New insights into the effects of organometallic ruthenium complexes on nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) are expressed in excitable and non-excitable cells of the organism. Extensive studies suggest that nAChR ligands have therapeutic potential, notably for neurological and psychiatric disorders. Organometallic ruthenium complexes are known to inhibit several medically important enzymes such as cholinesterases. In addition, they can also interact with muscle- and neuronal-subtype nAChRs. The present study aimed to investigate the direct effects of three organometallic ruthenium complexes, [(η6-p-cymene)Ru(II)(5-nitro-1,10-phenanthroline)Cl]Cl (C1-Cl), [(η6-p-cymene)Ru(II)(1-hydroxypyridine-2(1H)-thionato)Cl] (C1a) and [(η6-p-cymene)Ru(II)(1-hydroxy-3-methoxypyridine-2(1H)-thionato)pta]PF6 (C1), on muscle-subtype (Torpedo) nAChRs and on the two most abundant human neuronal-subtype nAChRs in the CNS (α4ß2 and α7) expressed in Xenopus laevis oocytes, using the two-electrode voltage-clamp. The results show that none of the three compounds had agonistic activity on any of the nAChR subtypes studied. In contrast, C1-Cl reversibly blocked Torpedo nAChR (half-reduction of ACh-evoked peak current amplitude by 332 nM of compound). When tested at 10 µM, C1-Cl was statistically more potent to inhibit TorpedonAChR than α4ß2 and α7 nAChRs. Similar results of C1 effects were obtained on Torpedo and α4ß2 nAChRs, while no action of the compound was detected on α7 nAChRs. Finally, the effects of C1a were statistically similar on the three nAChR subtypes but, in contrast to C1-Cl and C1, the inhibition was hardly reversible. These results, together with our previous studies on isolated mouse neuromuscular preparations, strongly suggest that C1-Cl is, among the three compounds studied, the only molecule that could be used as a potential myorelaxant drug.

Probing Alzheimer's pathology: Exploring the next generation of FDDNP analogues for amyloid ß detection.

Fluorescent probes are a powerful tool for imaging amyloid ß (Aß) plaques, the hallmark of Alzheimer's disease (AD). Herein, we report the synthesis and comprehensive characterization of 21 novel probes as well as their optical properties and binding affinities to Aß fibrils. One of these dyes, 1Ae, exhibited several improvements over FDDNP, an established biomarker for Aß- and Tau-aggregates. First, 1Ae had large Stokes shifts (138-213 nm) in various solvents, thereby reducing self-absorption. With a high quantum yield ratio (φ(dichloromethane/methanol) = 104), 1Ae also ensures minimal background emission in aqueous environments and high sensitivity. In addition, compound 1Ae exhibited low micromolar binding affinity to Aß fibrils in vitro (Kd = 1.603 µM), while increasing fluorescence emission (106-fold) compared to emission in buffer alone. Importantly, the selective binding of 1Ae to Aß1-42 fibrils was confirmed by an in cellulo assay, supported by ex vivo fluorescence microscopy of 1Ae on postmortem AD brain sections, allowing unequivocal identification of Aß plaques. The intermolecular interactions of fluorophores with Aß were elucidated by docking studies and molecular dynamics simulations. Density functional theory calculations revealed the unique photophysics of these rod-shaped fluorophores, with a twisted intramolecular charge transfer (TICT) excited state. These results provide valuable insights into the future application of such probes as potential diagnostic tools for AD in vitro and ex vivo such as determination of Aß1-42 in cerebrospinal fluid or blood.

Exploring pta Alternatives in the Development of Ruthenium-Arene Anticancer Compounds.

Organoruthenium pyrithione (1-hydroxypyridine-2-thione) complexes have been shown in our recent studies to be a promising family of compounds for development of new anticancer drugs. The complex [(η6-p-cymene)Ru(pyrithionato)(pta)]PF6 contains phosphine ligand pta (1,3,5-triaza-7-phosphaadamantane) as a functionality that improves the stability of the complex and its aqueous solubility. Here, we report our efforts to find pta alternatives and discover new structural elements to improve the biological properties of ruthenium anticancer drugs. The pta ligand was replaced by a selection of phosphine, phosphite, and arsine ligands to identify new functionalities, leading to improvement in inhibitory potency towards enzyme glutathione S-transferase. In addition, cytotoxicity in breast, bone, and colon cancers was investigated.

Novel Organoruthenium(II) Complex C1 Selectively Inhibits Butyrylcholinesterase without Side Effects on Neuromuscular Transmission.

Enzyme butyrylcholinesterase (BChE) shows increased activity in some brain regions after progression of Alzheimer's disease and is therefore one of the therapeutic targets for symptomatic treatment of this neurodegenerative disorder. The organoruthenium(II) complex [(η6-p-cymene)Ru(II)(1-hydroxy-3-methoxypyridine-2(1H)-thionato)pta]PF6 (C1) was designed based on the results of our previous structure-activity studies. Inhibitory activity toward cholinesterase enzymes shows that this complex selectively, competitively, and reversibly inhibits horse serum BChE (hsBChE) with an IC50 value of 2.88 µM. When tested at supra-pharmacological concentrations (30, 60, 90, and 120 µM), C1 had no significant effect on the maximal amplitude of nerve-evoked and directly elicited single-twitch and tetanic contractions. At the highest tested concentration (120 µM), C1 had no effect on resting membrane potential, but significantly decreased the amplitude of miniature end-plate potentials (MEPP) without reducing their frequency. The same concentration of C1 had no effect on the amplitude of end-plate potentials (EPP), however it shortened the half-decay time of MEPPs and EPPs. The decrease in the amplitude of MEPPs and shortening of the half-decay time of MEPPs and EPPs suggest a possible weak inhibitory effect on muscle-type nicotinic acetylcholine receptors (nAChR). These combined results show that, when applied at supra-pharmacological concentrations up to 120 µM, C1 does not importantly affect the physiology of neuromuscular transmission and skeletal muscle contraction.

Zinc pyrithione is a potent inhibitor of PLPro and cathepsin L enzymes with ex vivo inhibition of SARS-CoV-2 entry and replication.

Zinc pyrithione (1a), together with its analogues 1b-h and ruthenium pyrithione complex 2a, were synthesised and evaluated for the stability in biologically relevant media and anti-SARS-CoV-2 activity. Zinc pyrithione revealed potent in vitro inhibition of cathepsin L (IC50=1.88 ± 0.49 µM) and PLPro (IC50=0.50 ± 0.07 µM), enzymes involved in SARS-CoV-2 entry and replication, respectively, as well as antiviral entry and replication properties in an ex vivo system derived from primary human lung tissue. Zinc complexes 1b-h expressed comparable in vitro inhibition. On the contrary, ruthenium complex 2a and the ligand pyrithione a itself expressed poor inhibition in mentioned assays, indicating the importance of the selection of metal core and structure of metal complex for antiviral activity. Safe, effective, and preferably oral at-home therapeutics for COVID-19 are needed and as such zinc pyrithione, which is also commercially available, could be considered as a potential therapeutic agent against SARS-CoV-2.

Nanosecond electric pulses are equally effective in electrochemotherapy with cisplatin as microsecond pulses.

BACKGROUND: Nanosecond electric pulses showed promising results in electrochemotherapy, but the underlying mechanisms of action are still unexplored. The aim of this work was to correlate cellular cisplatin amount with cell survival of cells electroporated with nanosecond or standardly used 8 × 100 µs pulses and to investigate the effects of electric pulses on cisplatin structure. MATERIALS AND METHODS: Chinese hamster ovary CHO and mouse melanoma B16F1 cells were exposed to 1 × 200 ns pulse at 12.6 kV/cm or 25 × 400 ns pulses at 3.9 kV/cm, 10 Hz repetition rate or 8 × 100 µs pulses at 1.1 (CHO) or 0.9 (B16F1) kV/cm, 1 Hz repetition rate at three cisplatin concentrations. Cell survival was determined by the clonogenic assay, cellular platinum was measured by inductively coupled plasma mass spectrometry. Effects on the structure of cisplatin were investigated by nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. RESULTS: Nanosecond pulses equivalent to 8 × 100 µs pulses were established in vitro based on membrane permeabilization and cell survival. Equivalent nanosecond pulses were equally efficient in decreasing the cell survival and accumulating cisplatin intracellularly as 8 × 100 µs pulses after electrochemotherapy. The number of intracellular cisplatin molecules strongly correlates with cell survival for B16F1 cells, but less for CHO cells, implying the possible involvement of other mechanisms in electrochemotherapy. The high-voltage electric pulses did not alter the structure of cisplatin. CONCLUSIONS: Equivalent nanosecond pulses are equally effective in electrochemotherapy as standardly used 8 × 100 µs pulses.

Pyrithione metal (Cu, Ni, Ru) complexes as photo-catalysts for styrene oxide production.

Selective photochemical oxidation of styrene was performed in an active acetonitrile medium, using H2O2 with or without ultraviolet (UV) light radiation. Pyrithione metal complexes (M-Pth: M = Cu(II), Ni(II), Ru(II); Pth = 2-mercaptopyridine-N-oxide) were used as catalysts. Catalytic testing measurements were done by varying the time, chemical reaction temperature and H2O2 concentration with or without UV energy. Epoxide styrene oxide (SO), benzaldehyde and acetophenone were the major synthesized products. A high batch rate, conversion and selectivity towards SO was shown in the presence of UV. A minor constant formation of CO2 was observed in the stream. Coordinated Ru-based compounds demonstrated the highest process productivity of SO at 60 °C. The effect of the functional alkyl substituent on the ligand Pth, attached to the specific ruthenium(II) centre, decreased the activity of the substance. Ni-Pth selectively yielded benzaldehyde. The stability of the catalysts was examined by applying nuclear magnetic resonance (NMR) spectroscopy and thermogravimetric analysis coupled with mass spectrometry. Tested metal complexes with pyrithione (M-Pth) exhibited excellent reuse recyclability up to 3 cycles.

Fine Tuning of Cholinesterase and Glutathione-S-Transferase Activities by Organoruthenium(II) Complexes.

Cholinesterases (ChEs) show increased activities in patients with Alzheimer's disease, and remain one of the main therapeutic targets for treatment of this neurodegenerative disorder. A library of organoruthenium(II) complexes was prepared to investigate the influence of their structural elements on inhibition of ChEs, and on another pharmacologically important group of enzymes, glutathione S-transferases (GSTs). Two groups of organoruthenium(II) compounds were considered: (i) organoruthenium(II) complexes with p-cymene as an arene ligand, and (ii) organoruthenium(II) carbonyl complexes as CO-releasing molecules. Eight organoruthenium complexes were screened for inhibitory activities against ChEs and GSTs of human and animal origins. Some compounds inhibited all of these enzymes at low micromolar concentrations, while others selectively inhibited either ChEs or GSTs. This study demonstrates the importance of the different structural elements of organoruthenium complexes for their inhibitory activities against ChEs and GSTs, and also proposes some interesting compounds for further preclinical testing as ChE or GST inhibitory drugs.

Organoruthenium Complexes with Benzo-Fused Pyrithiones Overcome Platinum Resistance in Ovarian Cancer Cells.

Drug resistance to existing anticancer agents is a growing clinical concern, with many first line treatments showing poor efficacy in treatment plans of some cancers. Resistance to platinum agents, such as cisplatin, is particularly prevalent in the treatment of ovarian cancer, one of the most common cancers amongst women in the developing world. Therefore, there is an urgent need to develop next generation of anticancer agents which can overcome resistance to existing therapies. We report a new series of organoruthenium(II) complexes bearing structurally modified pyrithione ligands with extended aromatic scaffold, which overcome platinum and adriamycin resistance in human ovarian cancer cells. The mechanism of action of such complexes appears to be unique from that of cisplatin, involving G1 cell cycle arrest without generation of cellular ROS, as is typically associated with similar ruthenium complexes. The complexes inhibit the enzyme thioredoxin reductase (TrxR) in a model system and reduce cell motility towards wound healing. Importantly, this work highlights further development in our understanding of the multi-targeting mechanism of action exhibited by transition metal complexes.