Mitsuo Yano

Biological profiles of highly potent novel endothelin antagonists selective for the ETA receptor

We describe novel potent endothelin (ET) antagonists that are highly potent and selective for the ETA receptor (selective to ET-1). Of the synthetic analogs based on ETA antagonist BE-18257A isolated from Streptomyces misakiensis (IC50 value for ETA receptor on porcine aortic smooth muscle cells (VSMCs); 1.4 microM), the compounds BQ-123 and BQ-153 greatly improved the binding affinity of [125I]ET-1 for ETA receptors on VSMCs (IC50; 7.3 and 8.6 nM, respectively), whereas they barely inhibited [125I]ET-1 binding to ETB receptors (nonselective with respect to isopeptides of ET family) in the cerebellar membranes (IC50; 18 and 54 microM, respectively). Associated with the increased affinity for ETA receptors, these peptides antagonized ET-1-induced constriction of isolated porcine coronary artery. However, there was a small amount of ET-1-induced vasoconstriction resistant to these antagonists, which paralleled the incomplete inhibition of [125I]ET-1 binding in the membrane of the aortic smooth muscle layer. These data suggest that the artery has both ETA and ETB receptors responsible for ET-1-induced vasoconstriction. The antagonists shifted the concentration-response curve to the right for ET-1 in the coronary artery, and increased the apparent dissociation constant in the Scatchard analysis of [125I]ET-1 binding on the VSMCs without affecting the binding capacity, indicative of the competitive antagonism for ETA receptor. In conscious rats, pretreatment with the antagonists markedly antagonized ET-1-induced sustained pressor responses in dose-dependent fashion without affecting ET-1-induced transient depressor action, suggesting that the pressor action is mediated by ETA receptors, while the depressor action is mediated by ETB receptors. In addition, pretreatment with the potent antagonists prevented ET-1-induced sudden death in mice. Thus, these potent ETA antagonists should provide a powerful tool for exploring the therapeutic uses of ETA antagonists in putative ET-1-related disorders.

An endothelin receptor (ETA) antagonist isolated from Streptomyces misakiensis.

A competitive endothelin (ET) antagonist, BE-18257B, was isolated from the fermentation products of Streptomyces misakiensis. It is a novel cyclic pentapeptide, cyclo(-D-Glu-L-Ala-allo-D-Ile-L-Leu-D-Trp-), and binds to ETA receptors (ET-1 selective) in cardiovascular tissues, but not to ETB receptors (equally sensitive to isopeptides of ET family) in kidney, adrenal gland and cerebellum tissues. BE-18257B also antagonizes ET-1-induced vasoconstriction in rabbit iliac artery and pressor action in rats. Thus it is a selective ETA antagonist and should provide a valuable tool for elucidation of the pharmacological and pathophysiological roles of ET-1.

Inhibition of biological actions of big endothelin-1 by phosphoramidon

Endothelin (ET)-1 and big ET-1 both caused contraction of isolated porcine coronary arteries, but the potency of big ET-1 was 1/100-1/200 that of ET-1. These responses were independent of the vascular endothelium. Phosphoramidon blocked the vasoconstriction caused by 30 nM big ET-1, but was ineffective on the action of 0.3 nM ET-1. Also in vivo, phosphoramidon had no effect on the ET-1-induced pressor actions, but blocked the pressor and airway-contractile responses to big ET-1 in rats and/or guinea pigs. Thus, it is likely that the vascular responses to exogenous big ET-1 are at least in part due to its conversion to ET-1 by a phosphoramidon-sensitive ET converting enzyme(s) in the vascular smooth muscle in vitro and in vivo.

[Ala1,3,11,15]endothelin-1 analogs with ETB agonistic activity

A linear peptide analog of endothelin (ET)-1, [Ala1,3,11,15]ET-1 (4AlaET-1), and its truncated peptide analogs were synthesized to study the structural requirements of ET-1 for the recognition of ETs-nonselective ETB receptors. ET-1 exhibited sub-nanomolar binding to two distinct ET receptor subtypes (ETA and ETB), but 4AlaET-1 bound to ETB with an affinity 1,700 times higher than that seen during binding to ETA. The truncated linear peptides 4AlaET-1(6-21), 4AlaET-1(8-21) and N-acetyl-4AlaET-1(10-21) still had high affinity for ETB, whereas 4AlaET-1(6-20) and 4AlaET-1(11-21) displayed remarkably reduced affinity for ETB. Therefore, ET-1 requires the Glu10-Trp21 sequence for ETB binding, but not the disulfide bridges. These ETB-binding peptides elicit endothelium-dependent vasorelaxation of porcine pulmonary arteries in parallel with the binding affinity for ETB, suggesting that they are ETB agonists.

In vitro biological profile of a highly potent novel endothelin (ET) antagonist BQ-123 selective for the ETA receptor

Summary: The novel endothelin (ET) receptor antagonists BE‐18257A and BE‐18257B were isolated from the fermentation products of Streptomyces misakiensis. The above‐mentioned compounds inhibited [125I]ET‐1 binding to ETA receptors (selective for ET‐1) on porcine aortic vascular smooth muscle cells (VSMCs) with IC50 values of 1.4 and 0.47 μM, respectively. [125I]ET‐1 binding to ETB receptors (nonselective to ET isopeptides) in cerebellar membranes was not inhibited by either of these compounds even at 100 μM. The synthesized analogue BQ‐123 induced extremely potent inhibition of [125I]ET‐1 binding to ETA receptors (IC50 of 7.3 nM), but it barely inhibited [125I]ET‐1 binding to ETB receptors (IC50 of 18 μM) and binding of various other peptides to their receptors. BQ‐123 shifted the concentration‐response curve for ET‐1 toward the right in porcine isolated coronary arteries, indicative of competitive antagonism for the ETA receptor. However, there was a small amount of BQ‐123‐insensitive vasocontraction that paralleled the incomplete inhibition of [125I]ET‐1 binding in the membrane of the vascular smooth muscle layer. These data suggest that the artery contracts via both ETA and ETB receptors and that BQ‐123 selectively inhibits ETA‐mediated contraction. Furthermore, BQ‐123 revealed large tissue and species differences in the distribution of ETA receptors. Thus, the potent ETA antagonist BQ‐123 should be useful in clarifying the (patho)physiological roles of ETA receptors.

Antihypertensive effect of a newly synthesized endothelin antagonist, BQ-123, in a genetic hypertensive model

A newly synthesized ET(A)-selective antagonist, BQ-123, was examined with respect to its anti-endothelin(ET) action in vitro and in vivo and its effect on blood pressure in Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR) and stroke-prone spontaneously hypertensive rats (SHRSP). In isolated porcine coronary arteries, BQ-123 (0.07 microM to 6.0 microM) shifted the concentration-response curve for ET-1 to the right without affecting the maximal response of ET-1, its pA2 value being 7.35. Intravenous infusion of BQ-123 at a rate of 1.2 and 30 mg/kg/hr produced a significant decrease in blood pressure in 20- to 29-week-old SHRSP, but did not alter blood pressure in 13- to 16-week-old WKY or in 18- to 19-week-old and 40-week-old SHR. The hypotensive effect of BQ-123 depended on the pretreatment blood pressure level. These results suggest that ET-1 is involved in part in the maintenance of high blood pressure in malignant hypertension, as exemplified by SHRSP.

Synergistic inhibition by BQ-123 and BQ-788 of endothelin-1-induced contractions of the rabbit pulmonary artery.

In the rabbit isolated pulmonary artery, neither the ETA receptor antagonist, BQ‐123 (10 μm), nor the ETB receptor antagonist, BQ‐788 (10 μm), inhibited the contractions induced by 1 nm endothelin‐1 (ET‐1). However, the combination of BQ‐123 and BQ‐788 completely inhibited the ET‐1‐induced contraction. In contrast, the ETB‐selective agonist, sarafotoxin S6c (1 nm)‐induced contraction was completely inhibited by BQ‐788 but not by BQ‐123. In receptor binding assays, [125I]‐ET‐1 specific binding to pulmonary arterial membranes was inhibited by BQ‐123 (1 μm) by approximately 20% and additive treatment with BQ‐788 (1 μm) completely inhibited the BQ‐123‐resistant component of [125I]‐ET‐1 specific binding. The present study demonstrates synergistic inhibition by BQ‐123 and BQ‐788 of ET‐1‐induced contraction of the rabbit pulmonary artery and the coexistence of ETA and ETB receptors, suggesting that the activation of either only ETA or only ETB receptors may be sufficient to cause complete vasoconstriction. Therefore, blockade of both receptor subtypes would be necessary for the inhibition of some ETA/ETB composite types of responses.

A novel radioligand [125I]BQ-3020 selective for endothelin (ETB) receptors

A linear endothelin (ET) analog, N-acetyl-LeuMetAspLysGluAlaValTyrPheAlaHisLeu-AspIleIleTrp (BQ-3020), is highly selective for ETB receptors. BQ-3020 displaces [125I]ET-1 binding to ETB receptors (nonselective to ET isopeptides) in porcine cerebellar membranes (IC50: 0.2nM) at a concentration 4,700 times lower than that to ETA receptors (selective to ET-1) on aortic vascular smooth muscle cells (VSMC) (IC50: 940nM). BQ-3020 as well as ET-1 and ET-3 elicits vasoconstriction in the rabbit pulmonary artery. The ETA antagonist BQ-123 failed to inhibit this BQ-3020-induced vasoconstriction. Furthermore, BQ-3020 elicits endothelium-dependent vasodilation. These data indicate that BQ-3020 has ETB agonistic activity. The radioligand [125I]BQ-3020 binds to cerebellar membranes at single high affinity sites (Kd = 34.4pM), whereas it scarcely binds to VSMC. [125I]BQ-3020 binding to the cerebellum was displaced by BQ-3020, ET-1 and ET-3 in a nonselective manner (IC50: 0.07-0.17nM). However, the binding of [125I]BQ-3020 was insensitive to the ETA antagonist BQ-123 and other bioactive peptides. Both [125I]ET-1 and [125I]BQ-3020 show slow onset and offset binding kinetics to ETB receptors. These data indicate that the radioligand [125I]BQ-3020 selectively labels ETB receptors and that the slow binding kinetics of ET-1 are dependent on the peptide sequence from Leu6 to Trp21, but not on the structure formed by its two disulfide bridges.

Different distribution of endothelin receptor subtypes in pulmonary tissues revealed by the novel selective ligands BQ-123 and [Ala1,3,11,15]ET-1

We have demonstrated the different distribution of two distinct endothelin (ET) receptor subtypes in porcine pulmonary tissues using a radioligand binding assay. The clear differentiation of the subtypes was made possible by the discovery of two compounds, BQ-123 and [Ala1,3,11,15]ET-1 (4AlaET-1), that are highly selective for ETA and ETB receptors, respectively. In the bronchus and lung parenchyma, BQ-123 inhibited 65% and 30% of [125I]ET-1 binding on the sensitive sites, while 4AlaET-1 displaced 25% and 60%, respectively. The combination of the two compounds completely inhibited ET-1 binding in both tissues. An autoradiographic study of [125I]ET-1 binding using BQ-123 and 4AlaET-1 also supported the different localization of two ET receptor subtypes in pulmonary tissues. In particular, the blood vessels and bronchi are rich in ETA, but the lung parenchyma is rich in ETB.

Necessity of dual blockade of endothelin ETA and ETB receptor subtypes for antagonism of endothelin-1-induced contraction in human bronchi

1 Endothelin (ET)‐1 has been postulated to be involved in the development of obstructive airway diseases in man. In the present study, we attempted to characterize ET receptor subtypes mediating ET‐1‐induced contraction in human isolated bronchi. The ET receptor antagonists used in the present study were BQ‐123 (ETA receptor‐selective), BQ‐788 (ETB receptor‐selective) and BQ‐928 (ETA/ETB dual). Sarafotoxin S6c (S6c) was also used as an ETB receptor‐selective agonist. 2 In human bronchi, ET‐1 and S6c (10−12 m to 10−7 M) produced concentration‐dependent contraction with almost equal potency (pD2: 8.88± 0.16 for ET‐1 and 9.42±0.15 for S6c). The contraction induced by S6c was competitively antagonized by BQ‐788 alone (1 and 10 μm) with a pKB value of 7.49±0.21, suggesting that the stimulation of ETB receptors causes a contraction of human bronchi. However, contrary to expectation, the concentration‐response curves for ET‐1 were not affected by BQ‐788. The ET‐1‐ and S6c‐induced contractions were not affected by BQ‐123 (10 μm). Thus, ET‐1‐induced contraction of human bronchi is not antagonized by BQ‐123 alone or by BQ‐788 alone. 3 Combined treatment with 10 μm BQ‐123 and 10 μm BQ‐788 significantly antagonized the contraction induced by ET‐1 with a dose‐ratio of 11. BQ‐928 also significantly antagonized ET‐1‐induced contraction with a pKB value of 6.32±0.24. 4The specific binding of [125I]‐ET‐1 to human bronchial membrane preparations was inhibited by BQ‐123 (100 pM to 1 μm) by approximately 40%. Combination treatment with BQ‐788 (100 pM to 1 μm) completely inhibited the BQ‐123‐resistant component of [125I]‐ET‐1 specific binding. 5 In conclusion, the present study demonstrates that BQ‐788 alone cannot inhibit ET‐1‐induced contractions in human bronchi, although human bronchial ETB receptors are BQ‐788‐sensitive. Furthermore, it was shown that blockade of both receptor subtypes antagonizes ET‐1‐induced contraction, and that both receptor subtypes co‐exist in human bronchial smooth muscles. These findings suggest that ETA receptors as well as ETB receptors are involved in ET‐1‐induced contraction in human bronchi. If ET‐1 is involved in human airway diseases, dual blockade of ETA and ETB receptors may be necessary to treat the diseases.