Eddie C.-K. Liu

The endothelin receptor antagonist, BQ-123, inhibits angiotensin II-induced contractions in rabbit aorta

The purpose of this study was to examine the specificity of the cyclic pentapeptide ET(A) receptor antagonist BQ-123. BQ-123 competitively antagonized endothelin-1-induced contractions in rabbit aorta, increases in inositol phosphates in cultured rat vascular smooth muscle A10 cells, and binding of [125I]endothelin-1 to the cloned ETA receptor cDNA expressed in Cos 7 cells. In contrast, BQ-123 was a weak antagonist of [125I]endothelin-3 binding to rat cerebellar membranes and to membranes from Cos 7 cells transfected with the cloned ETB receptor cDNA. BQ-123 shifted concentration-response curves in isolated rabbit aorta elicited by angiotensin II, but did not bind to angiotensin II receptors nor affect angiotensin II-induced increases in inositol phosphates. BQ-123 also did not affect contractions induced by KCl or norepinephrine. These data suggest that endothelin may play a role in angiotensin II-induced contractions of rabbit aorta.

DuP 753, the selective angiotensin II receptor blocker, is a competitive antagonist to human platelet thromboxane A2/prostaglandin H2 (TP) receptors

DuP 753 is a potent, selective angiotensin II type 1 (AT1) receptor antagonist. The possibility was investigated that DuP 753 may crossreact with thromboxane A2/prostaglandin H2 (TP) receptors. DuP 753 inhibited the specific binding of the TP receptor antagonist [3H]SQ 29,548 (5 nM) in human platelets with kd/slope factor values of 9.6 +/- 1.4 microM/1.1 +/- 0.02. The AT2-selective angiotensin receptor ligand, PD 123,177 was a very weak inhibitor of specific [3H]SQ 29,548 binding in platelets (Kd/slope factor:200 microM/0.86). [3H]SQ 29,548 saturation binding in the absence and presence of DuP 753 resulted in an increase in equilibrium affinity constant (Kd: 9.3, 22, 33 nM, respectively) without a concentration-dependent reduction in binding site maxima (Bmax: 3597, 4597, 3109 fmol/mg protein, respectively). Platelet aggregation induced by the TP receptor agonist U 46,619 was concentration-dependently inhibited by DuP 753 (IC50 = 46 microM). These data indicate for the first time that DuP 753 is a weak but competitive antagonist at human platelet TP receptors.

Aspartate mutation distinguishes ETA but not ETB receptor subtype-selective ligand binding while abolishing phospholipase C activation in both receptors

The endothelin receptors, ETA and ETB, are G protein‐coupled receptors (GPCR) that show distinctively different binding profiles for the endothelin peptides and other ligands. We recently reported that Tyr129 in the second transmembrane region (TM2) of the ETA receptor was critical for subtype‐specific ligand binding [Krystek, S.R. et al. (1994) J. Biol. Chem. 269, 12383–12386]. Receptor models indicated that aspartic acids located one helical turn above (Asp133) and below (Asp126) Tyr129 in ETA had their side chains directed toward the putative binding cavity. Similarly in ETB, Asp147 and Asp154 are located one turn below and above His150, the residue that corresponds to Tyr129. Asp126 in ETA and Asp147 in ETB corresponds to the highly conserved aspartate present in TM2 of many GPCR that has frequently been shown to be crucial for agonist efficacy. Mutagenesis of Asp126 of the human ETA receptor to alanine resulted in an unaltered affinity for ET‐1, a 160‐fold increase in ET‐3 affinity and a decrease in affinity for the ETA selective naphthalenesulfonamide, BMS‐182874. ET‐1 activation of phospholipase C was abolished. In addition, despite the gain in binding affinity, ET‐3 failed to activate phospholipase C, suggesting that Asp126 is required for signal transduction. Mutagenesis of Asp133 to alanine indicated that it was critical only for the binding of BMS‐182874. In the ETB receptor, mutation of His150 to alanine or tyrosine indicated that it plays a minor role in ETB subtype‐selective ligand binding; mutation of the aspartates in TM2 of ETB did not alter ligand binding. As in the Asp126Ala ETA variant, ET‐1 and ET‐3 failed to increase intracellular levels of inositol phosphates in the Asp147Ala ETB mutant. Taken together, these data support the hypothesis that Asp126 and Asp133 flanking Tyr129 in TM2 of the ETA receptor play a role in defining ETA subtype‐selective ligand binding but Asp147 and Asp154 that flank the His150 in TM2 of the ETB receptor do not. Furthermore, these data indicate that Asp126 in ETA and Asp147 in ETB are important for transmembrane signaling via phospholipase C.

Structure-activity relationships of monocyclic endothelin analogs

Abstract Analogs of monocyclic ET-1 (Ala3,11, Nl7) containing substitutions were prepared and assayed for vasoconstrictor and receptor binding activity. Analogs substituted at residues Glu10, Phe14, Leu17 and Asp18 showed significant affinity for the receptor at concentrations below those at which agonism was observed. To the extent that structure-activity relationships of monocyclic and bicyclic ETs can be compared, this study suggests that residues 10, 14, 17 and 18 are candidate sites for exploration of peptides which show ET receptor antagonism.

Conformationally Constrained ortho-Anilino Diaryl Ureas: Discovery of 1-(2-(1′-Neopentylspiro[indoline-3,4′-piperidine]-1-yl)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea, a Potent, Selective, and Bioavailable P2Y1 Antagonist

Preclinical antithrombotic efficacy and bleeding models have demonstrated that P2Y1 antagonists are efficacious as antiplatelet agents and may offer a safety advantage over P2Y12 antagonists in terms of reduced bleeding liabilities. In this article, we describe the structural modification of the tert-butyl phenoxy portion of lead compound 1 and the subsequent discovery of a novel series of conformationally constrained ortho-anilino diaryl ureas. In particular, spiropiperidine indoline-substituted diaryl ureas are described as potent, orally bioavailable small-molecule P2Y1 antagonists with improved activity in functional assays and improved oral bioavailability in rats. Homology modeling and rat PK/PD studies on benchmark compound 3l will also be presented. Compound 3l was our first P2Y1 antagonist to demonstrate a robust oral antithrombotic effect with mild bleeding liability in the rat thrombosis and hemostasis models.

Comparison of a Novel ETA Receptor Antagonist and Phosphoramidon in Renal Ischemia

Infusion (0.46 mumol/kg/min) of the endothelin (ET)-converting-enzyme inhibitor, phosphoramidon (P), protected function and structure after 30 min renal ischemia in rats more than treatment (5 mumol/kg/min) with the ETA receptor antagonist, BMS-182874 (B). The glomerular filtration rate (GFR; 0.7 +/- 0.12 ml/min) and renal plasma flow (RPF) decreased approximately 40% at 2 h reflow versus controls (C: 1.2 +/- 0.12). B weakly protected the GFR (0.8 +/- 0.07 ml/min); P restored it (1.1 +/- 0.05). Both compounds reduced tubular injury at 2 h reflow; P ameliorated glomerular changes. At 24 h the GFR (0.6 +/- 0.06 ml/min) and RPF decreased 67% versus C (1.8 +/- 0.08). B did not protect the GFR and RPF. P partially protected the GFR (0.9 +/- 0.07 ml/min) but not RPF, and reduced tubular injury. The results suggest that both ETA and non-ETA receptors mediate ET-induced changes in ischemic renal failure.

Design, structure-activity relationships, X-ray crystal structure, and energetic contributions of a critical P1 pharmacophore: 3-chloroindole-7-yl-based factor Xa inhibitors.

An indole-based P1 moiety was incorporated into a previously established factor Xa inhibitor series. The indole group was designed to hydrogen-bond with the carbonyl of Gly218, while its 3-methyl or 3-chloro substituent was intended to interact with Tyr228. These interactions were subsequently observed in the X-ray crystal structure of compound 18. SAR studies led to the identification of compound 20 as the most potent FXa inhibitor in this series (IC(50) = 2.4 nM, EC(2xPT) = 1.2 microM). An in-depth energetic analysis suggests that the increased binding energy of 3-chloroindole-versus 3-methylindole-containing compounds in this series is due primarily to (a) the more hydrophobic nature of chloro- versus methyl-containing compounds and (b) an increased interaction of 3-chloroindole versus 3-methylindole with Gly218 backbone. The stronger hydrophobicity of chloro- versus methyl-substituted aromatics may partly explain the general preference for chloro- versus methyl-substituted P1 groups in FXa, which extends beyond the current series.

Role of endothelin receptor subtype B(ET-B) in myocardial ischemia

Previous work indicated that endothelin (ET) may be involved in the pathogenesis of myocardial ischemia, although the relative importance of the ET receptor subtypes is presently not clear. The purpose of this study was to determine the role of myocardial ET-B receptors in mediating ischemic/reperfusion damage in isolated rat hearts. Saturation binding analyses were conducted with [125I]ET-1 and [125I]IRL-1620 to assess changes in ET-A and ET-B receptor binding. Total ET receptor density (Bmax) was greater in atrial versus ventricular tissue. ET-A Bmax was 8 to 10-fold greater than ET-B Bmax. In ischemic and ischemic/reperfused atrial tissue neither the equilibrium dissociation constant (Kd) nor Bmax for ET-B receptors was changed. The ET-B receptor Kd in ischemic or ischemic/reperfused ventricular tissue was also unchanged. In ischemic ventricular tissue there was a trend towards an increased ET-B Bmax, which was accentuated after ischemia/reperfusion. No changes were found in ET-A Bmax or Kd in ischemic ventricular or atrial tissue. The physiological importance of this receptor subtype in ischemic myocardium was determined using the selective ET-B agonist, sarafotoxin S6c. In non-ischemic tissue no effect on coronary flow or function were observed with sarafotoxin S6c. Furthermore, no changes were seen in ischemic time to contracture or any of the reperfusion indexes of myocardial damage. The sarafotoxin S6c utilized was active as it inhibited [125I]ET-3 binding to ET-B receptors (Ki = 0.1 nM). Thus, the pro-ischemic effect of ET-1 seems to be mediated by ET-A receptors. ET-B receptors do not appear to play a role in the pathogenesis of myocardial ischemia.

VASCULAR A10 CELL MEMBRANES CONTAIN AN ENDOTHELIN METABOLIZING NEUTRAL ENDOPEPTIDASE

We have investigated the possible presence of endothelin-metabolizing neutral endopeptidase (NEP, EC 3.4.24.11) on A10 cell membranes using [125I]-ET-1 binding and direct measurements of NEP. NEP activity of A10 cell membranes has been compared to that of solubilized rat kidney brush border membranes (KNEP). Specific [125I]-ET-1 (50 pM) binding (defined with 100 nM ET-1) to A10 cell membranes was increased in a concentration dependent manner by the selective NEP inhibitors thiorphan, phosphoramidon, and SQ 28,603 [(+/-)-N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-beta-alanine] with EC50 values of 9.4, 28.4, and 5.7 nM respectively. At equilibrium (150 min), 70% more specific binding was apparent in the presence of these inhibitors. Phosphoramidon (2 microM) did not alter Bmax values, but it decreased the apparent KD for [125I] ET-1 from 63 (+/- 3) to 27 (+/- 2) pM. Thiorphan, phosphoramidon, and SQ 28,603 inhibited A10 cell NEP activity with IC50 values of 5.3, 36.5, and 6.0 nM respectively, which was similar to values obtained with KNEP (3.6, 22.6, and 3.5 nM). ET-1 inhibited A10 cell NEP, and KNEP with IC50 values of 30 and 21.3 microM respectively. The order of inhibitory potencies: ET-3 greater than ET-1 = ET-2 greater than or equal to sarafotoxin-6b was similar for both systems. These data suggest A10 cell membranes contain a NEP which has similar characteristics to NEP 24.11, and which actively metabolizes [125I]-ET-1.

Dissecting the Activation of Thienopyridines by Cytochromes P450 Using a Pharmacodynamic Assay In Vitro

The thienopyridine antiplatelet drugs, such as ticlopidine, clopidogrel, and prasugrel, require activation by cytochromes P450 in vivo to effectively block platelet aggregation. The study of the metabolic activation of these compounds has been hampered by the lability and reactivity of the ring-opened active metabolite (AM) and by the numerous metabolites that can be formed in such a transformation. We have developed a novel method whereby platelets are incubated with the cytochrome P450 and the thienopyridine of interest for various amounts of time, and the effects on ADP-driven platelet aggregation are directly examined. In this way, the platelet is used as a biosensor for detection of the AM. Using this method, cytochromes P450 capable of converting clopidogrel, prasugrel, and 2-oxo-clopidogrel to metabolites that inhibit ADP-induced platelet aggregation were identified as well as which cytochromes P450 were capable of catalyzing partial reactions (e.g., conversion of 2-oxo-clopidogrel to the AM). These studies show that, in vitro, CYP3A4/5, 2C19, and 2B6 are individually capable of converting clopidogrel and prasugrel to the AM and that the cytochrome P450 preference for these two thienopyridines is very similar.