From calcium entry blockers to ryanodine receptors: a new therapeutic target in heart diseases.

Even if, calcium entry blockers were introduced many years ago in the treatment of cardiovascular disorders such as hypertension, atrial arrhythmia, and angina pectoris, they still remain key drugs. At present, isoform tissue -selective L-type calcium channel (LTCC) modulators might extend their pharmacotherapeutic potential as well as their established use in cardiovascular diseases. In particular, negative inotropic selective compounds could be useful in the treatment of hypertrophic obstructive cardiomyopathy (HOCM), otherwise known as idiopathic hypertrophic subaortic stenosis. In addition, compounds that can directly modulate RyR2 (ryanodine receptor type 2) or indirectly through voltage-gated calcium channels, are claimed as a new therapeutic approach to heart failure and lethal arrhythmia. With the aim to improve and mapping the ligand site of diltiazem and benzothiazepines to the α1 pore forming subunit of L-type calcium channel, we discovered a series of thiazinoxadiazolone derivatives structurally related to diltiazem (fig. 1) with selective negative inotropic activity. Recently we have applied a multidisciplinary approach based on a virtual screening procedure to discover novel and cardioselective compounds able to bind the benzothiazepine site in the LTCCs. Three novel chemotypes were characterized as LTCC blockers: different forms of the benzosulfonamide scaffold, the 3-phenyl-4-isoxazolecarboxylate and, the 4-phenyl-thiomorpholine-3,5-dione. Compound M8, that is [(4-chlorophenyl)sulfonyl]-2-(2-thienyl)pyrrolidine (fig. 1), was considered the most interesting compound for its negative inotropic effect and vasorelaxant properties. First, M8 was studied as racemate, then the two enantiomers were separated to evaluate their own biological proprieties. As expected, the functional profile observed for the two enantiomers was significantly different. Both the enantiomers have negative inotropic activity in the guinea-pig left atria, R-(+) being twice more potent than S-(–). On the contrary, the vasorelaxing properties tested in guinea pig aortic strips 80 mM K+-depolarized, is uniquely shown by S-(–)-M8, with a potency comparable to (±)-M8. No variation in activity between the two enantiomers was found in binding studies in rat cardiomyocytes and patch-clamp experiments in isolated myocytes from rat tail artery. Such different behavior suggested us an additional mechanism of action for the S-(–)-M8 enatiomer possessing vasorelaxant effect. To verify this hypothesis we began a series of experiments on guinea-pig aortic strips. In this model both (±)-M8 and the two enantiomers did not modify the concentration-response curve to noradrenalin in guinea-pig aortic strips. Otherwise, only S-(–)-M8 inhibited the aortic strip contraction evoked by noradrenalin in absence of extracellular Ca2+. These preliminary findings confirmed that S-(–)-M8 does not interfere with adrenergic receptors and, might exploit its vasorelaxat activity through the modulation of the intracellular Ca2+ release. This latter phenomenon is probably due to the opening of RyRs in the sarcoplasmic reticulum. Voltage-clamp and Ca2+ transient experiments indicate that (±)-M8 interferes with the intracellular Ca2+ handling probably owing to its activity at LTCC level that are coupled to RyRs. As above mentioned, it is well established that RyR2 leakage in cardyomyocites is the main cause of heart failure and lethal arrhythmia, consequently the compounds able to modulate RyRs prevent the intracellular Ca2+ leak and arrhythmias.