Expression of endothelin 1 and endothelin A receptor in ovarian carcinoma: evidence for an autocrine role in tumor growth.

In the present study, we have investigated the expression of endothelin 1 (ET-1) and the ET(A) receptor (ET(A)R) and ET(B) receptor (ET(B)R) in primary (n = 30) and metastatic (n = 8) ovarian carcinomas and their involvement in tumor growth. By reverse transcription-PCR and Northern blot analysis, we detected ET-1 mRNA in 90% of primary and 100% of metastatic ovarian carcinomas. ET-1 mRNA expression was significantly higher in tumors than in normal ovarian tissues (n = 12; P < 0.01). ET(A)R mRNA was also detected in 84% of the carcinomas examined, whereas ET(B)R mRNA was expressed in 50% of the tumors. The in vivo presence of mature ET-1 and ET(A)R was confirmed by immunohistochemistry, demonstrating a higher expression in primary and metastatic cells. Ten primary cultures of ovarian tumors secreted ET-1 and were positive for ET-1 and ET(A)R mRNA, whereas only 40% expressed ET(B)R mRNA. Radioligand binding studies showed that ET-1-producing cells also expressed functional ET(A)R, whereas no specific ET(B)R could be demonstrated. ET-1 stimulated dose-dependent [3H]thymidine incorporation and enhanced the mitogenic effect of epidermal growth factor. The ET(A)R-selective antagonist BQ 123 strongly inhibited ET-1-stimulated growth and substantially reduced the basal growth rate of unstimulated cells, whereas the ET(B)R-selective antagonist BQ 788 had no effect. In conclusion, the present data demonstrate a novel mechanism in the growth control of ovarian carcinoma in vivo mediated by the ET-1 autocrine loop that selectively occurs via the ET(A)R.

Identification and characterization of A-105972, an antineoplastic agent.

A high-throughput screening assay was designed to select compounds that inhibit the growth of cultured mammalian cells. After screening more than 60,000 compounds, A-105972 was identified and selected for further testing. A-105972 is a small molecule that inhibits the growth of breast, central nervous system, colon, liver, lung, and prostate cancer cell lines, including multidrug-resistant cells. The cytotoxic IC50 values of A-105972 were between 20 and 200 nM, depending on the specific cell type. The potency of A-105972 is similar in cells expressing wild-type or mutant p53. A majority of cells treated with A-105972 were trapped in the G2-M phases, suggesting that A-105972 inhibits the progression of the cell cycle. Using [3H]A-105972, we found that A-105972 bound to purified tubulin. Unlabeled A-105972 competed with [3H]A-105972 binding with an IC50 value of 3.6 microL. Colchicine partially inhibited [3H]A-105972 binding with an IC50 value of approximately 90 microM, whereas paclitaxel and vinblastine had no significant effect. Tumor cells treated with A-105972 were observed to contain abnormal microtubule arrangement and apoptotic bodies. DNA ladder studies also indicated that A-105972 induced apoptosis. A-105972 caused a mobility shift of bcl-2 on SDS-PAGE, suggesting that A-105972 induced bcl-2 phosphorylation. A-105972 treatment increased the life span of mice inoculated with B16 melanoma, P388 leukemia, and Adriamycin-resistant P388. These results suggest that A-105972 is a small molecule that interacts with microtubules, arrests cells in G2-M phases, and induces apoptosis in both multidrug resistance-negative and multidrug resistance-positive cancer cells. A-105972 and its analogues may be useful for treating cell proliferative disorders such as cancer.

A-204197, a new tubulin-binding agent with antimitotic activity in tumor cell lines resistant to known microtubule inhibitors.

Drug resistance is a prevalent problem in the treatment of neoplastic disease, and the effectiveness of many clinically useful drugs is limited by the fact that they are substrates for the efflux pump, P-glycoprotein. Because there is a need for new compounds that are effective in treating drug-resistant tumors, we tested A-204197 (4-[4-acetyl-4,5-dihydro-5-(3,4,5-trimethoxyphenyl)-1,3,4-oxadiazol-2-yl]-N,N-dimethylbenzeneamine), a novel oxadiazoline derivative with antiproliferative properties, on cell lines that were either sensitive or resistant to known microtubule inhibitors. Cell lines that were resistant to paclitaxel, vinblastine, or colchicine were equally sensitive to A-204197 (proliferation IC50s ranging from 36 to 48 nM) despite their expression levels of P-glycoprotein. The effect of A-204197 on cell growth was associated with cell cycle arrest in G2-M, increased phosphorylation of select G2-M checkpoint proteins, and apoptosis. In competition-binding assays, A-204197 competed with [3H]-labeled colchicine for binding to tubulin (K(i) = 0.75 microM); however, it did not compete with [3H]-labeled paclitaxel. A-204197 prevented tubulin polymerization in a dose-dependent manner (IC50 = 4.5 microM) in vitro and depolymerized microtubules in a time-dependent manner in cultured cells. These findings indicate A-204197 is a promising new tubulin-binding compound with antimitotic activity that has potential for treating neoplastic diseases with greater efficacy than currently used antimitotic agents.

SV40 virus transformation down-regulates endothelin receptor.

Simian virus 40 (SV40) is an oncogenic DNA virus that induces malignant transformation. Endothelin (ET), a 21 amino acid peptide with mitogenic and anti-apoptotic effects, binds to G-protein coupled ETA and ETB receptors. This report examines the effect of SV40 transformation on the expression of ET receptors. Results from receptor binding and reverse transcription (RT)-polymerase chain reaction (PCR) studies show that human lung fibroblasts IMR90 and WI38 express both ETA and ETB receptors, and that the expression of both receptors is significantly down-regulated in IMR90-SV40 and WI38-SV40, cell lines derived from IMR90 and WI38 with SV40 virus transformation. Receptor binding and RT-PCR analysis of 3A(tPA-30-1), a cell line derived from human placenta that expresses a higher level of SV40 large T-antigen at the permissive temperature (33 degrees C) than at the restrictive temperature (40 degrees C), further demonstrates that there is an inverse correlation between the expression of SV40 T-antigen and the expression of ET receptor. ET-1 and fetal bovine serum stimulate DNA synthesis in non-transformed cells; however, proliferation of transformed cells is independent of either fetal bovine serum or ET-1. We conclude that SV40 transformation down-regulates the expression of ET receptors, and that expression of ET receptors is inversely correlated with expression of SV40 large T-antigen.

Endothelin receptor agonists and antagonists exhibit different dissociation characteristics.

Endothelins (ETs) are vasoconstricting peptides that bind to membrane receptors to initiate their physiological effects. This report compares the dissociation characteristics of selected ET agonists and antagonists, and studies the effects of any difference in dissociation characteristics on the potency of antagonists. Competition studies using various ET receptor ligands against [125I]ET-1 or [125I]ET-3 binding demonstrated that porcine cerebellum membranes contain predominantly ETB receptor. [125I]IRL1620 associated with the receptors in a time-dependent manner. Although bound [125I]IRL1620 was easier to dissociate than bound [125I]ET-3, both agonists exhibited a dissociation half life > 20 h. For non-radiolabeled ligands, bind-and-wash studies were employed in which membranes were pre-incubated with unlabeled ligand followed by extensive washing before assaying for [125I]ET-1 binding. Results from bind-and-wash studies confirmed that bound non-radiolabeled IRL1620 and ET were as difficult to dissociate as [125I]ligands. In contrast, bound PD142893 and Ro46-2005 were easily dissociated from ETB receptors. Consequently, the inhibitory effects of PD142893 and Ro46-2005 on [125I]agonist binding diminished following incubation time. In cloned human ETA and ETB receptors, bound ET-1 was also more difficult to dissociate than bound antagonists. These results suggest that the differences in the dissociation characteristics of ET receptor agonists vs. antagonists may account for the diminished potency of Ro46-2005 and PD142893 as a function of incubation time.

Human astrocytoma U138MG cells express predominantly type-A endothelin receptor.

Endothelin-1 (ET-1) binding to human astrocytoma U138MG cells was time-dependent, and bound [125I]ET-1 was difficult to dissociate. The B(max) and Kd values of [125I]ET-1 binding were 70 fmol/mg and 0.07 nM, respectively. Interestingly, different from other astrocytoma cells and astrocytes, the U138MG cells expressed predominantly ETA receptor as shown by RT-PCR results and binding studies. ET-1, FR139317, BQ123, PD142893 and Ro46-2005 inhibited specific [125I]ET-1 binding with Ki values of 0.10, 0.53, 4.3, 22, and 320 nM, respectively. ETB selective ligands ET-3 and IRL1620 were much less potent. The inhibitory effects of antagonists BQ123 and PD142893 on [125I]ET-1 binding diminished following the incubation time. ET-1 binding caused a modest stimulation in phosphatidylinositol hydrolysis with an EC50 value of 24 nM. In comparison to the human U373MG cells, ET-1-induced receptor internalization in U138MG cells was less efficient with 42% of bound ET-1 internalized after 30 min of incubation. These results imply that human astrocytoma cells/astrocytes are able to express either ETA or ETB receptor under different pathophysiological conditions.

Azole endothelin antagonists. 1. A receptor model explains an unusual structure-activity profile.

The pseudotetrapeptide FR-139317 is a potent and highly selective antagonist of the endothelin-A (ET(A)) receptor; however, its peptidic nature leads to poor oral absorption characteristics which make it an unlikely drug candidate. In an attempt to improve these properties, we have replaced a portion of the amide bond framework of FR-139317 with a heterocyclic surrogate. The resultant analogs are also ET(A)-selective antagonists, but show a structure-activity profile substantially different from that of the peptidic series, particularly with regard to the requirements for the side chain group that has been incorporated into the heterocycle. The nature of the heterocycle itself also has profound effects on the activity of the compounds. Both of these surprising results can be rationalized through examination of a 3D model of ET ligand--receptor binding that has previously been developed in our laboratories.

Azole endothelin antagonists. 2. Structure-activity studies.

Structure-activity studies have been performed in an attempt to improve the potency of a novel series of azole-based endothelin-A (ET(A)) selective antagonists. Modifications of the hydrophobic group on the terminal urea produced substantial effects on receptor affinity; in particular, the choice of cyclohexyl- or arylureas led to substantial improvements in activity. Conformational restriction of these groups provides an additional benefit. N-Methylation of the indole moiety which is part of the heterocyclic dipeptide surrogate also improves potency. The effects of these two modifications appear to be synergistic, with the best of the resultant doubly modified analogs (e.g. 14q, 15y, and 15ff) exhibiting an 80-200-fold improvement over the original leads.

Potent and selective inhibitors of an aspartyl protease-like endothelin converting enzyme identified in rat lung.

Two structurally distinct series of potent and selective inhibitors of an aspartyl protease-like endothelin converting enzyme (ECE) activity identified in the rat lung have been developed. Pepstatin A, which potently inhibits the rat lung ECE, served as the basis for the first series. Alternatively, selected renin inhibitors containing the dihydroxyethylene moiety were shown to be inhibitors of rat lung activity. Subsequent modifications improved inhibition of the rat lung ECE while eliminating renin activity. Both series of ECE inhibitors demonstrated a range of selectivity over Cathepsin D. Water-solubilizing moieties were appended onto selected compounds to facilitate in vivo testing. Partial reduction of the pressor response to exogenously administered Big ET-1 was observed with selected rat lung ECE inhibitors.

Pyrrolidine-3-carboxylic acids as endothelin antagonists. 3. Discovery of a potent, 2-nonaryl, highly selective ETA antagonist (A-216546).

Previously we have reported the discovery of ABT-627 (1, A-147627, active enantiomer of A-127722), a 2,4-diaryl substituted pyrrolidine-3-carboxylic acid based endothelin receptor-A antagonist. This compound binds to the ETA receptor with an affinity (Ki) of 0. 034 nM and with a 2000-fold selectivity for the ETA receptor versus the ETB receptor. We have expanded our structure-activity studies in this series, in an attempt to further increase the ETA selectivity. When the p-anisyl group of 1 was replaced by an n-pentyl group, the resultant antagonist 3 exhibited substantially increased ETB/ETA activity ratio, but a decreased ETA affinity. Structure-activity studies revealed that substitution and geometry of this alkyl group, and substitution on the benzodioxolyl ring, are important in optimizing this series of highly ETA selective antagonists. In particular, the combination of a (E)-2,2-dimethyl-3-pentenyl group and a 7-methoxy-1,3-benzodioxol-5-yl group provided hydrophobic compound 10b with subnanomolar affinity for human ETA receptor subtype and with an ETB/ETA activity ratio of over 130000. Meanwhile, synthetic efforts en route to olefinic compounds led to the discovery that 2-pyridylethyl (9o) and 2-(2-oxopyrrolidinyl)ethyl (9u) replacement of the p-anisyl group of 1yielded very hydrophilic ETA antagonists with potency and selectivity equal to those of 10b. On the basis of overall superior affinity, high selectivity for the ETA receptor (Ki, 0.46 nM for ETA and 13000 nM for ETB), and good oral bioavailability (48% in rats), A-216546 (10a) was selected as a potential clinical backup for 1.

Azole endothelin antagonists. 3. Using delta log P as a tool to improve absorption.

The oral absorption profile of a family of azole-based ET(A)-selective antagonists has been improved through a rational series of structural modifications which were suggested by analysis of the physicochemical parameter delta log P. Comparison of urea 2 with a series of well-absorbed compounds using delta log P analysis suggested that 2 has an excess capacity for forming hydrogen bonds with solvent. A series of urea modifications were explored as a means of reducing H-bonding capacity while maintaining affinity for the ET(A)-receptor. The correlation between delta log P values and absorption in an intraduodenal (id) bioavailability model was good; this strategy uncovered replacements for each of the urea NH groups which simultaneously improve both potency and drug absorption. A combination of these optimized modifications produces carbamate 16h, a highly-selective ET(A) antagonist with a potency/bioavailability profile consistent with an oral route of administration.

2,4-Diarylpyrrolidine-3-carboxylic acids--potent ETA selective endothelin receptor antagonists. 1. Discovery of A-127722.

We have discovered a novel class of endothelin (ET) receptor antagonists through pharmacophore analysis of the existing non-peptide ET antagonists. On the basis of this analysis, we determined that a pyrrolidine ring might replace the indian ring in SB 209670. The resultant compounds were readily prepared and amenable to extensive SAR studies. Thus a series of N-substituted trans,trans-2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)pyrroli din e-3- carboxylic acids (8) have been synthesized and evaluated for binding at ET(A) and ET(B) receptors. Compounds with N-acyl and simple N-alkyl substituents had weak activity. Compounds with N-alkyl substituents containing ethers, sulfoxides, or sulfones showed increased activity. Much improved activity resulted from compounds where the N-substituents were acetamides. Compound 17u (A-127722) with the N,N-dibutylacetamide substituent is the best of the series. It has an IC(50)=0.36 nM for inhibition of ET-1 radioligand binding at the ET(A) receptor, with a 1000-fold selectivity for the ET(A) vs the ET(B) receptor. It is also a potent inhibitor (IC(50)=0.16 nM) of phosphoinositol hydrolysis stimulated by ET-1, and it antagonized the ET-1-induced contraction of the rabbit aorta with a pA(2)=9.20. The compound has 70% oral bioavailability in rats.

New antimitotic agents with activity in multi-drug-resistant cell lines and in vivo efficacy in murine tumor models.

During a screen for compounds that could inhibit cell proliferation, a series of new tubulin-binding compounds was identified with the discovery of oxadiazoline 1 (A-105972). This compound showed good cytotoxic activity against non-multi-drug-resistant and multi-drug-resistant cancer cell lines, but its utility in vivo was limited by a short half-life. Medicinal chemistry efforts led to the discovery of indolyloxazoline 22g (A-259745), which maintained all of the in vitro activity seen with oxadiazoline 1, but also demonstrated a better pharmacokinetic profile, and dose-dependent in vivo activity. Over a 28 day study, indolyloxazoline 22g increased the life span of tumor-implanted mice by up to a factor of 3 upon oral dosing. This compound, and others of its structural class, may prove to be useful in the development of new chemotherapeutic agents to treat human cancers.

Sitaxsentan (ICOS-Texas Biotechnology).

ICOS-Texas Biotechnology is developing the endothelin A (ETA) receptor antagonist, sitaxsentan, for the potential treatment of pulmonary hypertension, congestive heart failure (CHF), chronic obstructive pulmonary disease and subarachnoid hemorrhage [205713], [302200]. The compound is in phase IIa trials as an iv formulation for CHF and has completed phase I safety trials as an oral formulation [272071]. Phase II/III trials for pulmonary hypertension are planned for the first quarter of 2001 [3945711]. In June 2000, ICOS and Texas Biotechnology established a joint venture to develop and commercialize endothelin antagonists [370007]. US-05591761 was issued to Texas in January 1997, covering TBC-11251 and several related isomers [2309301.

Dissociation characteristics of endothelin receptor agonists and antagonists in cloned human type-B endothelin receptor.

The human type-B endothelin receptor (h-ETB) was cloned from human lung poly A+RNA and stably expressed in CHO cells. Endothelin (ET) receptor binding and stimulation of PI hydrolysis demonstrated that the cloned h-ETB receptor is functional and linked to intracellular signal transduction pathways in CHO cells. The molecular mass of the h-ETB receptor was determined to be 65 KDa, and Bmax and Kd were 0.36 pmol/mg and 80 pM, respectively. Competition studies employing receptor ligands revealed that the potencies of the test ligands (IRL1620, PD142893, and Ro46-2005) were dependent on the length of the incubation time, whereas the natural agonists (ET-1 and ET-3) were not. When competing with ET-1 in the h-ETB receptor binding, the IC50 increased from 1.2 nM to 8.2 nM for IRL1620, 0.068 microM to 1.9 microM for PD142893, and 0.76 microM to 12.7 microM for Ro46-2005, as the incubation time increased from 1 hr to 24 hr. These time-induced changes are likely due to differences in the dissociation characteristics between the artificial ligands and the natural ligands.

New bone formation in an osteoblastic tumor model is increased by endothelin-1 overexpression and decreased by endothelin A receptor blockade.

OBJECTIVES: The osteoblastic response of bone to metastatic prostate cancer is both characteristic and enigmatic. The potent vasoconstrictor endothelin-1 (ET-1), produced by prostate cancer, has been identified as a potential factor in new bone formation. METHODS: Using a novel method to quantitate new bone formation induced by the WISH tumor, we examined the effects of ET-1 overexpression and endothelin receptor antagonists on the osteoblastic response. RESULTS: WISH, a human tumor cell line derived from amnion, produces ET-1 mRNA and protein and induces abundant new bone formation and splenomegaly in vivo. Stable transfection of WISH with an ET-1 overexpression cDNA construct produced clones that secreted 18-fold more bioactive ET-1 than vector-only controls. After 14 days of growth in the lower leg of nu/nu mice, ET-1 overexpressing tumors produced significantly more new bone than vector-only controls. Conversely, areas of new bone formation were significantly less in animals treated with a selective endothelin A (ET(A)) receptor antagonist A127722. CONCLUSIONS: The activity of ET-1 in this osteoblastic model provides a unique target for therapy.

Pharmacology of A-216546: a highly selective antagonist for endothelin ET(A) receptor.

Endothelins, 21-amino acid peptides involved in the pathogenesis of various diseases, bind to endothelin ET(A) and ET(B) receptors to initiate their effects. Here, we characterize the pharmacology of A-216546 ([2S-(2,2-dimethylpentyl)-4S-(7-methoxy-1,3-benzodioxol-5-yl )-1-(N,N-di(n-butyl) aminocarbonylmethyl)-pyrrolidine-3R-carboxylic acid), a potent antagonist with > 25,000-fold selectivity for the endothelin ET(A) receptor. A-216546 inhibited [125I]endothelin-1 binding to cloned human endothelin ET(A) and ET(B) receptors competitively with Ki of 0.46 and 13,000 nM, and blocked endothelin-1-induced arachidonic acid release and phosphatidylinositol hydrolysis with IC50 of 0.59 and 3 nM, respectively. In isolated vessels, A-216546 inhibited endothelin ET(A) receptor-mediated endothelin-1-induced vasoconstriction, and endothelin ET(B) receptor-mediated sarafotoxin 6c-induced vasoconstriction with pA2 of 8.29 and 4.57, respectively. A-216546 was orally available in rat, dog and monkey. In vivo, A-216546 dose-dependently blocked endothelin-1-induced pressor response in conscious rats. Maximal inhibition remained constant for at least 8 h after dosing. In conclusion, A-216546 is a potent, highly endothelin ET(A) receptor-selective and orally available antagonist, and will be useful for treating endothelin-1-mediated diseases.

Characterization of two endothelin converting enzymes and their preference for big endothelin-1 and -2 as substrates.

Two proteolytic activities that convert big ET to ET at neutral pH were identified in solubilized membranes prepared from rat lung. The endothelin-converting activities were partially purified by using A80227 ((2S,3R,4S)-2-([N-acetylcyclohexylalanyl-isoleucyl]amino)-1-(2-nap hthyl)-3,4-dihydroxy-6-methylheptane) coupled to an affinity-gel column (Affigel), and subsequently by concanavalin-A immobilized gel chromatography. An endothelin-converting activity was identified in the fraction containing proteins that did not bind to A80227-Affigel. This protease was sensitive to phosphoramidon, soybean trypsin inhibitor, and chymostatin, and preferred big ET-1 or big ET-2 as its substrate over bit ET-3. A second endothelin-converting activity was identified in the fraction containing proteins that bound to the A80227-coupled gel and was eluted by raising the pH. This protease exhibited activities throughout a range of pH 5.5-9.5, was inhibited by pepstatin A and A80227, and also preferred big ET-1 or big ET-2 over big ET-3 as its substrate. Both enzymes were glycoproteins based on their binding to concanavalin-A immobilized gel and were readily eluted by a buffer containing 0.5 M manopyranoside. The results from the pH and protease inhibitor profiles suggesting that these two ET-converting activities extracted from rat lung membranes are distinct and are different from the previously reported endothelin-converting enzymes.

Endothelin receptor antagonists exhibit diminishing potency following incubation with agonist.

Endothelins (ET) are 21-amino acid peptides that bind to membrane receptors to initiate a wide range of pathophysiological effects. ET binding to receptors has been shown to be almost irreversible because bound ET is difficult to dissociate. This report studies the dissociation characteristics of receptor antagonists and further examines the effects of ET's difficult-to-dissociate binding on the potency of antagonists. In membranes prepared from porcine cerebellum, [125I]ET-1 binding was effectively blocked by ET-1 and ET-3 with similar IC50 values (0.08 nM vs. 0.17 nM), suggesting that porcine cerebellum contains predominantly the ETB receptor subtype. [125I]ET-3 binding was inhibited by Ro46-2005 and PD142893, two non-selective antagonists, with IC50 values of 570 +/- 50 nM and 410 +/- 100 nM, respectively. Consistent with previous observations, bound [125I]ET-1 in porcine cerebellum membranes was also difficult to dissociate. In contrast, bound Ro46-2005 or PD142893, but not bound ET-1, could be readily washed away from membranes, suggesting that antagonist binding was more reversible than ET-1 binding. Although Ro46-2005 or PD142893 at 0.5 microM inhibited 0.1 nM [125I]ET-1 binding by > 80% after 15 min of incubation, the inhibitory effect decreased to approximately 50% after 3 h of incubation, and further decreased to < 10% at 24 h. This decrease in antagonizing potency was further confirmed by the results that the IC50 values of the two antagonists against [125I]ET-3 binding increased with increasing incubation time. Control experiments indicate that the observed decrease in the potency of Ro46-2005 and PD142893 was not the result of ligand degradation. These results suggest that the potency of antagonists is critically dependent on the incubation time because antagonist binding is more reversible than ET binding.

Direct determination of endothelin receptor antagonist levels in plasma using a scintillation proximity assay.

An assay using scintillation proximity bead technology has been developed suitable for the quantitation of endothelin (ET) receptor antagonists in preclinical and clinical samples of plasma. The assay measures the competitive inhibition of radiolabelled ET-1 binding to ET(A) receptor membranes bound to wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads in the presence of plasma containing A-127722, a potent orally active, ET(A) selective ET antagonist. The assay requires as little as 50 microl plasma and no extraction procedure is needed. The SPA methodology eliminates the need for the separation of bound from free ligand. Using this method, A-127722 could be directly quantified in rat plasma with a detection limit of 1 ng/ml.