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.

A-series agent A-234: initial in vitro and in vivo characterization.

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 (HssAChE; EC 3.1.1.7) and butyrylcholinesterase (HssBChE; 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 (HssAChE IC50 = 0.101 ± 0.003 µM and HssBChE IC50 = 0.036 ± 0.002 µM), whereas the five marketed oximes have negligible reactivation ability toward A-234-inhibited HssAChE and HssBChE. 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.

Neurotoxicity evoked by organophosphates and available countermeasures.

Organophosphorus compounds (OP) are a constant problem, both in the military and in the civilian field, not only in the form of acute poisoning but also for their long-lasting consequences. No antidote has been found that satisfactorily protects against the toxic effects of organophosphates. Likewise, there is no universal cure to avert damage after poisoning. The key mechanism of organophosphate toxicity is the inhibition of acetylcholinesterase. The overstimulation of nicotinic or muscarinic receptors by accumulated acetylcholine on a synaptic cleft leads to activation of the glutamatergic system and the development of seizures. Further consequences include generation of reactive oxygen species (ROS), neuroinflammation, and the formation of various other neuropathologists. In this review, we present neuroprotection strategies which can slow down the secondary nerve cell damage and alleviate neurological and neuropsychiatric disturbance. In our opinion, there is no unequivocal approach to ensure neuroprotection, however, sooner the neurotoxicity pathway is targeted, the better the results which can be expected. It seems crucial to target the key propagation pathways, i.e., to block cholinergic and, foremostly, glutamatergic cascades. Currently, the privileged approach oriented to stimulating GABAAR by benzodiazepines is of limited efficacy, so that antagonizing the hyperactivity of the glutamatergic system could provide an even more efficacious approach for terminating OP-induced seizures and protecting the brain from permanent damage. Encouraging results have been reported for tezampanel, an antagonist of GluK1 kainate and AMPA receptors, especially in combination with caramiphen, an anticholinergic and anti-glutamatergic agent. On the other hand, targeting ROS by antioxidants cannot or already developed neuroinflammation does not seem to be very productive as other processes are also involved.

A-agents, misleadingly known as "Novichoks": a narrative review.

"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.

Silver distribution in chronic wounds and the healing dynamics of chronic wounds treated with dressings containing silver and octenidine.

Although silver is an efficient antimicrobial and is a widely used antiseptic in wound healing, previous studies have reported the cytotoxic in vitro effects of silver dressings. Moreover, few studies have addressed the distribution of silver in chronic wounds. The study compares the healing of chronic wounds treated with a standard-of-care silver dressing (Ag-CMC) and a dressing containing antiseptic octenidine (OCT-HA). Biopsies were taken from two wound areas before the commencement of treatment (baseline), after 2 weeks and after 6 weeks (the end of the study). We analyzed the histopathologic wound-healing score, silver distribution, and expression of selected genes. The wound-healing score improved significantly in the wounded area treated with OCT-HA after 2 weeks compared to the baseline and the Ag-CMC. The Ag-CMC wound areas improved after 6 weeks compared to the baseline. Moreover, collagen maturation and decreases in the granulocyte and macrophage counts were faster in the OCT-HA parts. Treatment with OCT-HA resulted in less wound slough. The silver, visualized via autometallography, penetrated approximately 2 mm into the wound tissue and associated around capillaries and ECM fibers, and was detected in phagocytes. The metallothionein gene expression was elevated in the Ag-CMC wound parts. This exploratory study determined the penetration of silver into human chronic wounds and changes in the distribution thereof during treatment. We observed that silver directly affects the cells in the wound and elevates the metallothionein gene expression. Octenidine and hyaluronan dressings provide a suitable alternative to silver and carboxymethyl cellulose dressings without supplying silver to the wound.

A systematic evaluation of the cucurbit[7]uril pharmacokinetics and toxicity after a single dose and short-term repeated administration in mice.

Cucurbit[n]urils are macrocyclic compounds capable of forming host-guest complexes with different molecules. In this study, we focused on cucurbit[7]uril (CB[7]) safety and pharmacokinetics. We investigated CB[7] cytotocixity in human renal cells ACHN using the xCELLigence system. We also determined maximum tolerated doses (MTD) and no observed adverse effect levels (NOAEL) after intramuscular (i.m.), intraperitoneal (i.p.), and intragastric (i.g.) administration in mice using clinical observation, blood biochemistry, and histopathology. At NOAELs, we studied its pharmacokinetics in plasma and kidneys. Finally, we performed a 7 day repeated-dose toxicity study at 50% of NOAEL after i.p. administration, assaying CB[7] concentration in plasma, brain, kidney, and liver; we also assessed the liver and kidney histopathology. In vitro, CB[7] did not show toxicity up to 0.94 mg/mL. MTDs in vivo were set at 300, 350, and 600 mg/kg, and NOAEL were established at 150, 100, and 300 mg/kg after i.m., i.p., and i.g. administration, respectively. Parenteral administration produced tissue damage mainly to the kidney, while i.g. administration caused only minor liver damage. Parenteral CB[7] administration led to fast elimination from blood, accompanied with kidney accumulation; absorption from the gastrointestinal tract was minimal. Short repeated i.p. administration was well tolerated. After initial CB[7] accumulation in blood and kidney, the concentrations stabilised and decreased during the experiment. Approximately 3.6% of animals showed signs of nephrotoxicity. Although CB[7] appears to be a promising molecule, nephrotoxicity may be the most critical drawback of its parenteral use, because the kidney represents the main organ of its elimination.

In vitro stress response induced by sulfur mustard in lung fibroblasts NHLF and human pulmonary epithelial cells A-549.

Sulfur mustard [bis(2-chloroethyl) sulfide; SM] is a highly poisonous chemical warfare agent. The mechanism of its cytotoxicity affects several pathways, which cause cell damage or death. The main organ affected in case of exposure to both aerosol and vapor is lungs. The present study focuses on time- and concentration-dependent changes in human lung fibroblasts NHLF and lung epithelial cell line A-549. The cells were treated with SM at the concentrations of 5, 10 and 100 µM and signs of stress response were evaluated during 1-72 h post-treatment. Parameters for testing included cell viability and morphology, loss of transmembrane mitochondrial potential, apoptosis, oxidative stress, changes in the cell cycle, and ATM kinase activation. The cytotoxic effect of SM resulted in a time-dependent decrease in viability of A-459 associated with apoptosis more markedly than in NHLF. We did not observe any generation of reactive oxygen species by SM. SM at concentrations of 5 and 10 µM induced the S-phase cell cycle arrest at both cell lines. On the other hand, 100 µM caused nonspecific cell cycle arrest. ATM kinase was activated transiently. The results indicate that NHLF cells are less prone to toxic damage by SM in case of cell viability, apoptosis and loss of transmembrane mitochondrial potential. The analysis provides a time-related cytotoxic profile of A-549 and NHLF cells for further investigation into the prevention of SM toxic effects and their potential treatment.

Substituted Piperazines as Novel Potential Radioprotective Agents.

The increasing risk of radiation exposure underlines the need for novel radioprotective agents. Hence, a series of novel 1-(2-hydroxyethyl)piperazine derivatives were designed and synthesized. Some of the compounds protected human cells against radiation-induced apoptosis and exhibited low cytotoxicity. Compared to the previous series of piperazine derivatives, compound 8 exhibited a radioprotective effect on cell survival in vitro and low toxicity in vivo. It also enhanced the survival of mice 30 days after whole-body irradiation (although this increase was not statistically significant). Taken together, our in vitro and in vivo data indicate that some of our compounds are valuable for further research as potential radioprotectors.

Pharmacological and toxicological in vitro and in vivo effect of higher doses of oxime reactivators.

The major function of compounds with an oxime moiety attached to a quarternary nitrogen pyridinium ring is to reactivate acetylcholinesterase inhibited by organophosphorus agent (OP). However, other oxime mechanisms (e.g. modulation of cholinergic or glutamatergic receptor) may be involved in the recovery. The main disadvantage of positively charged reactivators is their low ability to penetrate into the brain although crossing the blood brain barrier could be supported via increasing the dose of administered oxime. Thus, this study presents maximal tolerated doses (MTD) for marketed oximes (TMB-4, MMB-4, LüH-6, HI-6, 2-PAM) and the most promising K-oximes (K027, K048, K203) which can be used in OP therapy in the future. No signs of sarin intoxication were observed in mice treated with 100% MTD of HI-6 in contrast to those treated with atropine and only 5% LD50 of HI-6. 100% MTD of HI-6 resulted in levels of 500 µM and 12 µM in plasma and brain, respectively. This concentration is by a far margin safe with respect to direct effects on neuronal cell viability and, on the other hand, does not have any effects on central NMDA receptors or central nACh receptors. However, a weak antimuscarinic activity in case of LüH-6 and a weak peripheral antinicotinic action in case of TMB-4 and 2-PAM could be observed at their respective 100% MTD dose. These high doses, represented by MTD, are, however, irrelevant to clinical practice since they led to mild to moderate toxic side effects. Therefore, we conclude that clinically used doses of marketed oxime reactivators have no significant direct pharmacological effect on the tested receptors.

Cytotoxicity of acetylcholinesterase reactivators evaluated in vitro and its relation to their structure.

The development of acetylcholinesterase reactivators, i.e., antidotes against organophosphorus poisoning, is an important goal of defense research. The aim of this study was to compare cytotoxicity and chemical structure of five currently available oximes (pralidoxime, trimedoxime, obidoxime, methoxime, and asoxime) together with four perspective oximes from K-series (K027, K074, K075, and K203). The cytotoxicity of tested substances was measured using two methods - colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and impedance based real-time cytotoxicity assay - in three different cell lines (HepG2, ACHN, and NHLF). Toxicity was subsequently expressed as toxicological index IC50. The tested compounds showed different cytotoxicity ranging from 0.92 to 40.06 mM. In HepG2 cells, K027 was the least and asoxime was the most toxic reactivator. In ACHN and NHLF cell lines, trimedoxime was the compound with the lowest adverse effects, whereas the highest toxicity was found in methoxime-treated cells. The results show that at least five structural features affect the reactivators' toxicity such as the number of oxime groups in the molecule, their position on pyridinium ring, the length of carbon linker, and the oxygen substitution or insertion of the double bond into the connection chain. Newly synthetized oximes with IC50 ≥ 1 mM evaluated in this three cell lines model might appear suitable for further testing.

A Potent Autophagy Inhibitor (Lys05) Enhances the Impact of Ionizing Radiation on Human Lung Cancer Cells H1299.

Autophagy inhibition through small-molecule inhibitors is one of the approaches to increase the efficiency of radiotherapy in oncological patients. A new inhibitor-Lys05-with the potential to accumulate within lysosomes and to block autophagy was discovered a few years ago. Several studies have addressed its chemosensitizing effects but nothing is known about its impact in the context of ionizing radiation (IR). To describe its role in radiosensitization, we employed radioresistant human non-small cell lung carcinoma cells (H1299, p53-negative). Combined treatment of H1299 cells by Lys05 together with IR decreased cell survival in the clonogenic assay and real-time monitoring of cell growth more than either Lys05 or IR alone. Immunodetection of LC3 and p62/SQSTM1 indicated that autophagy was inhibited, which correlated with increased SQSTM1 and decreased BNIP3 gene expression determined by qRT-PCR. Fluorescence microscopy and flow cytometry uncovered an accumulation of lysosomes. Similarly, transmission electron microscopy demonstrated the accumulation of autophagosomes confirming the ability of Lys05 to potentiate autophagy inhibition in H1299 cells. We report here for the first time that Lys05 could be utilized in combination with IR as a promising future strategy in the eradication of lung cancer cells.

Screening of the chemoprotective effect of 13 compounds and their mixtures with sodium 2-mercaptoethanesulfonate against 2-chloroethyl ethyl sulfide.

2-chloroethyl ethyl sulfide (CEES) is a vesicant agent, commonly referred to half mustard due to its ability to form monofunctional adducts with DNA. In this study, we evaluated the chemoprotective potential of 13 compounds and their mixtures with sodium 2-mercaptoethanesulfonate (MESNA) against CEES-induced geno- and cytotoxicity in human lung cell line A-549. MESNA, L-glutathione (GSH), thiourea, sodium thiosulfate, hexamethylenetetramine, 4-acetamidophenol, asoxime dichloride (HI-6), N-acetyl-L-cysteine (NAC), sodium pyruvate, myo-inositol, 3-aminobenzamide (3-AB), nicotinamide, and Nω-nitro-L-arginine methyl ester hydrochloride and combinations of these compounds with MESNA were applied 30 min before CEES. DNA alkylation was measured using modified comet assay 1 and 24 h after the exposure. Cell viability was determined using MTT assay at 24 and 72 h. The mono-therapeutical approach identified MESNA and GSH to provide significant chemoprotection. NAC and 3-AB supported DNA damage repair, while cell viability remained unaffected. Mixtures of GSH or NAC with MESNA showed protective synergism against DNA damage. Other compounds or their combinations with MESNA failed due to the potentiation of CEES-induced cytotoxicity. The chemoprotection against CEES remains limited; however, the combination of substances can provide protective synergy and may represent a promising strategy in the treatment of accidental exposure to monoalkylating agents.

Mitotic and apoptotic activity in colorectal neoplasia.

BACKGROUND: Colorectal cancer (CRC) is third most commonly diagnosed cancer worldwide. The aim of the prospective study was to evaluate mitosis and apoptosis of epithelial cells at each stage of colorectal neoplasia. METHODS: A total of 61 persons were enrolled into the study: 18 patients with non-advanced colorectal adenoma (non-a-A), 13 patients with advanced colorectal adenoma (a-A), 13 patients with CRC and 17 controls: individuals with normal findings on colonoscopy. Biopsy samples were taken from pathology (patients) and healthy mucosa (patients and healthy controls). Samples were formalin-fixed paraffin-embedded and stained with haematoxylin-eosin. Mitotic and apoptotic activity were evaluated in lower and upper part of the crypts and in the superficial compartment. Apoptotic activity was also assessed using detection of activated caspase-3. RESULTS: In controls, mitotic activity was present in lower part of crypts, accompanied with low apoptotic activity. Mitotic and apoptotic activity decreased (to almost zero) in upper part of crypts. In superficial compartment, increase in apoptotic activity was observed. Transformation of healthy mucosa into non-a-A was associated with significant increase of mitotic activity in lower and upper part of the crypts and with significant increase of apoptotic activity in all three compartments; p < 0.05. Transformation of non-a-A into a-A did not lead to any further significant increase in apoptotic activity, but was related to significant increase in mitotic activity in upper part of crypts and superficial compartment. A significant decrease in apoptotic activity was detected in all three comparments of CRC samples compared to a-A; p < 0.05. No differences in mitotic and apoptotic activity between biopsies in healthy controls and biopsy samples from healthy mucosa in patients with colorectal neoplasia were observed. Detection of activated caspase-3 confirmed the above findings in apoptotic activity. CONCLUSIONS: Significant dysregulation of mitosis and apoptosis during the progression of colorectal neoplasia, corresponding with histology, was confirmed. In patients with sporadic colorectal neoplasia, healthy mucosa does not display different mitotic and apoptotic activity compared to mucosa in healthy controls and therefore adequate endoscopic/surgical removal of colorectal neoplasia is sufficient.

Oxidative stress in organophosphate poisoning: role of standard antidotal therapy.

Despite the main mechanism of organophosphate (OP) toxicity through inhibition of acetylcholinesterase (AChE) being well known over the years, some chronic adverse health effects indicate the involvement of additional pathways. Oxidative stress is among the most intensively studied. Overstimulation of cholinergic and glutamatergic nervous system is followed by intensified generation of reactive species and oxidative damage in many tissues. In this review, the role of oxidative stress in pathophysiology of OP poisoning and the influence of commonly used medical interventions on its levels are discussed. Current standardized therapy of OP intoxications comprises live-saving administration of the anticholinergic drug atropine accompanied by oxime AChE reactivator and diazepam. The capability of these antidotes to ameliorate OP-induced oxidative stress varies between both therapeutic groups and individual medications within the drug class. Regarding oxidative stress, atropine does not seem to have a significant effect on oxidative stress parameters in OP poisoning. In a case of AChE reactivators, pro-oxidative and antioxidative properties could be found. It is assumed that the ability of oximes to trigger oxidative stress is rather associated with their chemical structure than reactivation efficacy. The data indicating the potency of diazepam in preventing OP-induced oxidative stress are not available. Based on current knowledge on the mechanism of OP-mediated oxidative stress, alternative approaches (including antioxidants or multifunctional drugs) in therapy of OP poisoning are under consideration.

Simultaneous determination of malondialdehyde and 3-nitrotyrosine in cultured human hepatoma cells by liquid chromatography-mass spectrometry.

Although reactive oxygen/nitrogen species (ROS/RNS) have a fundamental role in physiological processes, enhanced ROS/RNS production induced by exogenous sources, including drugs and other xenobiotics, may result in serious damage to biomolecules. Oxidative/nitrosative stress is being intensively investigated and might be responsible for a variety of health side effects. The present liquid chromatography-tandem mass spectrometry (LC-MS/MS) method provides reliable and accurate simultaneous measurement of malondialdehyde (MDA) and 3-nitrotyrosine (3-NT) in cultured human hepatoma (HepG2) cells. Sample preparation process involving ultrasonic homogenization, alkaline hydrolysis of protein-bound MDA and 3-NT, deproteination, derivatization of MDA by 2,4-dinitrophenylhydrazine and solid-phase extraction was optimized, ensuring the isolation and purification of desired analytes. Additionally, nonprotein thiols and nonprotein disulfides were measured using HPLC-UV. The established lower limit of quantification (0.025 nmol/mL for MDA; 0.0125 nmol/mL for 3-NT) allowed their LC-MS/MS determination in HepG2 cells exposed to model oxidizing agent, tert-butyl hydroperoxide (t-BOOH). The results show significant changes in MDA and 3-NT concentrations and alterations in thiol redox-state in dependence on the t-BOOH concentration and duration of its incubation in HepG2 cells. Concurrent evaluation of oxidative/nitrosative stress biomarkers in the in vitro model may significantly facilitate assessment of toxicity of newly developed drugs in preclinical trials and thus improve their safety profile.

Gnotobiotic mouse model's contribution to understanding host-pathogen interactions.

This brief review is dedicated to the legacy of Prof. Jaroslav Sterzl and his colleagues, who laid the foundation for gnotobiology in the former Czechoslovakia 55 years. Prof. Sterzl became one of the founders of modern Czechoslovak immunology, which was characterized by work on a wide range of problems needing to be solved. While examining the mechanisms of innate immunity, he focused his studies on the induction of antibody production by immunocompetent cells involved in adaptive immune transmission while using the model of pig fetuses and germ-free piglets and characterizing immunoglobulins in the sera of these piglets. Although not fully appreciated to this day, his experimental proof of the hypothesis focused on the common precursor of cell-forming antibodies of different isotypes was later confirmed in experiments at the gene level. Prof. Sterzl's work represented a true milestone in the development of not solely Czechoslovak but also European and global immunology. He collaborated closely with the World Health Organization for many years, serving there as leader of the Reference Laboratory for Factors of Innate Immunity.

The Evaluation of the Reactivating and Neuroprotective Efficacy of Two Newly Prepared Bispyridinium Oximes (K305, K307) in Tabun-Poisoned Rats-A Comparison with Trimedoxime and the Oxime K203.

The ability of two newly developed oximes (K305, K307) to protect tabun-poisoned rats from tabun-induced inhibition of brain acetylcholinesterase, acute neurotoxic signs and symptoms and brain damage was compared with that of the oxime K203 and trimedoxime. The reactivating and neuroprotective effects of the oximes studied combined with atropine on rats poisoned with tabun at a sublethal dose were evaluated. The reactivating efficacy of a newly developed oxime K305 is lower compared to the reactivating efficacy of the oxime K203 and trimedoxime while the ability of the oxime K307 to reactivate tabun-inhibited acetylcholinesterase (AChE) in the brain roughly corresponds to the reactivating efficacy of the oxime K203 and it is slightly lower compared to trimedoxime. In addition, only one newly developed oxime (K307) combined with atropine was able to markedly decrease tabun-induced neurotoxicity although it did not eliminate all tabun-induced acute neurotoxic signs and symptoms. These results correspond to the histopathological evaluation of tabun-induced brain damage. Therefore, the newly developed oximes are not suitable for the replacement of commonly used oximes (especially trimedoxime) in the treatment of acute tabun poisonings.

Radio-sensitization of human leukaemic MOLT-4 cells by DNA-dependent protein kinase inhibitor, NU7441.

We studied the effect of pre-incubation with NU7441, a specific inhibitor of DNA-dependent protein kinase (DNA-PK), on molecular mechanisms triggered by ionizing radiation (IR). The experimental design involved four groups of human T-lymphocyte leukaemic MOLT-4 cells: control, NU7441-treated (1 µM), IR-treated (1 Gy), and combination of NU7441 and IR. We used flow cytometry for apoptosis assessment, Western blotting and ELISA for detection of proteins involved in DNA repair signalling and epifluorescence microscopy for detection of IR-induced phosphorylation of histone H2A.X. We did not observe any major changes in the amount of DNA-PK subunits Ku70/80 caused by the combination of NU7441 and radiation. Their combination led to an increased phosphorylation of H2A.X, a hallmark of DNA damage. However, it did not prevent up-regulation of neither p53 (and its phosphorylation at Ser 15 and 392) nor p21. We observed a decrease in the levels of anti-apoptotic Mcl-1, cdc25A phosphatase, cleavage of PARP and a significant increase in apoptosis in the group treated with combination. In conclusion, the combination of NU7441 with IR caused increased phosphorylation of H2A.X early after irradiation and subsequent induction of apoptosis. It was efficient in MOLT-4 cells in 10× lower concentration than the inhibitor NU7026. NU7441 proved as a potent radio-sensitizing agent, and it might provide a platform for development of new radio-sensitizers in radiotherapy.

Potent and reversible open-channel blocker of NMDA receptor derived from dizocilpine with enhanced membrane-to-channel inhibition.

N-methyl-D-aspartate receptors (NMDARs) play a significant role in developing several central nervous system (CNS) disorders. Currently, memantine, used for treating Alzheimer's disease, and ketamine, known for its anesthetic and antidepressant properties, are two clinically used NMDAR open-channel blockers. However, despite extensive research into NMDAR modulators, many have shown either harmful side effects or inadequate effectiveness. For instance, dizocilpine (MK-801) is recognized for its powerful psychomimetic effects due to its high-affinity and nearly irreversible inhibition of the GluN1/GluN2 NMDAR subtypes. Unlike ketamine, memantine and MK-801 also act through a unique, low-affinity "membrane-to-channel inhibition" (MCI). We aimed to develop an open-channel blocker based on MK-801 with distinct inhibitory characteristics from memantine and MK-801. Our novel compound, K2060, demonstrated effective voltage-dependent inhibition in the micromolar range at key NMDAR subtypes, GluN1/GluN2A and GluN1/GluN2B, even in the presence of Mg2+. K2060 showed reversible inhibitory dynamics and a partially trapping open-channel blocking mechanism with a significantly stronger MCI than memantine. Using hippocampal slices, 30 µM K2060 inhibited excitatory postsynaptic currents in CA1 hippocampal neurons by ∼51 %, outperforming 30 µM memantine (∼21 % inhibition). K2060 exhibited No Observed Adverse Effect Level (NOAEL) of 15 mg/kg upon intraperitoneal administration in mice. Administering K2060 at a 10 mg/kg dosage resulted in brain concentrations of approximately 2 µM, with peak concentrations (Tmax) achieved within 15 minutes. Finally, applying K2060 with trimedoxime and atropine in mice exposed to tabun improved treatment outcomes. These results underscore K2060's potential as a therapeutic agent for CNS disorders linked to NMDAR dysfunction.

Phenoxytacrine derivatives: Low-toxicity neuroprotectants exerting affinity to ifenprodil-binding site and cholinesterase inhibition.

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.