From the library of newly synthesized organoruthenium(II) arene complexes, we identified biochemical characterization complexes that i) selectively inhibit butyrylcholinesterase (BChE); ii) inhibit cholinesterases; iii) inhibit cholinesterases and glutathione S-transferases (GSTs) or iv) selectively inhibit GSTs. Two selected organoruthenium(II) complexes ([(η6-p-cymene)Ru(II)(1-hydroxy-3-methoxypyridine-2(1H)-thionato)pta]PF6 (C1) and [(η6-p-cymene)Ru(II)(5-nitro-1,10-phenanthroline)Cl]Cl (C1-Cl) with anticholinesterase activity in the low micromolar range were studied with electrophysiological methods on mouse neuromuscular hemidiaphragm preparations for their effect on contractility and membrane potentials. We found that complex C1 selectively inhibited BChE in vitro but exhibited no effect on the amplitude of directly muscle-elicited and nerve-evoked single twitch and tetanic isometric contraction ex vivo at the highest concentration used (120 µM). Furthermore, C1 also had no effect on the resting membrane potential (rVm) of skeletal muscle fibres and the amplitudes of the end-plate potentials (EPPs). The 120 µM concentration of complex C1 decreased only the amplitude and shortened the half-decay time of the miniature end-plate potentials and the half-decay time of the EPPs. Complex C1 had no significant antagonistic effects on acetylcholinesterase (AChE) activity and the nicotinic acetylcholine receptors (nAChRs) present in neuromuscular junctions; thus, C1 did not affect the function of the peripheral neuromuscular system. For the C1-Cl complex, which inhibits cholinesterase activity in vitro, electrophysiological characterization showed that the complex reversibly inhibited nerve-evoked single twitch (IC50 = 19.44 μM) and tetanic (IC50 = 9.68 μM) isometric contraction at low, micromolar concentrations and decreases the amplitudes of EPPs (IC50 = 7.61 μM) without any significant change in the rVm of skeletal muscle fibres. The amplitude of muscle contraction recovered rapidly after applying the reversible AChE inhibitor neostigmine (3 µM), strongly suggesting that the C1-Cl complex exhibits a selective competitive antagonistic effect on postsynaptic muscle-type nAChRs. We also confirmed antagonistic activity by measuring ion currents through nAChRs in the presence of C1-Cl with a two-electrode voltage clamp on Xenopus laevis oocytes with nAChRs from Torpedo marmorata microimplanted in their membrane. We found that complex C1-Cl is a reversible antagonist of Torpedo nAChRs (IC50 = 332 nM). Electrophysiological characterization of the C1-Cl complex showed that at concentrations of pharmaceutical interest, the complex inhibits neuromuscular transmission ex vivo by a nondepolarizing mechanism through reversible binding on postsynaptic muscle-type nAChRs. Therefore, the C1-Cl complex may be interesting for further preclinical testing as a novel nondepolarizing myorelaxant for potential use in veterinary and human medicine.
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