ozagrel and dazoxiben

Thromboxane synthase (TXAS) is a P450 epoxygenase that synthesizes thromboxane A2 (TXA2), a potent mediator of platelet aggregation, vasoconstriction and bronchoconstriction. This enzyme plays an important role in several human diseases, including myocardial infarction, stroke, septic shock, asthma and cancer. Despite of the increasing interest on developing TXAS inhibitors, the structure and activity of TXAS are still not totally elucidated. In this study, we used a comparative molecular modeling approach to construct a reliable model of TXAS and analyze its interactions with Dazoxiben and Ozagrel, two competitive inhibitors. Our results were compatible with experimental published data, showing feasible cation-π interaction between the iron atom of the heme group of TXAS and the basic nitrogen atom of the imidazolyl group of those inhibitors. In the absence of the experimental structure of thromboxane synthase, this freely available model may be useful for designing new antiplatelet drugs for diseases related with TXA2.

Topics: Binding, Competitive; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Imidazoles; Ligands; Methacrylates; Molecular Docking Simulation; Structure-Activity Relationship; Thromboxane-A Synthase

Arachidonic acid (AA) is a potent inducer of platelet aggregation in vitro; this activity is due to its conversion to biologically active metabolites, prostaglandin (PG) endoperoxides and thromboxane A2 (TxA2). PG endoperoxides and TxA, are thought to act on the same receptor; however, at least two isoforms of this receptor have been identified. The aim of our work was to clarify whether endoperoxides and TxA2 activate the same or different receptor subtypes to induce aggregation and calcium movements in human platelets. AA-induced aggregation and calcium rises were still detectable in platelets preincubated with thromboxane synthase inhibitors, which suppress TxA2 formation and induce PGH2 accumulation, suggesting that PG endoperoxides can activate platelets. Exogenously added PGH2 was able to induce aggregation and calcium rises. Pretreatment of platelets with GR32191B or platelet activating factor, which desensitize one of the two receptor subtypes identified in platelets, did not prevent calcium rises induced by endogenously generated or by exogenouly added PGH2, indicating that TxA2 and PG endoperoxides share the same receptor subtype(s) to activate platelets. HEK-293 cells overexpressing either of the two thromboxane receptor isoforms cloned to date (TPalpha and TPbeta) and identified in human platelets, stimulated with PGH2, or with the stable endoperoxide analog U46619, formed inositol phosphates. These data show that endoperoxides and TXA2 mediate their effects on platelets acting on both, and the same, receptor isoform(s).

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Aspirin; Biphenyl Compounds; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Calcium Signaling; Cells, Cultured; Enzyme Inhibitors; Fatty Acids, Unsaturated; Heptanoic Acids; Humans; Hydrazines; Imidazoles; Inositol Phosphates; Kidney; Methacrylates; Phenylacetates; Platelet Activating Factor; Platelet Activation; Prostaglandin H2; Prostaglandins H; Protein Isoforms; Receptors, Thromboxane; Recombinant Fusion Proteins; Sulfonamides; Thromboxane A2; Thromboxane B2; Thromboxane-A Synthase

The in vitro properties of CS-518 (RS-5186; sodium 2-(1-imidazolylmethyl)- 4,5-dihydrobenzo[b]thiophene-6-carboxylate, CAS 113817-57-5), a new thromboxane (TX) synthetase inhibitor, as an antiplatelet agent were investigated. Incubation of clotting whole blood from man, rabbits, and dogs with CS-518 resulted in a concentration-dependent reduction of TXB2 production and an increase in 6-keto-PGF1 alpha. Similar properties were also observed for ozagrel and isbogrel, but both agents were less effective on TXB2 production. CS-518 inhibited arachidonic acid (AA)- or collagen-induced platelet aggregation in platelet rich plasma (PRP) from man, rabbits and dogs. In addition, antiaggregatory effects of CS-518 were confirmed in whole blood by two methods: impedance method and free platelet count method. TXA2 formation in washed canine platelets in response to AA (0.1 mmol/l) was dose-dependently inhibited by incubation with CS-518. This inhibition by CS-518 was gradually attenuated after platelets were subsequently washed with drug-free buffer, but a dose-dependent inhibition was still observed with platelets that had been washed three times. Ozagrel also inhibited TXB2 formation when incubated with platelets, whereas this inhibition disappeared with platelets only washed once. In contrast, platelets treated with acetylsalicylic acid, an irreversible inhibitor of cyclooxygenase showed a comparable inhibition before and after they were washed three times. These results indicate that CS-518 exerts antiplatelet effects in vitro via potent, selective, and long-lasting but reversible inhibition on TX synthetase.

Topics: 6-Ketoprostaglandin F1 alpha; Adult; Animals; Aspirin; Dogs; Humans; Imidazoles; In Vitro Techniques; Male; Methacrylates; Platelet Aggregation; Platelet Aggregation Inhibitors; Rabbits; Thiophenes; Thromboxane B2; Thromboxane-A Synthase; Thromboxanes