-is a major key player in the regulation of vascular tone and blood pressure. Tight control of this system is required to maintain homeostasis; otherwise, the endothelin system can cause severe CVDs like pulmonary artery hypertension. The high sequence homology between both receptor subtypes limits the development of novel and selective ligands. Identification of small differences in receptor-ligand interactions and determination of selectivity constraints are crucial to fine-tune ligand properties and subsequent signaling events. Here, we report on novel ET-1 analogs and their detailed pharmacological characterization. We generated simplified ET-1-derived monocyclic peptides to provide an accessible synthesis route. By detailed in vitro characterization, we demonstrated that both G protein signaling and the subsequent arrestin recruitment of activated ETB R remain intact, whereas activation of the ETA R depends on the intramolecular ring size. Increasing of the intramolecular ring structure reduces activity at the ETA R and shifts the peptide toward ETB R selectivity. All ET-1 analogs displayed efficient ETB R-mediated signaling by G protein activation and arrestin 3 recruitment. Our study provides in-depth characterization of the ET-1/ETA R and ET-1/ETB R interactions, which has the potential for future development of endothelin-based drugs for CVD treatment. By identification of Lys9 for selective labeling, novel analogs for peptide-mediated shuttling by ET-1 are proposed.

Chymase inhibitor-sensitive synthesis of endothelin-1 (1-31) by recombinant mouse mast cell protease 4 and human chymase.

Important structural differences imply that human and mouse mast cell chymases may differ with respect to their enzymatic properties. We compared in this study the catalytic efficiencies of recombinant human chymase (rCMA1) and its functional murine homologue recombinant mouse mast cell protease-4 (rmMCP-4) toward a fluorogenic chymase substrate (Suc-Ala-Ala-Pro-Phe-7-amino-4-methylcoumarin (AMC) and by their ability to convert Big-endothelin (ET)-1 into ET-1 (1-31) using a LC/MS/MS system. Activities toward a fluorogenic substrate (Suc-Leu-Leu-Val-Tyr-AMC) and Big ET-1 were also measured in extracts from mouse peritoneal mast cells, LUVA human mast cell-like cells and human aortas. The specificity of these activities was assessed with the chymase inhibitor TY-51469 (2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3-methanesulfonyl-phenyl]thiazole-4-carboxylic acid). For similar affinities, rmMCP-4 showed a higher activity toward the fluorogenic substrate and a higher ability to process Big ET-1 as compared to recombinant CMA1 (chymase activity (kcat/KM in µM(-1)s(-1)): 2.29 × 10(-4)vs. 6.41 × 10(-6); ET-1 (1-31) production: 2.19 × 10(-3)vs. 6.57 × 10(-5)), and both of these activities of mouse and human chymase were sensitive to TY-51469. Furthermore, extracts from mouse peritoneal mast cells, LUVA cells and human aorta homogenates contained processing activities toward the fluorogenic chymase substrate as well as Big ET-1, all of which were sensitive to TY-51469. Finally, the pressor responses to Big ET-1 but not to ET-1 were significantly reduced in conscious and free moving mMCP-4 KO mice when compared to wild type congeners. Our results suggest that both mouse and human chymases have potent ET-1 (1-31)-producing abilities, with the murine isoform being more efficient.

Biodistribution of a nonpeptidic fluorescent endothelin A receptor imaging probe.

Biodistribution studies are essential for understanding the biologic behavior of novel fluorochrome-based molecular imaging agents. In this study, the biodistribution of a recently developed fluorescent imaging probe with high affinity to the endothelin A (ET(A)) receptor was evaluated by fluorescence reflectance imaging (FRI). CD-1 mice were injected with 2 nmol of the probe intravenously and sacrificed at various time points. Tissue samples of the heart, spleen, lung, kidneys, liver, brain, and muscle were removed and imaged by FRI. Initially, the signal intensity (SI) was highest in lung, kidney, and liver tissue, followed by the heart, whereas spleen, muscle, and brain showed the lowest SI. In the kidneys, the SI decreased rapidly. In the heart, an initial SI increase was observed, followed by SI attenuation, whereas in the lung, the SI steadily increased. Competition experiments showed a significant (p < .005) degree of specific binding in the heart, with a reduction in SI of > 50%. In conclusion, FRI allows us to perform biodistribution studies of novel fluorescent tracers. The developed imaging probe can be exploited to image ET A receptor expression ideally 30 minutes to 3 hours after injection.

Effect of endothelin-1 (1-31) on the renal resistance vessels.

Human chymase produces not only angiotensin II but also endothelin(ET)-1(1-31). We previously reported that ET-1(1-31) had several biological activities in vascular smooth muscle cells. In this study, we investigated the vasoconstrictor effect of ET-1(1-31) on the renal resistance vessels using in vitro microperfused rabbit afferent and efferent arterioles. ET-1(1-31) decreased the lumen diameter of the afferent and efferent arterioles dose-dependently. ET-1(1-31)-induced afferent arteriolar vasoconstriction was not affected by phosphoramidon, an ET converting enzyme inhibitor. ET-1(1-31)-induced renal arteriolar vasoconstriction was inhibited by BQ123, an ETA receptor inhibitor, but not by BQ788, an ETB receptor inhibitor. These results suggest that ET-1(1-31)-induced renal arteriolar vasoconstriction may be mediated by ETA-like receptors.

The [Lys(-2)-Arg(-1)-des(17-21)]-endothelin-1 peptide retains the specific Arg(-1)-Asp8 salt bridge but reveals discrepancies between NMR data and molecular dynamics simulations.

The [des(17-21)]-endothelin-1 (CSH-ET) and [Lys(-)(2)-Arg(-)(1)-des(17-21)]-endothelin-1 (KR-CSH-ET) peptides, designed by removing the five-residue hydrophobic tail from the endothelin-1 (ET-1) and [Lys(-)(2)-Arg(-)(1)]-endothelin-1 (KR-ET-1) peptides, respectively, were synthesized. Previous studies on KR-ET-1 showed that, in contrast to ET-1, this engineered compound displays a pH-dependent conformational change related to the formation of a stabilizing salt bridge between the Arg(-)(1) and Asp(8) side chains. CD and NMR spectra indicate that CSH-ET and KR-CSH-ET display conformational behavior similar to those of ET-1 and KR-ET-1, respectively. The short salt bridge-stabilized KR-CSH-ET peptide therefore appears to be an attractive elementary scaffold for drug design. The solution structure of the salt-bridged form of KR-CSH-ET was determined by NMR at pH 4.5 and is very similar to the corresponding form of the parent KR-ET-1 peptide. Molecular dynamics simulations of the salt-bridged form of KR-CSH-ET were performed using both the GB/SA implicit solvation scheme or an explicit solvation and the particle-mesh Ewald method for long-range electrostatic calculation. Unexpectedly, the Arg(-)(1)-Asp(8) salt bridge does not display in the simulation the stability that could be expected from the experimental data. The cooperative involvement of a cation-pi interaction in formation of the salt bridge has been hypothesized. Difficulties in accurately simulating cation-pi interactions might be responsible for the lack of stability in the simulation. At this time, however, no definitive explanation for the observed discrepancy between experiments and simulations is available, and further experimental studies appear to be necessary to fully understand in atomic detail the pH-dependent conformational change observed in the KR-ET-1 series.

Hydrogen peroxide for disulfide bridge formation in methionine-containing peptides.

Two methionine-containing peptides, endothelin 1 and the 1-16 fragment of the receptor of the plasminogen activator 1 for human urokinase, were synthesized and cyclized by hydrogen peroxide. Endothelin 1 was obtained by using regioselective and random schemes of disulfide bond formation. The conditions of cyclization that provided the target products in high purity were found. The general potential of disulfide bond formation by means of hydrogen peroxide was demonstrated for methionine-containing peptides. The method resulted in target products containing insignificant quantities of the corresponding Met-sulfoxide derivatives.

[A novel synthetic approach of tryptophan-containing cystine peptides by regioselective disulfide bond-forming reaction using the silyl chloride-sulfoxide system].

A general scheme for the efficient synthesis of Trp-containing cystine peptide by the successive treatment with methyltrichlorosilane-diphenylsulfoxide in trifluoroacetic acid (TFA) and trifluoromethanesulfonic acid (TFMSA)-thioanisole in TFA, is described. A disulfide bond-forming reaction by silyl chloride-sulfoxide system is completed within 10-15 min without modifications at sensitive residues (Tyr, His, Met) in peptide chain, except for a Trp residue. In order to synthesize a Trp-containing cystine peptide using silyl chloride, the indole moiety of Trp has to be protected since the chlorination of the indole ring proceeded predominantly. A formyl group has been the only protecting group employed for this purpose in practical syntheses of cystine peptides, although it was clarified that a side reaction derived from the formyl group migration was inevitable in the synthesis of somatostatin. Firstly, we examined the application of the following Nin-protecting groups, mesitylene-2-sulfonyl (Mts), cyclohexyloxycarbonyl (Hoc), and 2,4-dimethylpent-3-yloxycarbonyl(Doc) for an efficient synthesis of the Trp-containing cystide peptide by the silyl chloride method. In order to find a feasible scheme of the successive treatment with CH3SiCl3-PhS(O)Ph/TFA and TFMSA-thianisole in TFA, we synthesized somatostatin using Trp(Mts), Trp(Hoc) or Trp(Doc) derivative. The Doc group was found to be the most suitable as an indole protecting group, since the protecting group was cleaved under mild conditions (4 degrees C, 30 min) via the corresponding Nin-carboxylic acid intermediate. We then applied the above procedure to the synthesis of endothelin-1 (ET-1), a peptide containing 21-amino acid residues having a C-terminal Trp residue and two disulfide bonds, by regioselective disulfide formation. The combination of the silyl chloride method with iodine oxidation using S-acetamidomethyl (Acm) and S-tBu groups for the regioselective double disulfide formation was successfully applied to give a highly purified ET-1. These results also show that the Nin-Doc group would be useful for the efficient syntheses of complex cystine-peptides by the silyl chloride method.

[Directed and spontaneous disulfide bond closure using hydrogen peroxide in the synthesis of endothelins-1 and -3]. / Napravlennoe i spontannoe zamykanie disul'fidnykh sviazei s pomoshch'iu perekisi vodoroda v sinteze éndotelinov-1 i -3.

The linear precursors of endothelin 1 and endothelin 3, natural vasoactive peptides, were obtained by using the Boc- and Fmoc-schemes of solid phase peptide synthesis. The methods of directed and spontaneous formation of two disulfide bonds in the molecules of these precursors were compared and shown to give comparable results. The conditions were found that provided the selective S-S-ring closure in the methionine-containing endothelin 1 by means of hydrogen peroxide without the undesired conversion of the Met residue into the corresponding sulfoxide.

Hybrid peptides constructed from RES-701-1, an endothelin B receptor antagonist, and endothelin; binding selectivity for endothelin receptors and their pharmacological activity.

Hybrid peptides were constructed from endothelin B receptor (ET(B)) selective antagonist RES-701-1 (1) and endothelin (ET-1). They have N-terminal 10 amino acids derived from 1 and C-terminal 10 amino acids derived from ET-1. RES-701-1(1-10)-[Ala15]ET-1(12-21) and its analogues substituted or truncated at the residues derived from RES-701-1 had proved to possess high receptor binding activity selective for ETB as well as 1. Substitutions at the residues derived from ET-1 had produced some analogues that possessed high affinity not only for ETB but for ETA. Although all analogues had antagonistic effects on ETA, some analogues had proved to function as agonist on ETB confirmed by the changes in intracellular calcium concentrations of ET receptor-transfected COS-7 cells. We have found four types of ET receptor-binding peptides: (1) ETB-selective agonist with weak ETA antagonism (3, KT7421); (2) ETB-selective antagonist with weak ETA antagonism (29, KT7539); (3) ETB agonist with potent ETA antagonism (27, KT7538); and (4) non-selective ETA/ETB antagonist (26, KT7540).