Reactivators of butyrylcholinesterase inhibited by organophosphorus compounds.

In this review, the current progress in the research and development of butyrylcholinesterase (BChE) reactivators is summarised and the advantages or disadvantages of these reactivators are critically discussed. Organophosphorus compounds such as nerve agents (sarin, tabun, VX) or pesticides (chlorpyrifos, diazinon) cause irreversible inhibition of acetylcholinesterase (AChE) and BChE in the human body. While AChE inhibition can be life threatening due to cholinergic overstimulation and crisis, selective BChE inhibition has presumably no adverse effects. Because BChE is mostly found in plasma, its activity is important for the scavenging of organophosphates before they can reach AChE in the central nervous system. Therefore, this enzyme in combination with its reactivator can be used as a pseudo-catalytic scavenger of organophosphates. Three structural types of BChE reactivators were found, i.e. bisquaternary salts, monoquaternary salts and uncharged compounds. Although the reviewed reactivators have certain limitations, the promising candidates for BChE reactivation were found in each structural group.

Brominated oxime nucleophiles are efficiently reactivating cholinesterases inhibited by nerve agents.

Six novel brominated bis-pyridinium oximes were designed and synthesized to increase their nucleophilicity and reactivation ability of phosphorylated acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Their pKa was valuably found lower to parent non-halogenated oximes. Stability tests showed that novel brominated oximes were stable in water, but the stability of di-brominated oximes was decreased in buffer solution and their degradation products were prepared and characterized. The reactivation screening of brominated oximes was tested on AChE and BChE inhibited by organophosphorus surrogates. Two mono-brominated oximes reactivated AChE comparably to non-halogenated analogues, which was further confirmed by reactivation kinetics. The acute toxicity of two selected brominated oximes was similar to commercially available oxime reactivators and the most promising brominated oxime was tested in vivo on sarin- and VX-poisoned rats. This brominated oxime showed interesting CNS distribution and significant reactivation effectiveness in blood. The same oxime resulted with the best protective index for VX-poisoned rats.

Direct Chiral Supercritical Fluid Chromatography-Mass Spectrometry Analysis of Monoacylglycerol and Diacylglycerol Isomers for the Study of Lipase-Catalyzed Hydrolysis of Triacylglycerols.

The fast and selective separation method of intact monoacylglycerol (MG) and diacylglycerol (DG) isomers using chiral supercritical fluid chromatography-mass spectrometry (SFC-MS) was developed and employed to study lipase selectivity in the hydrolysis of triacylglycerols (TGs). The synthesis of 28 enantiomerically pure MG and DG isomers was performed in the first stage using the most commonly occurring fatty acids in biological samples such as palmitic, stearic, oleic, linoleic, linolenic, arachidonic, and docosahexaenoic acids. To develop the SFC separation method, different chromatographic conditions such as column chemistry, mobile phase composition and gradient, flow rate, backpressure, and temperature were carefully assessed. Our SFC-MS method used a chiral column based on a tris(3,5-dimethylphenylcarbamate) derivative of amylose and neat methanol as a mobile phase modifier, which provides baseline separation of all the tested enantiomers in 5 min. This method was used to evaluate hydrolysis selectivity of lipases from porcine pancreas (PPL) and Pseudomonas fluorescens (PFL) using nine TGs differing in acyl chain length (14-22 carbon atoms) and number of double bonds (0-6) and three DG regioisomer/enantiomers as hydrolysis intermediate products. PFL exhibited preference of the fatty acyl hydrolysis from the sn-1 position of TG more pronounced for the substrates with long polyunsaturated acyls, while PPL did not show considerable stereoselectivity to TGs. Conversely, PPL preferred hydrolysis from the sn-1 position of prochiral sn-1,3-DG regioisomer, whereas PFL exhibited no preference. Both lipases showed selectivity for the hydrolysis of outer positions of DG enantiomers. The results show complex reaction kinetics of lipase-catalyzed hydrolysis given by different stereoselectivities for substrates.

Strategies for enhanced bioavailability of oxime reactivators in the central nervous system.

Oxime reactivators of acetylcholinesterase are commonly used to treat highly toxic organophosphate poisoning. They are effective nucleophiles that can restore the catalytic activity of acetylcholinesterase; however, their main limitation is the difficulty in crossing the blood-brain barrier (BBB) because of their strongly hydrophilic nature. Various approaches to overcome this limitation and enhance the bioavailability of oxime reactivators in the CNS have been evaluated; these include structural modifications, conjugation with molecules that have transporters in the BBB, bypassing the BBB through intranasal delivery, and inhibition of BBB efflux transporters. A promising approach is the use of nanoparticles (NPs) as the delivery systems. Studies using mesoporous silica nanomaterials, poly (L-lysine)-graft-poly(ethylene oxide) NPs, metallic organic frameworks, poly(lactic-co-glycolic acid) NPs, human serum albumin NPs, liposomes, solid lipid NPs, and cucurbiturils, have shown promising results. Some NPs are considered as nanoreactors for organophosphate detoxification; these combine bioscavengers with encapsulated oximes. This study provides an overview and critical discussion of the strategies used to enhance the bioavailability of oxime reactivators in the central nervous system.

Cysteine conjugates of acetaminophen and p-aminophenol are potent inducers of cellular impairment in human proximal tubular kidney HK-2 cells.

Acetaminophen (APAP) belong among the most used analgesics and antipyretics. It is structurally derived from p-aminophenol (PAP), a potent inducer of kidney toxicity. Both compounds can be metabolized to oxidation products and conjugated with glutathione. The glutathione-conjugates can be cleaved to provide cysteine conjugates considered as generally nontoxic. The aim of the present report was to synthesize and to purify both APAP- and PAP-cysteine conjugates and, as the first study at all, to evaluate their biological effects in human kidney HK-2 cells in comparison to parent compounds. HK-2 cells were treated with tested compounds (0-1000 µM) for up to 24 h. Cell viability, glutathione levels, ROS production and mitochondrial function were determined. After 24 h, we found that both APAP- and PAP-cysteine conjugates (1 mM) were capable to induce harmful cellular damage observed as a decrease of glutathione levels to 10% and 0%, respectively, compared to control cells. In addition, we detected the disappearance of mitochondrial membrane potential in these cells. In the case of PAP-cysteine, the extent of cellular impairment was comparable to that induced by PAP at similar doses. On the other hand, 1 mM APAP-cysteine induced even larger damage of HK-2 cells compared to 1 mM APAP after 6 or 24 h. We conclude that cysteine conjugates with aminophenol are potent inducers of oxidative stress causing significant injury in kidney cells. Thus, the harmful effects cysteine-aminophenolic conjugates ought to be considered in the description of APAP or PAP toxicity.

Inhibitors of phosphoinositide 3-kinase (PI3K) and phosphoinositide 3-kinase-related protein kinase family (PIKK).

Phosphoinositide 3-kinases (PI3K) and phosphoinositide 3-kinase-related protein kinases (PIKK) are two structurally related families of kinases that play vital roles in cell growth and DNA damage repair. Dysfunction of PIKK members and aberrant stimulation of the PI3K/AKT/mTOR signalling pathway are linked to a plethora of diseases including cancer. In recent decades, numerous inhibitors related to the PI3K/AKT/mTOR signalling have made great strides in cancer treatment, like copanlisib and sirolimus. Notably, most of the PIKK inhibitors (such as VX-970 and M3814) related to DNA damage response have also shown good efficacy in clinical trials. However, these drugs still require a suitable combination therapy to overcome drug resistance or improve antitumor activity. Based on the aforementioned facts, we summarised the efficacy of PIKK, PI3K, and AKT inhibitors in the therapy of human malignancies and the resistance mechanisms of targeted therapy, in order to provide deeper insights into cancer treatment.

Charged pyridinium oximes with thiocarboxamide moiety are equally or less effective reactivators of organophosphate-inhibited cholinesterases compared to analogous carboxamides.

The organophosphorus antidotes, so-called oximes, are able to restore the enzymatic function of acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) via cleavage of organophosphate from the active site of the phosphylated enzyme. In this work, the charged pyridinium oximes containing thiocarboxamide moiety were designed, prepared and tested. Their stability and pKa properties were found to be analogous to parent carboxamides (K027, K048 and K203). The inhibitory ability of thiocarboxamides was found in low µM levels for AChE and high µM levels for BChE. Their reactivation properties were screened on human recombinant AChE and BChE inhibited by nerve agent surrogates and paraoxon. One thiocarboxamide was able to effectively restore function of NEMP- and NEDPA-AChE, whereas two thiocarboxamides were able to reactivate BChE inhibited by all tested organophosphates. These results were confirmed by reactivation kinetics, where thiocarboxamides were proved to be effective, but less potent reactivators if compared to carboxamides.

Effects of Charged Oxime Reactivators on the HK-2 Cell Line in Renal Toxicity Screening.

Oxime cholinesterase reactivators (oximes) are used to counteract organophosphate intoxication. Charged oximes are administered via intramuscular or intravenous injection when the majority of dose is unmetabolized and is excreted as urine. In this study, the effects of selected double charged oximes were determined in the HK-2 cell line as a model for renal toxicity screening. Some effects on dehydrogenase activity were found for obidoxime, asoxime (syn. HI-6), K027, and K203. The effects of K868 and K869 were found to be unreliable due to rapid degradation of both chlorinated oximes in the assay medium, resulting for K868 in an isoxazole-pyridinium product.

Development of versatile and potent monoquaternary reactivators of acetylcholinesterase.

To date, the only treatments developed for poisoning by organophosphorus compounds, the most toxic chemical weapons of mass destruction, have exhibited limited efficacy and versatility. The available causal antidotes are based on reactivation of the enzyme acetylcholinesterase (AChE), which is rapidly and pseudo-irreversibly inhibited by these agents. In this study, we developed a novel series of monoquaternary reactivators combining permanently charged moieties tethered to position 6- of 3-hydroxypyridine-2-aldoxime reactivating subunit. Highlighted representatives (21, 24, and 27; also coded as K1371, K1374, and K1375, respectively) that contained 1-phenylisoquinolinium, 7-amino-1-phenylisoquinolinium and 4-carbamoylpyridinium moieties as peripheral anionic site ligands, respectively, showed efficacy superior or comparable to that of the clinically used standards. More importantly, these reactivators exhibited wide-spectrum efficacy and were minutely investigated via determination of their reactivation kinetics in parallel with molecular dynamics simulations to study their mechanisms of reactivation of the tabun-inhibited AChE conjugate. To further confirm the potential applicability of these candidates, a mouse in vivo assay was conducted. While K1375 had the lowest acute toxicity and the most suitable pharmacokinetic profile, the oxime K1374 with delayed elimination half-life was the most effective in ameliorating the signs of tabun toxicity. Moreover, both in vitro and in vivo, the versatility of the agents was substantially superior to that of clinically used standards. Their high efficacy and broad-spectrum capability make K1374 and K1375 promising candidates that should be further investigated for their potential as nerve agents and insecticide antidotes.

Pyridinium-2-carbaldoximes with quinolinium carboxamide moiety are simultaneous reactivators of acetylcholinesterase and butyrylcholinesterase inhibited by nerve agent surrogates.

The pyridinium-2-carbaldoximes with quinolinium carboxamide moiety were designed and synthesised as cholinesterase reactivators. The prepared compounds showed intermediate-to-high inhibition of both cholinesterases when compared to standard oximes. Their reactivation ability was evaluated in vitro on human recombinant acetylcholinesterase (hrAChE) and human recombinant butyrylcholinesterase (hrBChE) inhibited by nerve agent surrogates (NIMP, NEMP, and NEDPA) or paraoxon. In the reactivation screening, one compound was able to reactivate hrAChE inhibited by all used organophosphates and two novel compounds were able to reactivate NIMP/NEMP-hrBChE. The reactivation kinetics revealed compound 11 that proved to be excellent reactivator of paraoxon-hrAChE better to obidoxime and showed increased reactivation of NIMP/NEMP-hrBChE, although worse to obidoxime. The molecular interactions of studied reactivators were further identified by in silico calculations. Molecular modelling results revealed the importance of creation of the pre-reactivation complex that could lead to better reactivation of both cholinesterases together with reducing particular interactions for lower intrinsic inhibition by the oxime.

Design, synthesis, and in vitro evaluation of BP-1-102 analogs with modified hydrophobic fragments for STAT3 inhibition.

Twelve novel analogs of STAT3 inhibitor BP-1-102 were designed and synthesised with the aim to modify hydrophobic fragments of the molecules that are important for interaction with the STAT3 SH2 domain. The cytotoxic activity of the reference and novel compounds was evaluated using several human and two mouse cancer cell lines. BP-1-102 and its two analogs emerged as effective cytotoxic agents and were further tested in additional six human and two murine cancer cell lines, in all of which they manifested the cytotoxic effect in a micromolar range. Reference compound S3I-201.1066 was found ineffective in all tested cell lines, in contrast to formerly published data. The ability of selected BP-1-102 analogs to induce apoptosis and inhibition of STAT3 receptor-mediated phosphorylation was confirmed. The structure-activity relationship confirmed a demand for two hydrophobic substituents, i.e. the pentafluorophenyl moiety and another spatially bulky moiety, for effective cytotoxic activity and STAT3 inhibition.

Synthesis, in vitro screening and molecular docking of isoquinolinium-5-carbaldoximes as acetylcholinesterase and butyrylcholinesterase reactivators.

The series of symmetrical and unsymmetrical isoquinolinium-5-carbaldoximes was designed and prepared for cholinesterase reactivation purposes. The novel compounds were evaluated for intrinsic acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) inhibition, when the majority of novel compounds resulted with high inhibition of both enzymes and only weak inhibitors were selected for reactivation experiments on human AChE or BChE inhibited by sarin, VX, or paraoxon. The AChE reactivation for all used organophosphates was found negligible if compared to the reactivation ability of obidoxime. Importantly, two compounds were found to reactivate BChE inhibited by sarin or VX better to obidoxime at human attainable concentration. One compound resulted as better reactivator of NEMP (VX surrogate)-inhibited BChE than obidoxime. The in vitro results were further rationalized by molecular docking studies showing future directions on designing potent BChE reactivators.

Wide-Antimicrobial Spectrum of Picolinium Salts.

Nosocomial infections, which greatly increase morbidity among hospitalized patients, together with growing antibiotic resistance still encourage many researchers to search for novel antimicrobial compounds. Picolinium salts with different lengths of alkyl chains (C12, C14, C16) were prepared by Menshutkin-like reaction and evaluated with respect to their biological activity, i.e., lipophilicity and critical micellar concentration. Picolinium salts with C14 and C16 side chains achieved similar or even better results when in terms of antimicrobial efficacy than benzalkoniums; notably, their fungicidal efficiency was substantially more potent. The position of the methyl substituent on the aromatic ring does not seem to affect antimicrobial activity, in contrast to the effect of length of the N-alkyl chain. Concurrently, picolinium salts exhibited satisfactory low cytotoxicity against mammalian cells, i.e., lower than that of benzalkonium compounds, which are considered as safe.

Oxime K203: a drug candidate for the treatment of tabun intoxication.

For over 60 years, researchers across the world have sought to deal with poisoning by nerve agents, the most toxic and lethal chemical weapons. To date, there is no efficient causal antidote with sufficient effect. Every trialed compound fails to fulfil one or more criteria (e.g. reactivation potency, broad reactivation profile). In this recent contribution, we focused our attention to one of the promising compounds, namely the bis-pyridinium reactivator K203. The oxime K203 is very often cited as the best reactivator against tabun poisoning. Herein, we provide all the available literature data in comprehensive and critical review to address whether K203 could be considered as a new drug candidate against organophosphorus poisoning with the stress on tabun. We describe its development from the historical point of view and review all available in vitro as well as in vivo data to date. K203 is easily accessible by a relatively simple two-step synthesis. It is well accommodated in the enzyme active gorge of acetylcholinesterase providing suitable interactions for reactivation, as shown by molecular docking simulations. According to a literature survey, in vitro data for tabun-inhibited AChE are extraordinary. However, in vivo efficiency remains unconvincing. The K203 toxicity profile did not show any perturbations compared to clinically used standards; on the other hand versatility of K203 does not exceed currently available oximes. In summary, K203 does not seem to address current issues associated with the organophosphorus poisoning, especially the broad profile against all nerve agents. However, its reviewed efficacy entitles K203 to be considered as a backup or tentative replacement for obidoxime and trimedoxime, currently only available anti-tabun drugs.

Profiling donepezil template into multipotent hybrids with antioxidant properties.

Alzheimer's disease is debilitating neurodegenerative disorder in the elderly. Current therapy relies on administration of acetylcholinesterase inhibitors (AChEIs) -donepezil, rivastigmine, galantamine, and N-methyl-d-aspartate receptor antagonist memantine. However, their therapeutic effect is only short-term and stabilizes cognitive functions for up to 2 years. Given this drawback together with other pathological hallmarks of the disease taken into consideration, novel approaches have recently emerged to better cope with AD onset or its progression. One such strategy implies broadening the biological profile of AChEIs into so-called multi-target directed ligands (MTDLs). In this review article, we made comprehensive literature survey emphasising on donepezil template which was structurally converted into plethora of MTLDs preserving anti-cholinesterase effect and, at the same time, escalating the anti-oxidant potential, which was reported as a crucial role in the pathogenesis of the Alzheimer's disease.

Novel Group of AChE Reactivators-Synthesis, In Vitro Reactivation and Molecular Docking Study.

The acetylcholinesterase (AChE) reactivators (e.g., obidoxime, asoxime) became an essential part of organophosphorus (OP) poisoning treatment, together with atropine and diazepam. They are referred to as a causal treatment of OP poisoning, because they are able to split the OP moiety from AChE active site and thus renew its function. In this approach, fifteen novel AChE reactivators were determined. Their molecular design originated from former K-oxime compounds K048 and K074 with remaining oxime part of the molecule and modified part with heteroarenium moiety. The novel compounds were prepared, evaluated in vitro on human AChE (HssAChE) inhibited by tabun, paraoxon, methylparaoxon or DFP and compared to commercial HssAChE reactivators (pralidoxime, methoxime, trimedoxime, obidoxime, asoxime) or previously prepared compounds (K048, K074, K075, K203). Some of presented oxime reactivators showed promising ability to reactivate HssAChE comparable or higher than the used standards. The molecular modelling study was performed with one compound that presented the ability to reactivate GA-inhibited HssAChE. The SAR features concerning the heteroarenium part of the reactivator's molecule are described.

Rational Design of a New Class of Toll-Like Receptor 4 (TLR4) Tryptamine Related Agonists by Means of the Structure- and Ligand-Based Virtual Screening for Vaccine Adjuvant Discovery.

In order to identify novel lead structures for human toll-like receptor 4 (hTLR4) modulation virtual high throughput screening by a peta-flops-scale supercomputer has been performed. Based on the in silico studies, a series of 12 compounds related to tryptamine was rationally designed to retain suitable molecular geometry for interaction with the hTLR4 binding site as well as to satisfy general principles of drug-likeness. The proposed compounds were synthesized, and tested by in vitro and ex vivo experiments, which revealed that several of them are capable to stimulate hTLR4 in vitro up to 25% activity of Monophosphoryl lipid A. The specific affinity of the in vitro most potent substance was confirmed by surface plasmon resonance direct-binding experiments. Moreover, two compounds from the series show also significant ability to elicit production of interleukin 6.

Synthesis, antimicrobial evaluation and molecular modeling of 5-hydroxyisoquinolinium salt series; the effect of the hydroxyl moiety.

In the present paper, we describe the synthesis of a new group of 5-hydroxyisoquinolinium salts with different lengths of alkyl side-chain (C10-C18), and their chromatographic analysis and biological assay for in vitro activity against bacterial and fungal strains. We compare the lipophilicity and efficacy of hydroxylated isoquinolinium salts with the previously published (non-hydroxylated) isoquinolinium salts from the point of view of antibacterial and antifungal versatility and cytotoxic safety. Compound 11 (C18) had to be excluded from the testing due to its low solubility. Compounds 9 and 10 (C14, C16) showed only moderate efficacy against G+ bacteria, notably with excellent potency against Staphyloccocus aureus, but no effect against G- bacteria. In contrast, non-hydroxylated isoquinolinium salts showed excellent antimicrobial efficacy within the whole series, particularly 14 (C14) against G+ strains and 15 (C16) against fungi. The electronic properties and desolvation energies of 5-hydroxyisoquinolinium and isoquinolinium salts were studied by quantum-chemistry calculations employing B3LYP/6-311++G(d,p) method and an implicit water-solvent simulation model (SCRF). Despite the positive mesomeric effect of the hydroxyl moiety reducing the electron density of the quaternary nitrogen, it is probably the higher lipophilicity and lower desolvation energy of isoquinolinium salts, which is responsible for enhanced antimicrobial versatility and efficacy.

SAR study to find optimal cholinesterase reactivator against organophosphorous nerve agents and pesticides.

Irreversible inhibition of acetylcholinesterase (AChE) by organophosphates leads to many failures in living organism and ultimately in death. Organophosphorus compounds developed as nerve agents such as tabun, sarin, soman, VX and others belong to the most toxic chemical warfare agents and are one of the biggest threats to the modern civilization. Moreover, misuse of nerve agents together with organophosphorus pesticides (e.g. malathion, paraoxon, chlorpyrifos, etc.) which are annually implicated in millions of intoxications and hundreds of thousand deaths reminds us of insufficient protection against these compounds. Basic treatments for these intoxications are based on immediate administration of atropine and acetylcholinesterase reactivators which are currently represented by mono- or bis-pyridinium aldoximes. However, these antidotes are not sufficient to ensure 100 % treatment efficacy even they are administered immediately after intoxication, and in general, they possess several drawbacks. Herein, we have reviewed new efforts leading to the development of novel reactivators and proposition of new promising strategies to design novel and effective antidotes. Structure-activity relationships and biological activities of recently proposed acetylcholinesterase reactivators are discussed and summarized. Among further modifications of known oximes, the main attention has been paid to dual binding site ligands of AChE as the current mainstream strategy. We have also discussed new chemical entities as potential replacement of oxime functional group.

Synthesis and disinfection effect of the pyridine-4-aldoxime based salts.

A set of new quaternary ammonium compounds based on pyridine-4-aldoxime was synthesized, characterized with analytical data (NMR, EA, HPLC, MS) and tested for in vitro antimicrobial activity (antibacterial, antifungal) and cytotoxicity. Quaternary pyridinium-4-aldoxime salts with length of alkyl side chain from C8 to C20 and belonging to the group of cationic surfactants were investigated in this work. An HPLC experimental protocol for characterization of mixtures of all homologues has been found. Antimicrobial evaluation found that yeast-type fungi were most sensitive towards C14 and C16 analogues, whereas the C16 analogue was completely ineffective against filamentous fungi. Antibacterial assessment showed versatility of C14 and relatively high efficacy of C16 against G+ strains and C14 against G- strains. Notably, none of the studied compounds exceeded the efficacy and versatility of the benzalkonium C12 analogue, and benzalkonium analogues also exhibited lower cytotoxicity in the cell viability assay.