Carolyn Foster

Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs

ADP plays a critical role in modulating thrombosis and hemostasis. ADP initiates platelet aggregation by simultaneous activation of two G protein-coupled receptors, P2Y1 and P2Y12. Activation of P2Y1 activates phospholipase C and triggers shape change, while P2Y12 couples to Gi to reduce adenylyl cyclase activity. P2Y12 has been shown to be the target of the thienopyridine drugs, ticlopidine and clopidogrel. Recently, we cloned a human orphan receptor, SP1999, highly expressed in brain and platelets, which responded to ADP and had a pharmacological profile similar to that of P2Y12. To determine whether SP1999 is P2Y12, we generated SP1999-null mice. These mice appear normal, but they exhibit highly prolonged bleeding times, and their platelets aggregate poorly in responses to ADP and display a reduced sensitivity to thrombin and collagen. These platelets retain normal shape change and calcium flux in response to ADP but fail to inhibit adenylyl cyclase. In addition, oral clopidogrel does not inhibit aggregation responses to ADP in these mice. These results demonstrate that SP1999 is indeed the elusive receptor, P2Y12. Identification of the target receptor of the thienopyridine drugs affords us a better understanding of platelet function and provides tools that may lead to the discovery of more effective antithrombotic therapies.

Inhibition of cellular action of thrombin by N3-cyclopropyl-7-[[4-(1-methylethyl)phenyl]methyl]-7H-pyrrolo[3, 2-f]quinazoline-1,3-diamine (SCH 79797), a nonpeptide thrombin receptor antagonist.

A growing body of evidence suggests an important contribution of the cellular actions of thrombin to thrombosis and restenosis following angioplasty. Recently we reported on SCH 79797 (N3-cyclopropyl-7-¿[4-(1-methylethyl)phenyl]methyl¿-7H-pyrrolo[3, 2-f]quinazoline-1,3-diamine) and its analogs as new potent, nonpeptide thrombin receptor antagonists. This study further characterizes the biochemical and pharmacological actions of pyrroloquinazoline inhibitors of protease activated receptor-1 (PAR-1) in human platelets and coronary artery smooth muscle cells (hCASMC). SCH 79797 and its N-methyl analog (SCH 203099) inhibited binding of a high-affinity thrombin receptor-activating peptide ([(3)H]haTRAP, Ala-Phe(p-F)-Arg-ChA-HArg-[(3)H]Tyr-NH(2)) to PAR-1 with IC(50) values of 70 and 45 nM, respectively. SCH 79797 inhibited [(3)H]haTRAP binding in a competitive manner. SCH 79797 and SCH 203099 inhibited alpha-thrombin- and haTRAP-induced aggregation of human platelets, but did not inhibit human platelet aggregation induced by the tethered ligand agonist for protease-activated receptor-4 (PAR-4), gamma-thrombin, ADP, or collagen. SCH 203099 inhibited surface expression of P-selectin induced by haTRAP and thrombin, and it did not increase P-selectin expression or prevent thrombin cleavage of the receptor. Thrombin and TFLLRNPNDK-NH(2) (TK), a PAR-1-selective agonist, produced transient increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in hCASMC. This increase in [Ca(2+)](i) was inhibited effectively by SCH 79797. However, the Ca(2+) transients induced by SLIGKV-NH(2,) a PAR-2-selective agonist, were not inhibited by SCH 79797. Thrombin- and TK-stimulated [(3)H]thymidine incorporation also was inhibited completely by SCH 79797. The results of this study demonstrate that SCH 79797 and SCH 203099 are potent, selective antagonists of PAR-1 in human platelets and hCASMC. These data also suggest that the thrombin stimulation of Ca(2+) transients and mitogenesis in hCASMC is mediated primarily through activation of PAR-1.

Modulation of circulating endothelin levels in hypertension and endotoxemia in rats

We have developed separate radioimmunoassays to measure circulating ET-1 and ET-3 levels in normotensive and different hypertensive rat models so that the role of endothelin in the regulation of vasomotor function can be studied. We also assessed the stimulatory effects of endotoxin on plasma and liver lymph ET-1 and ET-3 levels. The circulating ET-1 levels in normotensive rats, SHRs, and DOCA-salt hypertensive rats were 2.3 ± 0.5, 2.1 ± 0.4, and 2.1 ± 0.9 pg/ml, respectively. Similarly; the plasma ET-3 levels in normotensive and different hypertensive rats were similar, ranging from 19.7 ± 1.5 to 24.7 ± 2.2 pg/ml. The data indicate that steadystate circulating levels of endothelins are a poor correlate of the hypertensive state. Endotoxin (30 mg/kg i.v. over 15 min) reduced blood pressure significantly and augmented plasma ET-1 levels by sevenfold (29.1 ± 3.7 vs. 4.1 ± 0.6 pg/ml in the vehicle group; p < 0.05) and ET-3 levels by twofold (47.7 ± 7.0 vs. 22.7 ± 4.0 pg/ml in the vehicle group; p < 0.05).Human TNF-α (30 ng/kg/min x 30 min), a putative mediator of endotoxin shock, enhanced plasma ET-1 (18.3 ± 1.0 vs: 2.7 ± 0.4 pg/ml in the vehicle group; p < 0.05) by sevenfold and ET-3 levels by twofold (45.7 ± 2.0 vs. 27.1 ± 4.0 pg/ml in the vehicle group; p < 0.05) without affecting blood pressure. In con-trash; PAF (50 ng/kg/min x 30 min); another mediator liberated during endotoxin shock; exerted asimilar hypotensive response to endotoxin but did not alter either plasma ET-1 or ET-3 levels. In a separate study, endotoxin augmented plasma and liver lymph ET-1 levels by eight- and twofold, respectively. In comparison, endotoxin caused a twofold increase in both plasma and lymph ET-3 levels. Pretreatment with indomethacin (10 mg/kg p.o.)significantly attenuated the endotoxin-induced in-creases in plasma ET-1 and ET-3 levels without affecting the depressor responses to endotoxin. The endotoxininduced increase in ET-1 levels in the plasma and liver lymph is most likely derived from the endothelial cells. However, since ET-3 is not produced by endothelial cells, the source of endotoxin-induced increases in plasma and liver lymph ET-3 levels remains to beidentified. The data suggest that the endotoxin-stimulated re-lease of ET-1 and ET-3 can be dissociated from blood pressure changes.Eicosanoids may also be involved in the release of endothelins due to endotoxin.

Structure-activity relationships of pyrroloquinazolines as thrombin receptor antagonists.

A series of pyrroloquinazolines has been discovered that represent novel small molecule inhibitors of the intramolecular ligand of the thrombin receptor. Analogs were prepared to study the structure-activity relationships of substitution at the N 1, N3, and N7 positions of the heterocycle. Compounds 4e and 4f have been identified with IC50s of 56 and 52 nM, respectively.

Development of Proteinase-Activated Receptor 1 Antagonists as Therapeutic Agents for Thrombosis, Restenosis and Inflammatory Diseases

Thrombin, a plasma serine protease, plays a key role not only in coagulation and hemostasis but in thrombosis, restenosis and atherosclerosis. Thrombin activates platelets, endothelium, inflammatory cells and smooth muscle cells. The cellular action of thrombin is mediated by specific G-protein coupled thrombin receptors called proteinase-activated receptors (protease-activated receptor or PARs). Among the three thrombin receptors, PAR1 is the primary thrombin receptor in human and animal cells with an exception of non-primate platelets. An increased thrombin generation and PAR1 expression are observed on cells within atherosclerotic plaque and thrombus and following vascular injury. Animal studies with PAR1 deficient mice and small molecule antagonists indicate an important role of PAR1 in thrombosis and restenosis and thus the therapeutic potential of a PAR1 antagonist in treating these diseases. Development of a thrombin receptor tethered ligand analog binding assay led to the discovery of several different series of potent, nonpeptide small molecular antagonists of PAR1. These antagonists are PAR1 selective and inhibit most of the cellular effects of thrombin. A PAR1 antagonist has an advantage over a direct thrombin inhibitor since it does not inhibit enzymatic action of thrombin in the coagulation cascade with the consequent minimal bleeding side-effects, unlike a direct thrombin inhibitor. In addition, the emerging evidence for the role of PAR1 in various inflammatory diseases suggests as yet unexplored therapeutic potentials of PAR1 antagonists in various inflammatory diseases.

Potent, low molecular weight thrombin receptor antagonists

Several benzimidazole derivatives have been identified as potent thrombin receptor (PAR-1) antagonists as represented by compound 1h, which showed an IC(50) of 33 nM.

Biaryl and heteroaryl derivatives of SCH 58261 as potent and selective adenosine A2A receptor antagonists.

SCH 58261 is a reported adenosine A(2A) receptor antagonist, which is active in rat in vivo models of Parkinsons Disease upon ip administration. However, it has poor selectivity versus the A(1) receptor and does not demonstrate oral activity. We report the design and synthesis of biaryl and heteroaryl analogs of SCH 58261 which improve the A(2A) receptor binding selectivity as well as the pharmacokinetic properties of SCH 58261. In particular, the quinoline 25 has excellent A(2A) receptor in vitro binding affinity and selectivity, sustained rat plasma levels upon oral dosing, and is active orally in a rat behavioral assay.

Potent Non-Peptide Thrombin Receptor Antagonists

Protease activated receptor-1 (PAR-1), also known as thrombin receptor, is present in a variety of cell types such as platelets and endothelial cells. PAR-1 is proteolytically activated by thrombin by cleavage at its extracellular domain, unmasking a new amino terminus, which internally binds to the proximal receptor, eliciting cellular activation. Inhibition of the cellular activation by thrombin is a potentially promising therapeutic approach for the treatment of thrombotic and vascular proliferative disorders such as atherosclerosis and restenosis. Reported herein is the pharmacology of potent, low molecular weight thrombin receptor antagonists from pyrroloquinazoline, benzimidazole, and himbacine series. In the radioligand binding assay, these compounds inhibited PAR-1 in a competitive manner. They also inhibited thrombin and agonist peptide induced human platelet aggregation in a dose-dependent manner. Additionally, these compounds showed dose-dependent inhibition of agonist-induced cytosolic Ca(+2) transients and thymidine incorporation in human coronary artery smooth muscle cells (hCASMC). The most potent compound among these antagonists showed a Ki of 12 nM in the radioligand binding assay and an IC50 of 70 nM in the platelet aggregation inhibition assay.

Partial characterization of a metalloendopeptidase activity produced by cultured endothelial cells that removes the COOH-terminal tripeptide from 125I-atrial natriuretic factor

The presence of the COOH-terminal region of human atrial natriuretic factor-(99-126) (hANF) is necessary for the full expression of its biological activity. Here, we report on the partial characterization of a proteolytic activity in the conditioned medium from cultured bovine aortic endothelial cells that cleaves the Ser123-Phe124 bond of 125I-hANF generating the COOH-terminal tripeptide. The concentrated conditioned medium was fractionated by gel filtration high performance liquid chromatography and fractions were assayed for the ability to generate the COOH-terminal tripeptide from 125I-hANF. This analysis indicated that the protein responsible for this activity had an approximate molecular weight of 200,000 daltons. Of 16 protease inhibitors tested, only 1,10 phenanthroline, EDTA, EGTA and N-ethylmaleimide significantly inhibited the endopeptidase activity. Thus, we conclude that cultured bovine aortic endothelial cells produce a potentially novel phosphoramidon-insensitive metalloendopeptidase that removes the COOH-terminal tripeptide from 125I-hANF.

Tryptic digestion of myosin light chain kinase produces an inactive fragment that is activated on continued digestion

Trypsin digestion of chicken gizzard myosin light chain kinase at limiting trypsin concentrations proceeds in stages. In the first stage, catalytic activity in the presence or absence of calcium and calmodulin decreases. In the second stage, activity in the absence of calcium increases, and the calcium-calmodulin complex no longer stimulates activity. The initial loss of activity is associated with the appearance of a 59,000-Da peptide that has been isolated and shown to have low catalytic activity. This peptide was further digested to a 55,000-Da peptide that has calcium-independent catalytic activity. This peptide has been isolated, and its affinities for the peptide substrate Kemptamide (Lys-Lys-Arg-Pro-Gln-Arg-Ala-Thr-Ser-Asn-Val-Phe-Ser-NH2) and ATP have been shown to be the same as those of the intact enzyme. Neither the 59,000-Da nor the 55,000-Da fragment binds calmodulin.