Tacrine, the first approved drug against Alzheimer's disease (AD), was withdrawn from clinical use due to serious adverse effects. The main concern was the human hepatotoxicity, stemming probably from the liver biotransformation and clinically manifested as hepatocellular necrosis, and lobular hepatitis. Concerning the biotransformation, 7-OH-tacrine metabolite is generally suspected of being a precursor of toxic quinone methide, which binds to intracellular -SH proteins and/or depletes intracellular glutathione, and by that probably causes the hepatotoxicity. However, to study these toxic effects, proper animal model is needed to monitor the interspecies differences of metabolism. To fully describe in vivo ADMET parameters of tacrine, five experimental pigs (Sus scrofa f. domestica), as the most physiologically human-like in vivo model showing similar tacrine biotransformation, were used. We studied tacrine and its metabolites ADMET characteristics after both acute i.g. single dose and chronic 42 days p.o daily dose administration of 200 mg of tacrine. Tacrine and its two major metabolites show Tmax in plasma of 360 min, so the absorption is much slower than in human (Tmax = 120 min) and are primarily distributed to the gastro-intestinal tract and CNS. Furthermore, due to the lower activity of CYP1A2 in pigs, tacrine is biotransformed much less efficiently than in humans. This study showed that tacrine accumulates only in adipose tissue, and organ histology and plasma biochemistry assessment revealed no signs of hepatotoxicity even after chronic tacrine administration. Pigs are therefore an unsuitable human-like animal model for evaluating tacrine toxicity.
- MeSH
- biotransformace MeSH
- cholinesterasové inhibitory toxicita farmakokinetika MeSH
- játra * účinky léků metabolismus patologie MeSH
- lékové postižení jater * metabolismus patologie MeSH
- lidé MeSH
- prasata MeSH
- Sus scrofa MeSH
- takrin * farmakokinetika toxicita metabolismus analogy a deriváty MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Alzheimer's disease (AD) is a multifaceted neurodegenerative disorder for which current treatments provide only symptomatic relief, primarily through cholinesterase (ChE) inhibition and N-methyl-d-aspartate receptor (NMDAR) antagonism. To improve therapeutic efficacy and safety, we designed and synthesized 16 novel tacrine derivatives modified at position 7 with various (hetero)aryl groups or deuterium substitution. Initially, in silico screening predicted favorable CNS permeability and oral bioavailability. Subsequent in vitro evaluations demonstrated significant inhibitory potency against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), with derivatives 5i and 5m displaying particularly promising profiles. Metabolic stability assessed using human liver microsomes revealed enhanced stability for compound 5e, whereas 5i and 5m underwent rapid metabolism. Notably, compound 7 showed improved metabolic stability attributed to deuterium incorporation. The newly synthesized compounds were further tested for antagonistic activity on the GluN1/GluN2B subtype of NMDAR, with compound 5m exhibiting the most potent and voltage-independent inhibition. The ability of these compounds to permeate the blood-brain barrier (BBB) was confirmed through in vitro PAMPA assays. In preliminary hepatotoxicity screening (HepG2 cells), most derivatives exhibited higher cytotoxicity than tacrine, emphasizing the ongoing challenge in hepatotoxicity management. Based on its overall favorable profile, compound 5m advanced to in vivo pharmacokinetic studies in mice, demonstrating efficient CNS penetration, with brain concentrations exceeding plasma levels (brain-to-plasma ratio 2.36), indicating active transport across the BBB. These findings highlight compound 5m as a promising tacrine-based multi-target-directed ligand, supporting further preclinical development as a potential therapeutic candidate for AD.
- MeSH
- acetylcholinesterasa metabolismus MeSH
- Alzheimerova nemoc * farmakoterapie metabolismus MeSH
- biologická dostupnost MeSH
- butyrylcholinesterasa metabolismus MeSH
- cholinesterasové inhibitory * farmakologie chemie chemická syntéza MeSH
- hematoencefalická bariéra metabolismus MeSH
- jaterní mikrozomy metabolismus MeSH
- lidé MeSH
- ligandy MeSH
- molekulární struktura MeSH
- myši MeSH
- receptory N-methyl-D-aspartátu * antagonisté a inhibitory metabolismus MeSH
- takrin * farmakologie chemie chemická syntéza MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Therapeutic options for Alzheimer's disease are limited. Dual compounds targeting two pathways concurrently may enable enhanced effect. The study focuses on tacrine derivatives inhibiting acetylcholinesterase (AChE) and simultaneously N-methyl-D-aspartate (NMDA) receptors. Compounds with balanced inhibitory potencies for the target proteins (K1578 and K1599) or increased potency for AChE (K1592 and K1594) were studied to identify the most promising pro-cognitive compound. Their effects were studied in cholinergic (scopolamine-induced) and glutamatergic (MK-801-induced) rat models of cognitive deficits in the Morris water maze. Moreover, the impacts on locomotion in the open field and AChE activity in relevant brain structures were investigated. The effect of the most promising compound on NMDA receptors was explored by in vitro electrophysiology. The cholinergic antagonist scopolamine induced a deficit in memory acquisition, however, it was unaffected by the compounds, and a deficit in reversal learning that was alleviated by K1578 and K1599. K1578 and K1599 significantly inhibited AChE in the striatum, potentially explaining the behavioral observations. The glutamatergic antagonist dizocilpine (MK-801) induced a deficit in memory acquisition, which was alleviated by K1599. K1599 also mitigated the MK-801-induced hyperlocomotion in the open field. In vitro patch-clamp corroborated the K1599-associated NMDA receptor inhibitory effect. K1599 emerged as the most promising compound, demonstrating pro-cognitive efficacy in both models, consistent with intended dual effect. We conclude that tacrine has the potential for development of derivatives with dual in vivo effects. Our findings contributed to the elucidation of the structural and functional properties of tacrine derivatives associated with optimal in vivo pro-cognitive efficacy.
- MeSH
- acetylcholinesterasa metabolismus MeSH
- antagonisté excitačních aminokyselin farmakologie MeSH
- bludiště - učení * účinky léků MeSH
- cholinesterasové inhibitory * farmakologie MeSH
- dizocilpinmaleát * farmakologie MeSH
- kognice * účinky léků MeSH
- krysa rodu rattus MeSH
- paměť účinky léků MeSH
- potkani Wistar * MeSH
- receptory N-methyl-D-aspartátu * antagonisté a inhibitory metabolismus MeSH
- skopolamin MeSH
- takrin * farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Tacrine (THA), a long withdrawn drug, is still a popular scaffold used in medicinal chemistry, mainly for its good reactivity and multi-targeted effect. However, THA-associated hepatotoxicity is still an issue and must be considered in drug discovery based on the THA scaffold. Following our previously identified hit compound 7-phenoxytacrine (7-PhO-THA), we systematically explored the chemical space with 30 novel derivatives, with a focus on low hepatotoxicity, anticholinesterase action, and antagonism at the GluN1/GluN2B subtype of the NMDA receptor. Applying the down-selection process based on in vitro and in vivo pharmacokinetic data, two candidates, I-52 and II-52, selective GluN1/GluN2B inhibitors thanks to the interaction with the ifenprodil-binding site, have entered in vivo pharmacodynamic studies. Finally, compound I-52, showing only minor affinity to AChE, was identified as a lead candidate with favorable behavioral and neuroprotective effects using open-field and prepulse inhibition tests, along with scopolamine-based behavioral and NMDA-induced hippocampal lesion models. Our data show that compound I-52 exhibits low toxicity often associated with NMDA receptor ligands, and low hepatotoxicity, often related to THA-based compounds.
- MeSH
- acetylcholinesterasa metabolismus MeSH
- Alzheimerova nemoc * farmakoterapie MeSH
- cholinesterasové inhibitory chemie MeSH
- cholinesterasy MeSH
- lékové postižení jater * MeSH
- lidé MeSH
- neuroprotektivní látky * farmakologie terapeutické užití MeSH
- piperidiny * MeSH
- receptory N-methyl-D-aspartátu MeSH
- takrin chemie MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Tacrine was the first drug used in the therapy of Alzheimer's disease (AD) and is one of the leading structures frequently pursued in the drug discovery of novel candidates for tackling AD. However, because tacrine has been withdrawn from the market due to its hepatotoxicity, ascribed to specific metabolites, concerns are high about the toxicity profile of newly developed compounds related to tacrine. From the point of view of drug safety, the formation of metabolites must be uncovered and analyzed. Bearing in mind that the main culprit of tacrine hepatotoxicity is its biotransformation to hydroxylated metabolites, human liver microsomes were used as a biotransformation model. Our study aims to clarify phase I metabolites of three potentially non-toxic tacrine derivatives (7-methoxytacrine, 6-chlorotacrine, 7-phenoxytacrine) and to semi-quantitatively determine the relative amount of individual metabolites as potential culprits of tacrine-based hepatotoxicity. For this purpose, a new selective UHPLC-Orbitrap method has been developed. Applying UHPLC-Orbitrap method, two as yet unpublished tacrine and 7-methoxytacrine monohydroxylated metabolites have been found and completely characterized, and the separation of ten dihydroxylated tacrine and 7-methoxytacrine metabolites was achieved for the first time. Moreover, the structures of several new metabolites of 7-phenoxytacrine and 6-chlorotacrine have been identified. In addition, the relative amount of these newly observed metabolites was determined. Based on the results and known facts about the toxicity of tacrine metabolites published so far, it appears that 7-phenoxytacrine and 6-chlorotacrine could be substantially less hepatotoxic compared to tacrine, and could potentially pave the way for metabolically safe molecules applicable in AD therapy.
- MeSH
- Alzheimerova nemoc * farmakoterapie metabolismus MeSH
- cholinesterasové inhibitory chemie MeSH
- jaterní mikrozomy metabolismus MeSH
- lékové postižení jater * metabolismus MeSH
- lidé MeSH
- takrin MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Tacrine was withdrawn from clinical use as a drug against Alzheimer's disease in 2013, mainly due to drug-induced liver injury. The culprit of tacrine-associated hepatotoxicity is believed to be the 7-OH-tacrine metabolite, a possible precursor of quinone methide (Qmeth), which binds to intracellular -SH proteins. In our study, several different animal and human models (liver microsomes, primary hepatocytes, and liver slices) were used to investigate the biotransformation and hepatotoxicity of tacrine and its 7-substituted analogues (7-methoxy-, 7-phenoxy-, and 7-OH-tacrine). Our goal was to find the most appropriate in vitro model for studying tacrine hepatotoxicity and, through rational structure modifications, to develop derivatives of tacrine that are less prone to Qmeth formation. Our results show that none of animal models tested accurately mimic human tacrine biotransformation; however, the murine model seems to be more suitable than the rat model. Tacrine metabolism was overall most accurately mimicked in three-dimensional (3D) spheroid cultures of primary human hepatocytes (PHHs). In this system, tacrine and 7-methoxytacrine were hydroxylated to 7-OH-tacrine, whereas 7-phenoxytacrine formed, as expected, only trace amounts. Surprisingly, however, our study showed that 7-OH-tacrine was the least hepatotoxic (7-OH-tacrine < tacrine < 7-methoxytacrine < 7-phenoxytacrine) even after doses had been adjusted to achieve the same intracellular concentrations. The formation of Qmeth-cysteine and Qmeth-glutathione adducts after human liver microsome incubation was confirmed by all of the studied tacrine derivatives, but these findings were not confirmed after incubation with 3D PHH spheroids. Therefore, the presented data call into question the suggested previously hypothesized mechanism of toxicity, and the results open new avenues for chemical modifications to improve the safety of novel tacrine derivatives.
- MeSH
- biotransformace MeSH
- indolochinony * MeSH
- krysa rodu rattus MeSH
- lékové postižení jater * MeSH
- lidé MeSH
- methamfetamin * MeSH
- myši MeSH
- takrin toxicita MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Twenty-four novel compounds bearing tetrahydroacridine and N-propargyl moieties have been designed, synthesised, and evaluated in vitro for their anti-cholinesterase and anti-monoamine oxidase activities. Propargyltacrine 23 (IC50 = 21 nM) was the most potent acetylcholinesterase (AChE) inhibitor, compound 20 (IC50 = 78 nM) showed the best inhibitory human butyrylcholinesterase (hBChE) profile, and ligand 21 afforded equipotent and significant values on both ChEs (human AChE [hAChE]: IC50 = 0.095 ± 0.001 μM; hBChE: IC50 = 0.093 ± 0.003 μM). Regarding MAO inhibition, compounds 7, 15, and 25 demonstrated the highest inhibitory potential towards hMAO-B (IC50 = 163, 40, and 170 nM, respectively). In all, compounds 7, 15, 20, 21, 23, and 25 exhibiting the most balanced pharmacological profile, were submitted to permeability and cell viability tests. As a result, 7-phenoxy-N-(prop-2-yn-1-yl)-1,2,3,4-tetrahydroacridin-9-amine hydrochloride (15) has been identified as a permeable agent that shows a balanced pharmacological profile [IC50 (hAChE) = 1.472 ± 0.024 μM; IC50 (hBChE) = 0.659 ± 0.077 μM; IC50 (hMAO-B) = 40.39 ± 5.98 nM], and consequently, as a new hit-ligand that deserves further investigation, in particular in vivo analyses, as the preliminary cell viability test results reported here suggest that this is a relatively safe therapeutic agent.
- MeSH
- acetylcholinesterasa metabolismus MeSH
- Alzheimerova nemoc * farmakoterapie MeSH
- aminy MeSH
- butyrylcholinesterasa * metabolismus MeSH
- cholinesterasové inhibitory farmakologie terapeutické užití MeSH
- inhibitory MAO farmakologie MeSH
- lidé MeSH
- ligandy MeSH
- monoaminoxidasa MeSH
- oxidoreduktasy MeSH
- racionální návrh léčiv MeSH
- takrin terapeutické užití MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Insecticides represent the most crucial element in the integrated management approach to malaria and other vector-borne diseases. The evolution of insect resistance to long-used substances and the toxicity of organophosphates (OPs) and carbamates are the main factors contributing to the development of new, environmentally safe pesticides. In our work, fourteen compounds of 7-methoxytacrine-tacrine heterodimers were tested for their insecticidal effect. Compounds were evaluated in vitro on insect acetylcholinesterase from Anopheles gambiae (AgAChE) and Musca domestica (MdAChE). The evaluation was executed in parallel with testing on human erythrocyte acetylcholinesterase (HssAChE) and human butyrylcholinesterase (HssBChE) using a modified Ellman's method. Compound efficacy was determined as IC50 values for the respective enzymes and selectivity indexes were expressed to compare the interspecies selectivity. Docking studies were performed to predict the binding modes of selected compounds. K1328 and K1329 provided high HssAChE/AgAChE selectivity outperforming standard pesticides (carbofuran and bendiocarb), and thus can be considered as suitable lead structure for novel anticholinesterase insecticides.
- MeSH
- acetylcholinesterasa metabolismus MeSH
- Anopheles * metabolismus MeSH
- butyrylcholinesterasa MeSH
- cholinesterasové inhibitory toxicita MeSH
- insekticidy * MeSH
- karbamáty MeSH
- karbofuran * MeSH
- komáří přenašeči MeSH
- lidé MeSH
- organofosfáty MeSH
- takrin MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Nowadays, advanced computational chemistry methods offer various strategies for revealing prospective hit structures in drug development essentially through accurate binding free energy predictions. After the era of molecular docking and quantitative structure-activity relationships, much interest has been lately oriented to perturbed molecular dynamic approaches like replica exchange with solute tempering and free energy perturbation (REST/FEP) and the potential of the mean force with adaptive biasing and accelerated weight histograms (PMF/AWH). Both of these receptor-based techniques can exploit exascale CPU&GPU supercomputers to achieve high throughput performance. In this fundamental study, we have compared the predictive power of a panel of supercomputerized molecular modelling methods to distinguish the major binding modes and the corresponding binding free energies of a promising tacrine related potential antialzheimerics in human acetylcholinesterase. The binding free energies were estimated using flexible molecular docking, molecular mechanics/generalized Born surface area/Poisson-Boltzmann surface area (MM/GBSA/PBSA), transmutation REST/FEP with 12 x 5 ns/λ windows, annihilation FEP with 20 x 5 ns/λ steps, PMF with weight histogram analysis method (WHAM) and 40 x 5 ns samples, and PMF/AWH with 10 x 100 ns replicas. Confrontation of the classical approaches such as canonical molecular dynamics and molecular docking with alchemical calculations and steered molecular dynamics enabled us to show how large errors in ΔG predictions can be expected if these in silico methods are employed in the elucidation of a common case of enzyme inhibition.Communicated by Ramaswamy H. Sarma.
A series of tacrine - benzothiazole hybrids incorporate inhibitors of acetylcholinesterase (AChE), amyloid β (Aβ) aggregation and mitochondrial enzyme ABAD, whose interaction with Aβ leads to mitochondrial dysfunction, into a single molecule. In vitro, several of 25 final compounds exerted excellent anti-AChE properties and interesting capabilities to block Aβ aggregation. The best derivative of the series could be considered 10w that was found to be highly potent and selective towards AChE with the IC50 value in nanomolar range. Moreover, the same drug candidate exerted absolutely the best results of the series against ABAD, decreasing its activity by 23% at 100 μM concentration. Regarding the cytotoxicity profile of highlighted compound, it roughly matched that of its parent compound - 6-chlorotacrine. Finally, 10w was forwarded for in vivo scopolamine-induced amnesia experiment consisting of Morris Water Maze test, where it demonstrated mild procognitive effect. Taking into account all in vitro and in vivo data, highlighted derivative 10w could be considered as the lead structure worthy of further investigation.
- MeSH
- 3-hydroxyacyl-CoA-dehydrogenasy antagonisté a inhibitory metabolismus MeSH
- acetylcholinesterasa metabolismus MeSH
- Alzheimerova nemoc farmakoterapie metabolismus MeSH
- amyloidní beta-protein antagonisté a inhibitory metabolismus MeSH
- benzothiazoly chemie farmakologie MeSH
- cholinergní látky chemická syntéza chemie farmakologie MeSH
- inhibitory enzymů chemická syntéza chemie farmakologie MeSH
- lidé MeSH
- mitochondrie účinky léků metabolismus MeSH
- molekulární struktura MeSH
- neuroprotektivní látky chemická syntéza chemie farmakologie MeSH
- proteinové agregáty účinky léků MeSH
- takrin chemie farmakologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
