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 [(n⁶-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.Centro de Química Inorgánic

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 [(n⁶-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.Centro de Química Inorgánic

Interactions of new ruthenium organometallic complexes with mammalian cholinergic system

Iz knjižnice novosintetiziranih organorutenijevih(II) arenskih kompleksov smo z biokemično karakterizacijo določili komplekse, ki: i) selektivno zavirajo butirilholinesterazo (BChE)ii) zavirajo holinesterazeiii) zavirajo holinesteraze in glutation S-transferaze (GST) in iv) selektivno zavirajo GST. Z elektrofiziološkimi metodami smo na živčno-mišičnih preparatih mišjih hemidiafragem proučili vpliv izbranih organorutenijevih(II) kompleksov ([(?6-p-cimen)Ru(II)(1-hidroksi-3-metoksipiridin-2(1H)-tionato)pta]PF6 (C1)) in ([(?6-p-cimen)Ru(II)(5-nitro-1,10-fenantrolin)Cl]Cl (C1-Cl) z ugotovljeno antiholinesterazno aktivnostjo v nizkem mikromolarnem območju na krčenje in membranske potenciale mišičnih vlaken. Ugotovili smo, da kompleks C1, ki v pogojih in vitro selektivno zavre encim BChE, ex vivo v najvišji uporabljeni koncentraciji (120 µM) ne vpliva na amplitudo posredno in neposredno izzvane enostavne in tetanične izometrične kontrakcije. Poleg tega tudi ne vpliva na mirovni membranski potencial (rVm) skeletnih mišičnih vlaken in na amplitudo potenciala motorične ploščice (PMP). Največja koncentracija (120 µM) kompleksa C1 značilno zmanjša le amplitudo ter skrajša razpolovni čas padajoče faze spontanih miniaturnih potencialov motorične ploščice in razpolovni čas padajoče faze PMP. C1 pri zelo visoki koncentraciji (120 µM) ne zavira pomembno acetilholinesteraze (AChE) in nikotinskih acetilholinskih receptorjev (nAChR), prisotnih v živčno-mišičnem stiku, in s tem ne vpliva na funkcijo perifernega živčno-mišičnega sistema. Za kompleks C1-Cl, ki in vitro zavira delovanje holinesteraz, je elektrofiziološka karakterizacija pokazala, da kompleks v nizkih mikromolarnih koncentracijah reverzibilno zavre posredno izzvano enostavno (IC50 = 19,44 µM) in tetanično (IC50 = 9,68 µM) izometrično kontrakcijo in zmanjša amplitude PMP (IC50 = 7,61 µM), pri čemer se rVm skeletnih mišičnih vlaken ne spremeni. Amplituda mišične kontrakcije se je po dodatku 3 µM neostigmina, ki je povratni zaviralec AChE, izjemno hitro povrnila, kar kaže selektivno kompetitivno zaviralno delovanje kompleksa C1-Cl na posinaptične nAChR mišičnega tipa. To smo potrdili tudi z metodo vpete napetosti na oocitih navadne krempljarke (Xenopus laevis) z meritvami ionskih tokov skozi v membrani mikroimplantirane nAChR iz navadnega električnega skata (Torpedo marmorata). Ugotovili smo, da C1-Cl deluje kot povratni antagonist nAChR iz navadnega električnega skata (IC50 = 332 nM). Elektrofiziološka karakterizacija kompleksa C1-Cl je tako pokazala, da kompleks ex vivo v farmakološko zanimivih koncentracijah s povratno vezavo na posinaptične nAChR prek nedepolarizirajočega mehanizma zavre živčno-mišični prenos. Zato je kompleks C1-Cl zanimiv za nadaljnja predklinična testiranja kot nov nedepolarizirajoč miorelaksant za potencialno uporabo v veterinarski in humani medicini.From the library of newly synthesized organoruthenium(II) arene complexes, we identified biochemical characterization complexes that i) selectively inhibit butyrylcholinesterase (BChE)ii) inhibit cholinesterasesiii) 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 junctionsthus, 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

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

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

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

Inhibition of butyrylcholinesterase activity by abamectin and boscalid: computational and in vitro studies

Rastući trend za poljoprivrednim proizvodima doveo je do pogrešne upotrebe pesticida. Butirilholinesteraza (BChE) sprečava određene pesticide da nepovratno inhibiraju acetilholinesterazu (AChE), drugi enzim ključan za neurotransmisiju. Za naša istraživanja smo izabrali često korišćene pesticide abamektin i boskalid. Naša in silico i in vitro istraživanja su pokazala da BChE iz Homo sapiens-a nije meta za abamektine B1A i B1B. Boskalid je pokazao gori Glide score (-6.88 kcal/mol) nego potvrđeni inhibitor BChE (2- ((1-(benzensulfonil)-1H-indol-4-il)oksi)etil)(benzil)amin sa IC50=0.473 μM (Glide score = -8.64 kcal/mol), tako da nije iznenađujuće da je njegov IC50 značajno viši. Ova studija naglašava zaštitnu ulogu BChE protiv određenih pesticida.The growing demand for agricultural products has led to the misuse of pesticides. Butyrylcholinesterase (BChE) prevents some pesticides from irreversibly inhibiting acetylcholinesterase (AChE), another enzyme crucial for neurotransmission. For our studies, we choose commonly used pesticides abamectin and boscalid. The Homo sapiens BChE was shown not to be a target for abamectins B1A and B1B either in silico or in vitro. Boscalid showed a worse Glide score (-6.88 kcal/mol) than the approved BChE inhibitor (2-((1-(benzenesulfonyl)-1H-indol-4-yl)oxy)ethyl)(benzyl)amine with an IC50 value of 0.473 μM (Glide score=-8.64 kcal/mol), so it is not surprising that its IC50 is significantly higher. This study highlights the protective role of BChE against certain pesticides.60th Meeting of the Serbian Chemical Society; June 8-9, 2024, Niš, Serbia

Development of potent cholinesterase inhibitors based on a marine pharmacophore

The management of neurological disorders such as dementia associated with Alzheimer’s or Parkinson’s disease includes the use of cholinesterase inhibitors. These compounds can slow down the progression of these diseases and can also be used in the treatment of glaucoma and myasthenia gravis. The majority of the cholinesterase inhibitors used in the clinic are derived from natural products and our current paper describes the use of a small marine pharmacophore to develop potent and selective cholinesterase inhibi- tors. Fourteen small inhibitors were designed based on recent discoveries about the inhibitory potential of a range of related marine secondary metabolites. The compounds were evaluated, in kinetic enzymatic assays, for their ability to inhibit three different cholinesterase enzymes and it was shown that compounds with a high inhibitory activity towards electric eel and human recombinant acetylcholinesterase (IC50 between 20–70 μM) could be prepared. It was also shown that this compound class was particularly active against horse serum butyrylcholinesterase, with IC50 values between 0.8–16 μM, which is an order of magnitude more potent than the clinically used positive control neostigmine. The compounds were further tested for off-target toxicity against both human umbilical vein endothelial cells and bovine and human erythrocytes and were shown to display a low mammalian cellular toxicity. Overall, the study illus- trates how the brominated dipeptide marine pharmacophore can be used as a versatile natural scaffold for the design of potent, and selective cholinesterase inhibitors

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

Using Virtual AChE Homology Screening to Identify Small Molecules With the Ability to Inhibit Marine Biofouling

The search for effective yet environmentally friendly strategies to prevent marine biofouling is hampered by the large taxonomic diversity amongst fouling organisms and a lack of well-defined conserved molecular targets. The acetylcholinesterase enzyme catalyses the breakdown of the neurotransmitter acetylcholine, and several natural antifouling allelochemicals have been reported to display acetylcholinesterase inhibitory activity. Our study is focussed on establishing if acetylcholinesterase can be used as a well-defined molecular target to accelerate discovery and development of novel antifoulants via sequential high-throughput in silico screening, in vitro enzymatic studies of identified compound libraries, and in vivo assessment of the most promising lead compounds. Using this approach, we identified potent cholinesterase inhibitors with inhibitory concentrations down to 3 μM from a 10,000 compound library. The most potent inhibitors were screened against five microfouling marine bacteria and marine microalgae and the macrofouling tunicate Ciona savignyi. No activity was seen against the microfoulers but a potent novel inhibitor of tunicate settlement and metamorphosis was discovered. Although only one of the identified active cholinesterase inhibitors displayed antifouling activity suggesting the link between cholinesterase inhibition and antifouling is limited to certain compound classes, the study highlights how in silico screening employed regularly for drug discovery can also facilitate discovery of antifouling leads