Biomedical subjects
Publications and source records attributed to E E Reynolds.
Novel non-nucleoside tricyclic platelet ADP receptor (P2Y(12)) antagonists have been discovered that bind reversibly and with high affinity to the platelet receptor. Condensation of various 2-aminobenzothiazoles with chlorosulfonylacetyl chloride affords these novel tricyclic heterocycles, which are novel and unpredicted products of this reaction.
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The protease-activated thrombin receptor-1 (PAR-1) can be activated by both the tethered ligand exposed by thrombin cleavage and a synthetic peptide having the tethered ligand sequence (thrombin receptor agonist peptide or TRAP). We conducted a mutational analysis of extracellular residues of the receptor potentially involved in interaction with both the tethered ligand and the soluble peptide agonist. Agonist-stimulated calcium efflux in X. laevis oocytes or inositol phosphate accumulation in COS-7 cells was used to assess receptor activation. We have also examined the binding of a radiolabeled TRAP for the wild-type and mutant PAR-1 receptors. Our results indicated that most of the mutations strongly affected TRAP-induced responses without significantly altering thrombin-induced responses or TRAP binding. Several point mutations and deletion of extracellular domains (DeltaEC3, DeltaNH3) drastically altered the ability of mutant receptors to respond to TRAP, but not to thrombin, and did not affect the affinity for the radiolabeled TRAP by these mutant receptors. Only mutations that disrupted the putative disulfide bond or substitution of multiple acidic residues in the second extracellular loop by alanine had a significant effect on both ligand binding and thrombin activation. These results suggest that although both agonists can activate PAR-1, there are profound differences in the ability of thrombin and TRAP to activate PAR-1. In addition, we have found PAR-1 mutants with the ability to dissociate receptor-specific binding from functional activity.
Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease alpha-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH(2), RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.
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The identity of the receptors mediating platelet activation by ADP remains elusive. To distinguish between platelet ADP receptor subtypes, the effects of antagonists on platelet responses and the cloned P2Y1 receptor, a putative platelet ADP receptor, have been investigated. 2-methylthio-AMP (2MeSAMP), an inhibitor of ADP-dependent platelet aggregation, antagonized ADP-mediated inhibition of adenylyl cyclase, competed with binding of [3H]2-methylthio-ADP and inhibited the stimulation of [35S]GTPgammaS binding. 2MeSAMP did not inhibit platelet shape change and was only a weak antagonist of intracellular calcium mobilization in platelets or in cells expressing the cloned human P2YI receptor. By contrast, the P2Y1 receptor antagonist adenosine 3',5'-diphosphate (A3P5P) inhibited ADP-induced platelet aggregation, completely abolished shape change, but did not antagonize ADP effects on cyclic AMP generation or [3H]2-methylthio-ADP binding. However, A3P5P antagonized intracellular calcium mobilization in platelets and cells expressing the cloned P2Y1 receptor. Furthermore, using a specific monoclonal antibody and flow cytometry, P2Y1 receptor protein was detected on human platelets. These results support the existence of two G protein-coupled ADP receptors mediating platelet aggregation, one of which is coupled to Gi proteins and blocked by 2MeSAMP, whereas the second receptor is similar or identical to P2Y1 and coupled to Gq.
We have described the pharmacologic profiles of endothelin B receptors in human endothelial cells and vascular and nonvascular smooth-muscle cells. First, by amplifying endothelin B receptor numbers through the use of phosphoramidon and intact cell-binding techniques, we demonstrated the presence of these receptors in human umbilical vein endothelial cells (100% endothelin B receptors), human aortic smooth-muscle cells (22% endothelin B, 78% endothelin A receptors), and human bronchial smooth-muscle cells (55% endothelin B, 45% endothelin A receptors) by using [125I]-endothelin-1 radioligand binding. The typical binding profiles of the endothelin B receptors were established through competition binding curve analysis with endothelin-1, endothelin-3, sarafotoxin 6c, and the endothelin A receptor-selective antagonist BQ-123. In the presence of BQ-123, a diverse group of antagonists, including PD 142893, BQ-788, SB 209670, and Ro 47-0203, were used to probe for binding differences indicative of multiple endothelin B-receptor subtypes. The results indicate a rank order of potency for the antagonists of BQ-788 > SB 209670 > PD 142893 > Ro 47-0203 for each cell line, and that between any of these human cell lines, measurements of [125I]-endothelin-1-binding antagonism for each of the four test compounds differed by less than twofold. Although this study cannot discount the possibility of more than one endothelin B-receptor subtype in humans, it does indicate that these tissues express receptors that show equivalent binding pharmacology.
The design of potent and selective non-peptide antagonists of endothelin-1 (ET-1) and its related isopeptides are important tools defining the role of ET in human diseases. In this report we will describe the detailed structure-activity relationship (SAR) studies that led to the discovery of a potent series of butenolide ETA selective antagonists. Starting from a micromolar screening hit, PD012527, use of Topliss decision tree analysis led to the discovery of the nanomolar ET(A) selective antagonist PD155080. Further structural modifications around the butenolide ring led directly to the subnanomolar ETA selective antagonist PD156707, IC50's = 0.3 (ET(A)) and 780 nM (ET(B)). This series of compounds exhibited functional activity exemplified by PD156707. This derivative inhibited the ETA receptor mediated release of arachidonic acid from rabbit renal artery vascular smooth muscle cells with an IC50 = 1.1 nM and also inhibited the ET-1 induced contraction of rabbit femoral artery rings (ETA mediated) with a pA2 = 7.6. PD156707 also displayed in vivo functional activity inhibiting the hemodynamic responses due to exogenous administration of ET-1 in rats in a dose dependent fashion. Evidence for the pH dependence of the open and closed tautomerization forms of PD156707 was demonstrated by an NMR study. X-ray crystallographic analysis of the closed butenolide form of PD156707 shows the benzylic group located on the same side of the butenolide ring as the gamma-hydroxyl and the remaining two phenyl groups on the butenolide ring essentially orthogonal to the butenolide ring. Pharmacokinetic parameters for PD156707 in dogs are also presented.
Intracellular signaling mechanisms affected by endothelin (ET), a hypertrophic agonist, and platelet-derived growth factor (PDGF)-BB, a proliferative agonist, in vascular smooth muscle cells were examined. PDGF-BB was a potent mitogen compared with untreated cultures, stimulating both [3H]thymidine incorporation and cell number. In contrast, ET was a poor mitogen, enhancing [3H]thymidine incorporation but not cell number. Simultaneous ET and PDGF-BB treatment was significantly more effective than either agonist alone at stimulating both [3H]thymidine uptake and cell number. Although either ET or PDGF-BB alone stimulated arachidonic acid release, phosphoinositide hydrolysis, protein kinase C activation, PDGF receptor phosphorylation, and mitogen-activated protein kinase activity, of these effectors, only arachidonic acid release was further enhanced by simultaneous ET and PDGF-BB treatment. These results link proliferative and hypertrophic signal transduction pathways in these cells and suggest that arachidonic acid or its metabolites mediate the observed effects of ET on PDGF-BB-stimulated vascular smooth muscle cell proliferation.
A series of novel drimane sesquiterpene esters (1-6) was isolated from fermentations of Aspergillus ustus var. pseudodeflectus and their structures elucidated by spectroscopic methods including the HMQC, HMBC and INADEQUATE NMR experiments. The major component of the fermentation, 1, was (2'E,4'E,6'E)-6-(1'-carboxy-2',4',6'-trien)-9-hydroxydrim-7-ene-11 ,12-olide. Compounds 1, 2, 3 and 5 exhibited endothelin receptor binding inhibitory activity against rabbit endothelin-A and rat endothelin-B receptors with IC50 values in the range 20-150 microM. These compounds had similar levels of activity in assays for binding to human endothelin A and endothelin B receptors. The isolation of 9,11-dihydroxy-6-oxodrim-7-ene, 7, a probable biosynthetic precursor to the drimane esters is also reported.
Episodes of myocardial ischemia are associated with increases in cardiac venous plasma endothelin (ET) concentrations, suggesting that ET may play a role in the development of myocardial infarction. The purpose of this study was to determine if selective blockade of ET(A) receptors by PD 156707 reduces infarct size caused by coronary artery occlusion and reperfusion in pentobarbital-anesthetized micropigs. A PD 156707 dose which selectively blocks the ET(A)-mediated vasopressor response, but not the ET(B)-mediated vasodepressor response to i.v. ET-1 challenges (0.3 nmol/kg), was established in dose ranging studies in anesthetized micropigs. In myocardial infarction studies, micropigs received either saline vehicle (n = 7) or PD 156707 (n = 8) at a loading dose of 10 mg/kg/1 hr, followed by a maintenance dose of 7 mg/kg/hr. Coinciding with the start of the maintenance dose, the left anterior descending coronary artery was occluded for 1 hr followed by 3 hr of reperfusion. PD 156707 caused a significant (29 mm Hg) decrease in arterial blood pressure before occlusion. PD 156707 had no effect on infarct size (61.1 +/- 5.6% of the region at risk in the PD 156707 treatment group vs. 70.1 +/- 3.9% in the control group). These results suggest that ET(A) receptor activation does not substantially contribute to coronary artery occlusion/reperfusion-induced myocardial infarction.
Cloned rat and human endothelin-B receptors (ETBR) were utilized to determine if there are significant pharmacological differences between highly homologous ETBR from different species. Recombinant rat and human ETBR were expressed in CHO-K1 cells, and radioligand binding studies were carried out with [125I]-ET-3 to determine the affinities of various ET receptor agonists and antagonists for rat and human ETBR. These receptors had similar affinities for a number of ETBR agonists (ET-1, ET-3, S6C, BQ 3020) and antagonists (Ro 47-0203, PD 142893). However, several peptide (PD 147452, PD 151583, BQ 788) and non-peptide (PD 156707, SB 209670) antagonists had different affinities for rat and human ETBR, with differences in Ki values between species ranging from 4.1- to 53.4-fold. The ETBR-selective agonist IRL 1620 also had a 5.7-fold higher affinity for rat ETBR than human ETBR. Thus despite their high degree of homology, rat and human ETBR show significant pharmacological differences with respect to both antagonist and agonist binding.
A series of azaphilones produced by Penicillium sclerotiorum (Xenova culture collection number X11853) active in assays for the detection of antagonists of the endothelin-A (ETA) and endothelin-B (ETB) receptors has been identified. The series includes two novel sclerotiorin analogues, (8S,8 alpha-R)-7-deacetyl-1,O8,8,8a-tetrahydro-7-epi-sclerotiorin, 1, and its 5-dechloro analogue, 2. It also includes 5-chloroisorotiorin, 6, previously unreported as a natural product, in addition to the major product of these fermentations, (+)-sclerotiorin, 5. Data for the inhibition of endothelin-1 (ET-1) and endothelin-3 (ET-3) binding in the ETA and ETB receptor assays respectively are reported for this series. Compounds 1 and 2 were more selective for the rabbit ETA receptor than for the rat ETB receptor. The IC50 values for 1 and 2 were 9 and 28 microM respectively in an assay based on binding of ET-1 to rabbit ETA receptors. In an assay based on the binding of ET-3 to the rat ETB receptor compounds 1 and 2 exhibited IC50's of 77 and 172 microM. Members of this series of compounds demonstrated antagonist behavior in a secondary assay based on blockade of ET-1 stimulated arachidonic acid release from rabbit renal artery smooth muscle cells, when present at concentrations of > or = 30 microM.
We describe the pharmacological characteristics of PD 156707 (sodium 2-benzo[1,3]dioxol-5-yl-4-(4-methoxy-phenyl)-4-oxo-3-(3,4,5- trimethoxy-benzyl)-but-2-enoate), a potent, orally active, nonpeptide antagonist of the endothelin A (ETA) receptor subtype. PD 156707 was designed on the basis of a compound identified by screening the Parke-Davis chemical library. PD 156707 is highly selective for the ETA receptor (ETAR) and inhibits the binding of [125I]-ET-1 to cloned human ETAR and ETBR with Ki values of 0.17 and 133.8 nM, respectively. PD 156707 antagonizes ET-1-stimulated phosphoinositide hydrolysis in Ltk- cells expressing cloned human ETAR with an IC50 value of 2.4 nM. PD 156707 inhibits vasoconstriction in isolated blood vessels mediated by ETAR (rabbit femoral artery) and ETBR (rabbit pulmonary artery) with pA2 values of 7.5 and 4.7, respectively. PD 156707 administered orally to rats blocked subsequent ETAR-mediated pressor responses in vivo but had no effect on ETBR-mediated dilator responses. As a potent and orally active ETA-selective antagonist, PD 156707 will be useful in defining the physiological and pathological roles of ETAR.
The effects of different amino acids incorporated into the 16 and 17 positions of the C-terminal hexapeptide of ET-1 were examined. Structure-activity relationships (SAR) of the ET receptor antagonists PD 142893 [Ac-(D-Dip16-L-Leu17-L-Asp-L-Ile-L-Ile-L-Trp) (D-Dip = 3,3-D-diphenylalanine)] and PD 145065 [Ac-(D-Bhg16-L-Leu17-L-Asp-L-Ile-L-Ile-L-Trp) (D-Bhg = 5H-dibenzyl[a,d]cycloheptene-10,11-dihydro-glycine)] uncovered certain requirements for high potency. The disodium salt of PD 145065 has 4.0 and 15 nM binding affinity (IC50 values) for the ETA (rabbit renal artery vascular smooth-muscle cells) and ETB receptor (rat cerebellum), respectively. The compound is also an antagonist of ET-1- and SRTX-6c-stimulated vasoconstrictor activity, with pA2 values of 6.9 (rabbit femoral artery, ETA assay) and 7.1 (rabbit pulmonary artery, ETB assay). The tripeptidic ETA antagonist FR 139317 was found to be less active in the rabbit femoral artery, with a pA2 value of 6.0, and inactive in the rabbit pulmonary artery. Substitution of acidic and basic residues at position 17 in PD 142893 and PD 145065 indicates differences in selectivity. Incorporation of bulky non-natural amino acids at position 16 has led to potent nonselective analogues, including Ac-D-Bheg16-L-Leu-L-Asp-L-Ile-L-Ile-L-Trp [D-Bheg (5H-dibenzo[a,d]cycloheptene glycine)]. The in vivo effects of single-bolus doses of selected ET antagonists on depressor and pressor responses to ET-1 in anesthetized ganglion-blocked rats were evaluated.(ABSTRACT TRUNCATED AT 250 WORDS)