Butyrylcholinesterase (BChE) is recognized as a promising therapeutic target for the late stages of Alzheimer's disease (AD) due to its role in the hydrolysis of acetylcholine (ACh), while acetylcholinesterase (AChE) activity declines during disease progression. Here, we have reported an efficient chemistry procedure for the naturally occurring Amaryllidaceae alkaloid carltonine B, along with the design and synthesis of 36 novel carltonine-based analogues to determine structure-activity relationship (SAR). Most of the synthesized compounds exhibited potent and selective human BChE (hBChE) inhibition, with IC50 values ranging from low micromolar to nanomolar concentrations. The drug-like properties of the molecules were assessed by in silico tools, using the blood-brain barrier (BBB) score algorithm, and subsequently validated by in vitro permeability assessment via parallel artificial membrane permeability assay (PAMPA). The derivatives exhibited potent hBChE inhibition in the low micromolar to submicromolar range, while their cytotoxicity against human neuroblastoma (SH-SY5Y) cells was observed only at higher micromolar concentrations, indicating a favorable safety profile. The synthesized alkaloid carltonine B (37) and its N-ethyl derivative (38) emerged as the most potent and selective hBChE inhibitors, with IC50 values of 0.014 ± 0.002 μM and 0.013 ± 0.001 μM, respectively. Enzyme kinetic studies were conducted to elucidate the inhibition mechanism toward hBChE enzyme. Compound 37 demonstrated competitive inhibition with Ki value of 0.055 μM. In contrast, compound 38 showed a noncompetitive inhibition profile, with a Ki value of 0.067 μM. Molecular modeling suggested that the superior potency of compounds 37 and 38 arises from their more optimal engagement of the BChE active-site gorge compared to compound 33. For the additional safety assessment, CYP inhibition assay revealed that compounds 37 and 38 may pose a risk of CYP3A4-mediated drug-drug interactions during chronic administration.

Sustained effort has been dedicated to the development of novel cholinesterase reactivators—the only causal antidotes—to counter organophosphorus (OP) intoxication. As more lethal nerve agents—such as A-agents—continue to emerge, the existing arsenal of causal antidotes remains unchanged. Approved oxime reactivators—2-PAM, HI-6, and LüH-6—are restricted by their limited efficacy spectrum, poor blood–brain barrier permeability, and suboptimal pharmacokinetics. The objective of this study is to design, synthesize, and characterize a new class of asymmetric monoquaternary bisoxime reactivators with broad-spectrum reactivation potential, favorable pharmacokinetics, and dual mechanisms of action—cholinesterase reactivation and direct OP compound degradation. In vitro and in vivo experiments identified LG-1795 as the lead candidate with the broadest OP spectrum. The averaged second-order reactivation constant (kr2) across five hAChE–OP and two hBChE-OP complexes was 16.8 mM−1 min−1, surpassing clinical standards. Notably, LG-1795 reactivated both AChE and BChE, a dual activity not previously reported for reactivators. In vivo, LG-1795 restored both cholinesterase enzymes and demonstrated prophylactic efficacy against GB, VX, and PXE following intramuscular administration, preventing symptoms in sarin-poisoned animals. These findings represent a significant advance in antidotal therapy, providing the first evidence that asymmetric monoquaternary bisoximes deliver broad-spectrum efficacy against nerve agents and pesticides while simultaneously targeting both OP-inhibited cholinesterases. The translational potential of LG-1795 supports its further preclinical development as a next-generation countermeasure for both clinical and regulatory use in chemical defense.

The nerve agent A-234, classified among the so-called A-series agents or "Novichoks", represents a significant challenge in the treatment of nerve agent poisoning due to its potent and irreversible inhibition of human recombinant acetylcholinesterase (HssAChE). In this study, we screened 22 structurally diverse oxime reactivators for their efficacy against A-234-inhibited HssAChE in vitro after a 10-min incubation. The initial screening was extended to GB- and VX-inhibited enzymes under the same conditions for comparison. Then, the 22 oximes were tested against A-234-inhibited HssAChE for up to 24 h. Finally, we evaluated the reactivation kinetics of the two most effective oximes over a 240-min period. While most reactivators demonstrated considerable reactivation against GB- and VX-inhibited enzymes within 10 min, none showed efficacy against A-234 in this time frame. Extended incubation over 24 h revealed that only HLö-7, MMB-4, HI-6, K027, K868, TMB-4, GM415, and LüH-6 achieved efficient reactivation (set at a 10 % threshold for a 24-h interval), with HLö-7 and methoxime (MMB-4) being the most effective. Kinetic analysis indicated that HLö-7 exhibited a superior second-order reactivation rate constant compared to MMB-4, highlighting differences in binding affinity and catalytic efficacy. In the case of A-234 poisonings, our findings indicate the most effective oxime antidotes and emphasize the need for prolonged therapy to improve clinical outcomes.

Currently available antidotes against toxic organophosphorus compounds suffer from poor permeability across the blood-brain barrier (BBB) and due to this, are limited in their ability to restore the inhibited acetylcholinesterase (AChE) in the central nervous system (CNS). We designed functionalized detonation nanodiamond nanocarrier platforms to transport quaternary oxime antidotes into CNS. We showed that the nanodiamonds with covalently attached 4-oximinopyridinium moiety, cross the layer of Madin-Darby Canine Kidney (MDCK) cells, the surrogate BBB model, and demonstrate a dose-independent reactivation in vitro towards human AChE inhibited by nerve agents GB and VX, and pesticide paraoxon. Confocal microscopy visualization of tight junctions and actin cytoskeleton in MDCK and Human Umbilical Vein Endothelial Cells (HUVEC) revealed temporary disruption of tight junctions at higher nanoparticle concentrations without compromising cell viability or cytoskeletal integrity. Although reactivation was modest, the nanodiamond platform showed promise for delivering quaternary oxime to the central nervous system (CNS) in vitro. The results reveal the potential of detonation nanodiamonds as a promising delivery platform for charged therapeutic agents to CNS aimed to enhance treatment outcomes in organophosphorus poisoning.

A-series agent A-234 belongs to a new generation of nerve agents. The poisoning of a former Russian spy Sergei Skripal and his daughter in Salisbury, England, in March 2018 led to the inclusion of A-234 and other A-series agents into the Chemical Weapons Convention. Even though five years have already passed, there is still very little information on its chemical properties, biological activities, and treatment options with established antidotes. In this article, we first assessed A-234 stability in neutral pH for subsequent experiments. Then, we determined its inhibitory potential towards human recombinant acetylcholinesterase ( Hss AChE; EC 3.1.1.7) and butyrylcholinesterase ( Hss BChE; EC 3.1.1.8), the ability of HI-6, obidoxime, pralidoxime, methoxime, and trimedoxime to reactivate inhibited cholinesterases (ChEs), its toxicity in rats and therapeutic effects of different antidotal approaches. Finally, we utilized molecular dynamics to explain our findings. The results of spontaneous A-234 hydrolysis showed a slow process with a reaction rate displaying a triphasic course during the first 72 h (the residual concentration 86.2%). A-234 was found to be a potent inhibitor of both human ChEs ( Hss AChE IC 50 = 0.101 ± 0.003 µM and Hss BChE IC 50 = 0.036 ± 0.002 µM), whereas the five marketed oximes have negligible reactivation ability toward A-234-inhibited Hss AChE and Hss BChE. The acute toxicity of A-234 is comparable to that of VX and in the context of therapy, atropine and diazepam effectively mitigate A-234 lethality. Even though oxime administration may induce minor improvements, selected oximes (HI-6 and methoxime) do not reactivate ChEs in vivo. Molecular dynamics implies that all marketed oximes are weak nucleophiles, which may explain the failure to reactivate the A-234 phosphorus-serine oxygen bond characterized by low partial charge, in particular, HI-6 and trimedoxime oxime oxygen may not be able to effectively approach the A-234 phosphorus, while pralidoxime displayed low interaction energy. This study is the first to provide essential experimental preclinical data on the A-234 compound.

The current study imposes a new class of organophosphorus (OP)-inhibited cholinesterase reactivators by conceptualizing a family of asymmetric bisoximes with various reactivating scaffolds. Several novel nucleophilic warheads were investigated, putting forward 29 novel reactivating options, by evaluating their nucleophilicity and ability to directly decompose OP compounds. Adopting the so-called zwitterionic strategy, 17 mono-oxime and nine bisoxime reactivators were discovered with major emphasis on the bifunctional-moiety approach. Compounds were compared with clinically used standards and other known experimentally highlighted reactivators. Our results clearly favor the concept of asymmetric bisoximes as leading reactivators in terms of efficacy and versatility. These top-ranked compounds were characterized in detail by reactivation kinetics parameters and evaluated for potential CNS availability. The highlighted molecules 55, 57, and 58 with various reactivating warheads, surpassed the reactivating potency of pralidoxime and several notable uncharged reactivators. The versatility of lead drug candidate 55 was also inspected on OP-inhibited butyrylcholinesterase, revealing a much higher rate compared to existing clinical antidotes.

Innovative chemotype design provided 41 novel h BChE inhibitors. CNS availability was predicted using the BBB score. Potent derivatives showed favorable drug-like properties and acceptable safety profiles in SH-SY5Y and HepG2 cells.

Organophosphorus compounds (OPs) involving life-threatening nerve agents (NA) have been known for several decades. Despite a clear mechanism of their lethality caused by the irreversible inhibition of acetylcholinesterase (AChE) and manifested via overstimulation of peripheral nicotinic and muscarinic acetylcholine (ACh) receptors, the mechanism for central neurotoxicity responsible for acute or delayed symptoms of the poisoning has not been thoroughly uncovered. One of the reasons is the lack of a suitable model. In our study, we have chosen the SH-SY5Y model in both the differentiated and undifferentiated state to study the effects of NAs (GB, VX and A234). The activity of expressed AChE in cell lysate assessed by Ellman's method showed 7.3-times higher activity in differentiated SH-SY5Y cells in contrast to undifferentiated cells, and with no involvement of BuChE as proved by ethopropazine (20 µM). The activity of AChE was found to be, in comparison to untreated cells, 16-, 9.3-, and 1.9-times lower upon A234, VX, and GB (100 µM) administration respectively. The cytotoxic effect of given OPs expressed as the IC50 values for differentiated and undifferentiated SH-SY5Y, respectively, was found 12 mM and 5.7 mM (A234), 4.8 mM and 1.1 mM (VX) and 2.6 mM and 3.8 mM (GB). In summary, although our results confirm higher AChE expression in the differentiated SH-SY5Y cell model, the such higher expression does not lead to a more pronounced NA cytotoxic effect. On the contrary, higher expression of AChE may attenuate NA-induced cytotoxicity by scavenging the NA. Such finding highlights a protective role for cholinesterases by scavenging Novichoks (A-agents). Second, we confirmed the mechanism of cytotoxicity of NAs, including A-agents, can be ascribed rather to the non-specific effects of OPs than to AChE-mediated effects.

We report a green chemistry approach for preparation of oxime-functionalized ILs as AChE reactivators: amide/ester linked IL, l-alanine, and l-phenylalanine derived salts bearing pyridinium aldoxime moiety. The reactivation capacities of the novel oximes were evaluated towards AChE inhibited by typical toxic organophosphates, sarin (GB), VX, and paraoxon (PON). The studied compounds are mostly non-toxic up to the highest concentrations screened (2 mM) towards Gram-negative and Gram-positive bacteria cell lines and both filamentous fungi and yeasts in the in vitro screening experiments as well as towards the eukaryotic cell (CHO-K1 cell line). Introduction of the oxime moiety in initially biodegradable structure decreases its ability to biodegradation. The compound 3d was shown to reveal remarkable activity against the AChE inhibited by VX, exceeding conventional reactivators 2-PAM and obidoxime. The regularities on antidotal activity, cell viability, plasma stability, biodegradability as well as molecular docking study of the newly synthesized oximes will be used for further improvement of their structures.

Butyrylcholinesterase (BChE) is one of the most frequently implicated enzymes in the advanced stage of Alzheimer's disease (AD). As part of our endeavors to develop new drug candidates for AD, we have focused on natural template structures, namely the Amaryllidaceae alkaloids carltonine A and B endowed with high BChE selectivity. Herein, we report the design, synthesis, and in vitro evaluation of 57 novel highly selective human BChE (hBChE) inhibitors. Most synthesized compounds showed hBChE inhibition potency ranging from micromolar to low nanomolar scale. Compounds that revealed BChE inhibition below 100 nM were selected for detailed biological investigation. The CNS-targeted profile of the presented compounds was confirmed theoretically by calculating the BBB score algorithm, these data were corroborated by determining the permeability in vitro using PAMPA-assay for the most active derivatives. The study highlighted compounds 87 (hBChE IC50 = 3.8 ± 0.2 nM) and 88 (hBChE IC50 = 5.7 ± 1.5 nM) as the top-ranked BChE inhibitors. Compounds revealed negligible cytotoxicity for the human neuroblastoma (SH-SY5Y) and hepatocellular carcinoma (HepG2) cell lines compared to BChE inhibitory potential. A crystallographic study was performed to inspect the binding mode of compound 87, revealing essential interactions between 87 and hBChE active site. In addition, multidimensional QSAR analyses were applied to determine the relationship between chemical structures and biological activity in a dataset of designed agents. Compound 87 is a promising lead compound with potential implications for treating the late stages of AD.

“Novichok” refers to a new group of nerve agents called the A-series agents. Their existence came to light in 2018 after incidents in the UK and again in 2020 in Russia. They are unique organophosphorus-based compounds developed during the Cold War in a program called Foliant in the USSR. This review is based on original chemical entities from Mirzayanov's memoirs published in 2008. Due to classified research, a considerable debate arose about their structures, and hence, various structural moieties were speculated. For this reason, the scientific literature is highly incomplete and, in some cases, contradictory. This review critically assesses the information published to date on this class of compounds. The scope of this work is to summarize all the available and relevant information, including the physicochemical properties, chemical synthesis, mechanism of action, toxicity, pharmacokinetics, and medical countermeasures used to date. The environmental stability of A-series agents, the lack of environmentally safe decontamination, their high toxicity, and the scarcity of information on post-contamination treatment pose a challenge for managing possible incidents.

One undescribed indole alkaloid together with twenty-two known compounds have been isolated from aerial parts of Vinca minor L. (Apocynaceae). The chemical structures of the isolated alkaloids were determined by a combination of MS, HRMS, 1D, and 2D NMR techniques, and by comparison with literature data. The NMR data of several alkaloids have been revised, corrected, and missing data have been supplemented. Alkaloids isolated in sufficient quantity were screened for their in vitro acetylcholinesterase (AChE; E.C. 3.1.1.7) and butyrylcholinesterase (BuChE; E.C. 3.1.1.8) inhibitory activity. Selected compounds were also evaluated for prolyl oligopeptidase (POP; E.C. 3.4.21.26), and glycogen synthase 3 beta-kinase (GSK-3 beta; E.C. 2.7.11.26) inhibition potential. Significant hBuChE inhibition activity has been shown by (- )-2-ethyl-3[2-(3-ethylpiperidinyl)-ethyl]-1Hindole with an IC50 value of 0.65 +/- 0.16 mu M. This compound was further studied by enzyme kinetics, along with in silico techniques, to reveal the mode of inhibition. This compound is also predicted to cross the blood-brain barrier (BBB) through passive diffusion.

ObjectiveThe PKD1 encodes polycystin-1, a large transmembrane protein that plays important roles in cell proliferation, apoptosis, and cation transport. Previous studies have identified PKD1 mutations in autosomal dominant polycystic kidney disease (ADPKD). However, the expression of PKD1 in the brain is much higher than that in the kidney. This study aimed to explore the association between PKD1 and epilepsy. MethodsTrios-based whole-exome sequencing was performed in a cohort of 314 patients with febrile seizures or epilepsy with antecedent febrile seizures. The damaging effects of variants was predicted by protein modeling and multiple in silico tools. The genotype-phenotype association of PKD1 mutations was systematically reviewed and analyzed. ResultsEight pairs of compound heterozygous missense variants in PKD1 were identified in eight unrelated patients. All patients suffered from febrile seizures or epilepsy with antecedent febrile seizures with favorable prognosis. All of the 16 heterozygous variants presented no or low allele frequencies in the gnomAD database, and presented statistically higher frequency in the case-cohort than that in controls. These missense variants were predicted to be damaging and/or affect hydrogen bonding or free energy stability of amino acids. Five patients showed generalized tonic-clonic seizures (GTCS), who all had one of the paired missense mutations located in the PKD repeat domain, suggesting that mutations in the PKD domains were possibly associated with GTCS. Further analysis demonstrated that monoallelic mutations with haploinsufficiency of PKD1 potentially caused kidney disease, compound heterozygotes with superimposed effects of two missense mutations were associated with epilepsy, whereas the homozygotes with complete loss of PKD1 would be embryonically lethal. ConclusionPKD1 gene was potentially a novel causative gene of epilepsy. The genotype-phenotype relationship of PKD1 mutations suggested a quantitative correlation between genetic impairment and phenotypic variation, which will facilitate the genetic diagnosis and management in patients with PKD1 mutations.

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 organo-phosphates (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 Ell -man'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 out-performing standard pesticides (carbofuran and bendiocarb), and thus can be considered as suitable lead structure for novel anticholinesterase insecticides.

The problem of the efficient treatment of acute organophosphorus (OP) poisoning needs more efforts in the development of a versatile antidote, applicable for treatment of the injuries of both peripheral and central nervous systems. A series of N-H, N-methyl, N-butyl, and N-phenyl derivatives of benzhydroxamic (1a-1d), 3-methoxybenzhydroxamic (2a-2d), 4-methoxybenzhydroxamic (3a-3d) acids, and corresponding salycilhydroxamates (4a-4d) was prepared. Their predicted hydrophobicity (log P) was evaluated as regards to ВВВ score by the open access cheminformatics tools; prediction of the passive transport across the BBB was found by means on the parallel artificial membrane permeability assay (PAMPA). The data on reactivation capacity of human acetylcholinesterase (HssAChE) inhibited by GB, VX, and paraoxon was supported by molecular docking study on binding to the active site of the AChE, viability study against mammalian cells (Chinese hamster ovary CHO-K1), and biodegradability (Closed Bottle test OECD 301D). Among the studied compounds, N-butyl derivatives have better balanced combination of properties; among them, N-butylsalicylhydroxamic acid is most promising. The studied compounds demonstrate modest reactivation capacity; change of N-H by N-Me ensures the reactivation capacity in studied concentrations on all studied OP substrates; among N-butyl derivatives, the N-butylsalicylhydroxamic acid demonstrates most promising results within the series. The found regularities may lead to selection of perspective structures to complement current formulations for medical countermeasures against poisoning by organophosphorus toxicants.

Natural products, especially alkaloids, are still a substantial resource for the drug development [1]. Based on the preliminary screening of alkaloidal extracts of various plants against Alzheimerʼs disease, Vinca minor L. (Apocynaceae) have been chosen for a detailed phytochemical and biological study. V. minor L. is an evergreen trailing subshrub common in Europe with rich content of monoterpene indole alkaloids [2]. In this study, we have isolated and identified active alkaloidal compounds and assessed their potential to inhibit hAChE, hBuChE, POP, and GSK-3β – enzymes that play a key role in the pathophysiology of Alzheimerʼs disease. Using chromatographic methods, we have isolated 23 alkaloids; 11 of them have been reported in this species for the first time. One alkaloidal structure was undescribed and was named as vincaminorudeine. The most active compound was (−)-2-ethyl-3[2-(3-ethylpiperidinyl)-ethyl]-1H-indole with IC50 = 0.65 µM for the inhibition of hBuChE, and with IC50 = 58 µM for the inhibition of POP. Other alkaloids that exhibited significant inhibition against hBuChE (IC50 < 30 µM) were vincaminoreine, minovine, 16-methoxyminovine, vincorine, and tubotaiwine. None of the isolated alkaloids was active against hAChE. The (−)-2-ethyl-3[2-(3-ethylpiperidinyl)-ethyl]-1H-indole was also further studied for its pharmacokinetic, revealing a reversible competitive type of inhibition for hBuChE with Ki = 55 nM. This compound can also penetrate the blood-brain barrier by passive diffusion, as was assessed by the PAMPA study. Additionally, the alkaloid on the panel of ten cell lines showed non-cytotoxicity. These compelling results open a possibility for further research on this indole alkaloid.

This dataset contains the digitized treatments in Plazi based on the original journal article Vrabec, Rudolf, Ma, Jana, ríkov, a, Loc, Miroslav, arek, Kor, Jan, abe, cný, Hulcova, Daniela, Ho, Anna, st, alkov, a, Ji, Kune, rí, s, Chlebek, Jakub, Toma, Ku, s, cera, Hrabinova, Martina, Jun, Daniel, Ond, Soukup, rej, Andrisano, Vincenza, Jen, Jaroslav, co, Marcela, Safratova, Nov, Lucie, akova, Opletal, Lubomír, Cahlíkova, Lucie (2022): Monoterpene indole alkaloids from Vinca minor L. (Apocynaceae): Identification of new structural scaffold for treatment of Alzheimer’s disease. Phytochemistry (113017) 194: 1-13, DOI: 10.1016/j.phytochem.2021.113017, URL: http://dx.doi.org/10.1016/j.phytochem.2021.113017

Multitarget-directed ligands (MTDLs) are considered a promising therapeutic strategy to address the multifactorial nature of Alzheimer's disease (AD). Novel MTDLs have been designed as inhibitors of human acetylcholinesterases/butyrylcholinesterases, monoamine oxidase A/B, and glycogen synthase kinase 3β and as calcium channel antagonists via the Biginelli multicomponent reaction. Among these MTDLs, (±)-BIGI-3h was identified as a promising new hit compound showing in vitro balanced activities toward the aforementioned recognized AD targets. Additional in vitro studies demonstrated antioxidant effects and brain penetration, along with the ability to inhibit the aggregation of both τ protein and β-amyloid peptide. The in vivo studies have shown that (±)-BIGI-3h (10 mg/kg intraperitoneally) significantly reduces scopolamine-induced cognitive deficits.

Alzheimer’s disease (AD) is a multifactorial neurodegenerative condition of the central nervous system (CNS) that is currently treated by cholinesterase inhibitors and the N-methyl-d-aspartate receptor antagonist, memantine. Emerging evidence strongly supports the relevance of targeting butyrylcholinesterase (BuChE) in the more advanced stages of AD. Within this study, we have generated a pilot series of compounds (1–20) structurally inspired from belladine-type Amaryllidaceae alkaloids, namely carltonine A and B, and evaluated their acetylcholinesterase (AChE) and BuChE inhibition properties. Some of the compounds exhibited intriguing inhibition activity for human BuChE (hBuChE), with a preference for BuChE over AChE. Seven compounds were found to possess a hBuChE inhibition profile, with IC50 values below 1 µM. The most potent one, compound 6, showed nanomolar range activity with an IC50 value of 72 nM and an excellent selectivity pattern over AChE, reaching a selectivity index of almost 1400. Compound 6 was further studied by enzyme kinetics, along with in-silico techniques, to reveal the mode of inhibition. The prediction of CNS availability estimates that all the compounds in this survey can pass through the blood-brain barrier (BBB), as disclosed by the BBB score.