Advances in cholinesterase inhibitor research

Cholinesterase (ChE) inhibitors are crucial therapeutic agents for the symptomatic treatment of certain chronic neurodegenerative diseases linked to functional disorders of the cholinergic system. Significant research efforts have been made to develop novel derivatives of classical ChE inhibitors and ChE inhibitors with novel scaffolds. Over the past decade, ruthenium complexes have emerged as promising novel therapeutic alternatives for the treatment of neurodegenerative diseases. Our research group has investigated a number of newly synthesized organoruthenium(II) complexes for their inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Three complexes (C1a, C1-C, and C1) inhibit ChE in a pharmacologically relevant range. C1a reversibly inhibits AChE and BChE without undesirable peripheral effects, making it a promising candidate for the treatment of Alzheimer’s disease. C1-Cl complex reversibly and competitively inhibits ChEs, particularly AChE. It inhibits nerve-evoked skeletal muscle twitch and tetanic contraction in a concentration-dependent manner with no effect on directly elicited twitch and tetanic contraction and is promising for further preclinical studies as a competitive neuromuscular blocking agent. C1 is a selective, competitive, and reversible inhibitor of BChE that inhibits horse serum BChE (hsBChE) without significant effect on the peripheral neuromuscular system and is a highly species-specific inhibitor of hsBChE that could serve as a species-specific drug target. This research contributes to the expanding knowledge of ChE inhibitors based on ruthenium complexes and highlights their potential as promising therapeutic candidates for chronic neurodegenerative diseases

Alzheimerʼs disease

Alzheimerjeva bolezen je kronična progresivna nevrodegenerativna bolezen, za katero oboleva vse večje število ljudi po vsem svetu. Je najpogostejši vzrok za demenco (pri 50–80 % vseh primerov) in obenem tudi najhujša oblika demence, za katero so značilne kognitivne in vedenjske motnje pri ljudeh, večinoma starejših od 65 let. Patogeneza bolezni še ni popolnoma pojasnjena. Obstaja več hipotez, ki vključujejo kombinacijo genetskih in okoljskih dejavnikov ter načina življenja. Glede na začetek bolezni lahko bolezen opredelimo kot obliko z zgodnjim začetkom, običajno pred 65. letom starosti, in obliko s poznim začetkom, običajno po 65. letu starosti. Redka oblika bolezni z zgodnjim začetkom je povezana z mutacijami v genu za amiloidni prekurzorski protein ter v genih za presenilin 1 in 2. Genetski dejavnik tveganja za nastanek bolezni s poznim začetkom je prisotnost alela ε4 za apolipoprotein E. Za nastanek oblike s poznim začetkom obstaja več vzročnih hipotez: a) holinergična hipoteza, b) amiloidna hipoteza, c) hipoteza hiperfosforilaciji proteina tau, č) hipoteza o mitohondrijski kaskadi, d) vnetna hipoteza, e) nevro-vaskularna hipoteza, f) hipoteza o kovinskih ionih ter g) hipoteza limfnega sistema. Poleg tega obstajajo tudi presnovni in drugi dejavniki tveganja za nastanek Alzheimerjeve bolezni, med katere sodijo: zvišana raven holesterola v krvi, debelost, zvišan krvni tlak, sladkorna bolezen tipa 2, motnje spanja idr. Kljub velikemu številu raziskav etiopatogeneza do danes še ni dokončno pojasnjena, prav tako ne poznamo zdravila, ki bi preprečilo nastanek bolezni ali ustavilo njeno napredovanje. Zdravljenje Alzheimerjeve bolezni trenutno temelji na farmakološkem zdravljenju, ki poteka na osnovi doslej znane etiologije bolezni. Veliko obeta tudi imunoterapija z antiamiloidnimi protitelesi

Cyanobacterial cyclic peptides can disrupt cytoskeleton organization in human astrocytes – a contribution to the understanding of the systemic toxicity of cyanotoxins

The systemic toxicity of cyclic peptides produced by cyanobacteria (CCPs) is not yet completely understood. Apart from the most known damages to the liver and kidneys, symptoms of their neurotoxicity have also been reported. Hepatotoxic CCPs, like microcystins, as well as non-hepatotoxic anabaenopeptins and planktopeptins, all exhibit cytotoxic and cytostatic effects on mammalian cells. However, responses of different cell types to CCPs depend on their specific modes of interaction with cell membranes. This study demonstrates that non-hepatotoxic planktopeptin BL1125 and anabaenopeptins B and F, at concentrations up to 10 µM, affect normal and tumor human astrocytes (NHA and U87-GM) in vitro by their almost immediate insertion into the lipid monolayer. Like microcystin-LR (up to 1 µM), they inhibit Ser/Thr phosphatases and reorganize cytoskeletal elements, with modest effects on their gene expression. Based on the observed effects on intermediate filaments and intermediate filament linkage elements, their direct or indirect influence on tubulin cytoskeletons via post-translational modifications, we conclude that the basic mechanism of CCP toxicities is the induction of inter- and intracellular communication failure. The assessed inhibitory activity on Ser/Thr phosphatases is also crucial since the signal transduction cascades are modulated by phosphorylation/dephosphorylation processes

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 [(n6-pcymene) 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 endplate 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

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

Comparative electrophysiological characterization of ammodytoxin A, a β-neurotoxin from the nose-horned viper venom, and its enzymatically inactive mutant

Presynaptic- or β-neurotoxicity of secreted phospholipases A$_2$ (sPLA$_2$) is a complex process. For full expression of β-neurotoxicity, the enzymatic activity of the toxin is essential. However, it has been shown that not all toxic effects of a β-neurotoxin depend on its enzymatic activity, for example, the inhibition of mitochondrial cytochrome c oxidase. The main objective of this study was to verify whether it is possible to observe and study the phospholipase-independent actions of β-neurotoxins by a standard ex vivo twitch-tension experimental approach. To this end, we compared the effects of a potent snake venom β-neurotoxin, ammodytoxin A (AtxA), and its enzymatically inactive mutant AtxA(D49S) on muscle contraction of the mouse phrenic nerve-hemidiaphragm preparation. While AtxA significantly affected the amplitude of the indirectly evoked isometric muscle contraction, the resting tension of the neuromuscular (NM) preparation, the amplitude of the end-plate potential (EPP), the EPP half decay time and the resting membrane potential, AtxA(D49S) without enzymatic activity did not. From this, we can conclude that the effects of AtxA independent of enzymatic activity cannot be studied with classical electrophysiological measurements on the isolated NM preparation. Our results also suggest that the inhibition of cytochrome c oxidase activity by AtxA is not involved in the rapid NM blockade by this β-neurotoxin, but that its pathological consequences are rather long-term. Interestingly, in our experimental setup, AtxA upon direct stimulation reduced the amplitude of muscle contraction and induced contracture of the hemidiaphragm, effects that could be interpreted as myotoxic

Cyanobacterial cyclic peptides can disrupt cytoskeleton organization in human astrocytes

The systemic toxicity of cyclic peptides produced by cyanobacteria (CCPs) is not yet completely understood. Apart from the most known damages to the liver and kidneys, symptoms of their neurotoxicity have also been reported. Hepatotoxic CCPs, like microcystins, as well as non-hepatotoxic anabaenopeptins and planktopeptins, all exhibit cytotoxic and cytostatic effects on mammalian cells. However, responses of different cell types to CCPs depend on their specific modes of interaction with cell membranes. This study demonstrates that non-hepatotoxic planktopeptin BL1125 and anabaenopeptins B and F, at concentrations up to 10 µM, affect normal and tumor human astrocytes (NHA and U87-GM) in vitro by their almost immediate insertion into the lipid monolayer. Like microcystin-LR (up to 1 µM), they inhibit Ser/Thr phosphatases and reorganize cytoskeletal elements, with modest effects on their gene expression. Based on the observed effects on intermediate filaments and intermediate filament linkage elements, their direct or indirect influence on tubulin cytoskeletons via post-translational modifications, we conclude that the basic mechanism of CCP toxicities is the induction of inter- and intracellular communication failure. The assessed inhibitory activity on Ser/Thr phosphatases is also crucial since the signal transduction cascades are modulated by phosphorylation/dephosphorylation processes

Structural and functional characterization of an organometallic ruthenium complex as a potential myorelaxant drug

In addition to antibacterial and antitumor effects, synthetic ruthenium complexes have been reported to inhibitseveral medicinally important enzymes, including acetylcholinesterase (AChE). They may also interact withmuscle-type nicotinic acetylcholine receptors (nAChRs) and thus affect the neuromuscular transmission andmuscle function. In the present study, the effects of the organometallic ruthenium complex of 5-nitro-1,10-phenanthroline (nitrophen) were evaluated on these systems. The organoruthenium-nitrophen complex [(η6-p-cymene)Ru(nitrophen)Cl]ClC22H21Cl2N3O2Ru (C1-Cl) was synthesized, structurally characterized and eval-uatedin vitrofor its inhibitory activity against electric eel acetylcholinesterase (eeAChE), human recombinantacetylcholinesterase (hrAChE), horse serum butyrylcholinesterase (hsBChE) and horse liver glutathione-S-transferase. The physiological effects of C1-Cl were then studied on isolated mouse phrenic nerve-hemi-diaphragm muscle preparations, by means of single twitch measurements and electrophysiological recordings.The compound C1-Cl acted as a competitive inhibitor of eeAChE, hrAChE and hsBChE with concentrationsproducing 50 % inhibition (IC50) of enzyme activity ranging from 16 to 26μM. Moreover, C1-Cl inhibited thenerve-evoked isometric muscle contraction (IC50= 19.44μM), without affecting the directly-evoked musclesingle twitch up to 40μM. The blocking effect of C1-Cl was rapid and almost completely reversed by neos-tigmine, a reversible cholinesterase inhibitor. The endplate potentials were also inhibited by C1-Cl in a con-centration-dependent manner (IC50= 7.6μM) without any significant change in the resting membrane potentialof musclefibers up to 40μM. Finally, C1-Cl (5–40μM) decreased (i) the amplitude of miniature endplatepotentials until a complete block by concentrations higher than 25μM and (ii) their frequency at 10μM or higherconcentrations. The compound C1-Cl reversibly blocked the neuromuscular transmissionin vitroby a non-de-polarizing mechanism and mainly through an action on postsynaptic nAChRs. The compound C1-Cl may betherefore interesting for further preclinical testing as a new competitive neuromuscular blocking, and thusmyorelaxant, dru

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, [(η$^6$-p-cymene)Ru(II)(5-nitro-1,10-phenanthroline)Cl]Cl (C1–Cl), [(η$^6$-p-cymene)Ru(II)(1-hydroxypyridine-2(1H)-thionato)Cl] (C1a) and [(η$^6$-p-cymene)Ru(II)(1-hydroxy-3-methoxypyridine-2(1H)-thionato)pta]PF$_6$ (C1), on muscle-subtype (Torpedo) nAChRs and on the two most abundant human neuronal-subtype nAChRs in the CNS (α4β2 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 α4β2 and α7 nAChRs. Similar results of C1 effects were obtained on Torpedo and α4β2 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