THROMBOXANE PROSTANOID RECEPTOR: FUNCTION, ACTIVATION AND POSSIBLE TARGET FOR CARDIOVASCULAR PROTECTION

Abstract In the last few years cardiovascular diseases are considered one of the major cause of death, and one of the main player is TXA2 (Thromboxane A2), a product of arachidonic acid metabolism generated from the activity of thromboxane synthase on prostaglandin H2 intermediate via cyclooxygenase (COX). TXA2 is responsible for platelets activation and aggregation, thrombus formation, and thus it can cause stroke and myocardial infarction. TXA2 exerts its actions through the TP receptor, a widely expressed GPCR (G protein coupled receptor) present in many cell types among different organ systems. During my thesis I worked to shed light on the mechanism of activation of TP receptor WT (wild type), and two of its mutants (TPαE129V and TPαR130V) of the highly conserved motif E/DRY, in order to assign each receptor state to the CTC (Cubic Ternary Complex) model. In particular, using the new technique SPASM (Systematic Protein Affinity Strength Modulation), the goal was to understand the conformational state of TPαWT and mutants in basal condition, i.e. their coupling or uncoupling states with G proteins. The results obtained suggest that TPαE129V (SAM-super active mutant) is in an ‘active-like’ conformation corresponding to the RG state (inactive, coupled to G protein), on the contrary, TPαR130V (loss of function mutant) seems to display an inactive R conformation (uncoupled to G protein), as envisioned by CTC model. The study of TPα receptor induced us to consider a second focus in my thesis: TPα receptor as a possible target for new chemical entities with both COX-2 selectivity (COXIB) and TP antagonist activity. New compounds were obtained modulating the structure of existing drugs (lumiracoxib and RC 0) to obtain novel multitarget NSAIDs (Nonsteroidal Anti-inflammatory Drug) endowed with balanced COXIB and TP receptor antagonist properties. Antagonistic activity on the TP receptor was examined for all compounds by evaluating the inhibition of platelets aggregation induced by the stable TXA2 receptor agonist U46619. COX-1 and COX-2 inhibition were assessed in human washed platelets (challenged by the calcium ionophoreA23187) and human lympho-monocytes suspension (stimulated with lipopolysaccharides), respectively. COX selectivity was determined by calculating IC50 values ratio (COX-2/COX-1) obtained from concentration-response curves. Among the lumiracoxib derivatives, the tetrazole compound 18 and the trifluoromethan sulfonamido-isoster 20 were the more active antagonists at the TP receptor, preventing human platelet aggregation and intracellular signalling, with pA2 values statistically higher than lumiracoxib. Comparative data regarding COX- 2/COX-1 selectivity showed that while compounds 18 and 7 were rather potent and selective COX-2 inhibitors, compound 20 was somehow less potent and selective for COX-2. Among the RC 0 derivatives, of particular interest resulted compounds SWE 74, CP 7 and CP 8, because they demonstrated to be fairly selective for COX-2 enzyme, but they appeared to be the weakest TP receptor antagonists among the 35 compounds tested. On the other hand, the last compound of interest was SWE 96, a molecule possessing a good activity on TPα receptor, but lacking selectivity in term of COX-2 inhibition, that is behaving like traditional NSAIDs. All the other derivatives tested were not selective COX-2 inhibitors and/or did not inhibit platelet aggregation. Taking advantage of what we learned in terms of structural requirements for COX-2 selective inhibition and TP antagonism, additional studies will certainly be carried out to improve the pharmacodynamic profile of these molecules before a careful evaluation can be considered in an in vivo animal model.