The search for new anticonvulsants in a group of (2,5-dioxopyrrolidin-1-yl)(phenyl)acetamides with hybrid structure - synthesis and in vivo/In vitro studies

Epilepsy belongs to the most common and debilitating neurological disorders with multifactorial pathophysiology and a high level of drug resistance. Therefore, with the aim of searching for new, more effective, and/or safer therapeutics, we discovered a focused series of original hybrid pyrrolidine-2,5-dione derivatives with potent anticonvulsant properties. We applied an optimized coupling reaction yielding several hybrid compounds that showed broad-spectrum activity in widely accepted animal seizure models, namely, the maximal electroshock (MES) test and the psychomotor 6 Hz (32 mA) seizure model in mice. The most potent anticonvulsant activity and favorable safety profile was demonstrated for compound 30 (median effective dose (ED50) MES = 45.6 mg/kg, ED50 6 Hz (32 mA) = 39.5 mg/kg, median toxic dose (TD50) (rotarod test) = 162.4 mg/kg). Anticonvulsant drugs often show activity in pain models, and compound 30 was also proven effective in the formalin test of tonic pain, the capsaicin-induced pain model, and the oxaliplatin (OXPT)-induced neuropathic pain model in mice. Our studies showed that the most plausible mechanism of action of 30 involves inhibition of calcium currents mediated by Cav1.2 (L-type) channels. Importantly, 30 revealed high metabolic stability on human liver microsomes, negligible hepatotoxicity, and relatively weak inhibition of CYP3A4, CYP2D6, and CYP2C9 isoforms of cytochrome P450, compared to reference compounds. The promising in vivo activity profile and drug-like properties of compound 30 make it an interesting candidate for further preclinical development

Diverse Stomatal Behaviors Mediating Photosynthetic Acclimation to Low Temperatures in Hordeum vulgare

Photosynthetic acclimation to cold conditions is an important factor influencing freezing tolerance of plants. Photosynthetic enzyme activities increase as part of a photochemical mechanism underlying photosynthetic acclimation to low temperatures. Additionally, a non-photochemical mechanism may be activated to minimize photooxidative damage. The aim of this study was to test the hypothesis that differences in stomatal conductance in Hordeum vulgare plants with contrasting freezing tolerances induce various strategies for photosynthetic acclimation to cold stress. Different stomatal behaviors during the prehardening step resulted in diverse plant reactions to low-temperature stress. Plants with a relatively low freezing tolerance exhibited decreased stomatal conductance, resulting in decreased photochemical activity, faster induction of the non-photochemical mechanism, and downregulated expression of two Rubisco activase (RcaA) splicing variants. In contrast, plants with a relatively high freezing tolerance that underwent a prehardening step maintained the stomatal conductance at control level and exhibited delayed photochemical activity and RcaA expression decrease, and increased Rubisco activity, which increased net photosynthetic rate. Thus, in barley, the induction of photoinhibition avoidance (i.e., non-photochemical photoacclimation mechanism) is insufficient for an effective cold acclimation. An increase in cold-induced net photosynthetic rate due to open stomata is also necessary

Identification of the Genetic Basis of Response to de-Acclimation in Winter Barley

Mechanisms involved in the de-acclimation of herbaceous plants caused by warm periods during winter are poorly understood. This study identifies the genes associated with this mechanism in winter barley. Seedlings of eight accessions (four tolerant and four susceptible to de-acclimation cultivars and advanced breeding lines) were cold acclimated for three weeks and de-acclimated at 12 C/5 C (day/night) for one week. We performed differential expression analysis using RNA sequencing. In addition, reverse-transcription quantitative real-time PCR and enzyme activity analyses were used to investigate changes in the expression of selected genes. The number of transcripts with accumulation level changed in opposite directions during acclimation and deacclimation was much lower than the number of transcripts with level changed exclusively during one of these processes. The de-acclimation-susceptible accessions showed changes in the expression of a higher number of functionally diverse genes during de-acclimation. Transcripts associated with stress response, especially oxidoreductases, were the most abundant in this group. The results provide novel evidence for the distinct molecular regulation of cold acclimation and de-acclimation. Upregulation of genes controlling developmental changes, typical for spring de-acclimation, was not observed during mid-winter de-acclimation. Mid-winter de-acclimation seems to be perceived as an opportunity to regenerate after stress. Unfortunately, it is competitive to remain in the coldacclimated state. This study shows that the response to mid-winter de-acclimation is far more expansive in de-acclimation-susceptible cultivars, suggesting that a reduced response to the rising temperature is crucial for de-acclimation tolerance

Utilisation of methylcellulose as a shaping agent in the fabrication of Ba_{0.95}Ca_{0.05}Ce_{0.9}Y_{0.1}O_3 proton-conducting ceramic membranes via the gelcasting method

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Genome-Wide Associations of Chlorophyll Fluorescence OJIP Transient Parameters Connected With Soil Drought Response in Barley

One hundred and nine accessions of spring barley seedlings were phenotyped under soil drought conditions. Chlorophyll fluorescence induction (OJIP) parameters, leaf water content, relative turgidity, net assimilation rate (PN), and water use efficiency (WUE) of plants were measured. All the tested lines were genotyped by means of DArT sequencing (DArTseq) technology. For association mapping a 11,780 polymorphic DArTseq and 4,725 DArTseq SNP markers were used. Our results revealed dissimilar patterns of the relationships between OJIP-parameters under control and drought conditions. A high level of correlation between parameters characterizing Photosystem's II (PSII) energy trapping efficiency (Fv/Fm) and photochemical events downstream of PSII reaction center (e.g., Performance Index—PICSo) was observed only in the case of drought-treated plants. Generally, OJIP parameters were correlated with leaf water content (less in control). This correlation was weaker with WUE, and absent with PN. Under drought stress, 6,252 genotype × phenotype associations, which passed false discovery rate (FDR) verification, were found between all the studied phenotypic characteristics (23, including 19 OJIP parameters) and 2,721 markers. On the other hand, only 282 associations passed FDR test in the control. They comprised 22 phenotypic parameters and 205 markers. Probing for gene annotations of sequences was performed for markers associated with Fv/Fm for both drought and control, markers were associated with studied traits in both control and drought, as well as for markers associated with both OJIP and other physiological parameters in drought. Our work allowed us to conclude that drought treatment differentiates the studied lines through the revealing of relationships between water content and the damages to PSII reaction centers or different components of PSII energy transfer chain. Moreover, the former was not connected with net photosynthesis rate

N-Benzyl-(2,5-dioxopyrrolidin-1-yl)propanamide (AS-1) with hybrid structure as a candidate for a broad-spectrum antiepileptic drug

In our recent studies, we identified compound N-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide (AS-1) as a broad-spectrum hybrid anticonvulsant which showed potent protection across the most important animal acute seizure models such as the maximal electroshock (MES) test, the subcutaneous pentylenetetrazole (s.c. PTZ) test, and the 6-Hz (32 mA) test in mice. Therefore, AS-1 may be recognized as a candidate for new anticonvulsant effective in different types of human epilepsy with a favorable safety margin profile determined in the rotarod test in mice. In the aim of further pharmacological evaluation of AS-1, in the current study, we examined its activity in the 6-Hz (44 mA) test, which is known as the model of drug-resistant epilepsy. Furthermore, we determined also the antiseizure activity in the kindling model of epilepsy induced by repeated injection of pentylenetetrazole (PTZ) in mice. As a result, AS-1 revealed relatively potent protection in the 6-Hz (44 mA) test, as well as delayed the progression of kindling induced by repeated injection of PTZ in mice at doses of 15 mg/kg, 30 mg/kg, and 60 mg/kg. Importantly, the isobolographic analysis showed that a combination of AS-1 and valproic acid (VPA) at the fixed ratio of 1:1 displayed a supra-additive (synergistic) interaction against PTZinduced seizures inmice. Thus, AS-1may be potentially used in an add-on therapy with VPA. Moreover, incubation of zebrafish larvae with AS-1 substantially decreased the number, cumulative but not the mean duration of epileptiform-like events in electroencephalographic assay. Finally, the in vitro ADME-Tox studies revealed that AS-1 is characterized by a very good permeability in the parallel artificial membrane permeability assay test, excellent metabolic stability on human liver microsomes (HLMs), no significant influence on CYP3A4/CYP2D6 activity, and moderate inhibition of CYP2C9 in a concentration of 10 $\mu$M, as well as no hepatotoxic properties in HepG2 cells (concentration of 10 $\mu$M)

Nowy wymiar konfliktów politycznych i społeczno-gospodarczych w XXI wieku

Praca recenzowana / peer-reviewed pape

Discovery of (R)-N-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide [(R)-AS-1], a novel orally bioavailable EAAT2 modulator with drug-like properties and potent antiseizure activity in vivo

[Image: see text] (R)-7 [(R)-AS-1] showed broad-spectrum antiseizure activity across in vivo mouse seizure models: maximal electroshock (MES), 6 Hz (32/44 mA), acute pentylenetetrazol (PTZ), and PTZ-kindling. A remarkable separation between antiseizure activity and CNS-related adverse effects was also observed. In vitro studies with primary glia cultures and COS-7 cells expressing the glutamate transporter EAAT2 showed enhancement of glutamate uptake, revealing a stereoselective positive allosteric modulator (PAM) effect, further supported by molecular docking simulations. (R)-7 [(R)-AS-1] was not active in EAAT1 and EAAT3 assays and did not show significant off-target activity, including interactions with targets reported for marketed antiseizure drugs, indicative of a novel and unprecedented mechanism of action. Both in vivo pharmacokinetic and in vitro absorption, distribution, metabolism, excretion, toxicity (ADME-Tox) profiles confirmed the favorable drug-like potential of the compound. Thus, (R)-7 [(R)-AS-1] may be considered as the first-in-class small-molecule PAM of EAAT2 with potential for further preclinical and clinical development in epilepsy and possibly other CNS disorders