Artur Wnorowski

Novel synthesis scheme and in vitro antimicrobial evaluation of a panel of (E)-2-aryl-1-cyano-1-nitroethenes

Abstract Drug resistance has become a major concern in the field of infection management, therefore searching for new antibacterial agents is getting more challenging. Our study presents an optimized and eco-friendly synthesis scheme for a panel of nitroalkenes bearing various functional groups in the aromatic moiety and bromine or cyano substituents in 1 position of nitrovinyl moiety. The presence of nitrolefine group outside the ring minimalizes genotoxic properties while conjugation of aryl group with nitrovinyl moiety increases stability of the compounds. Then our research focused on evaluation of biological properties of such obtained (E)-2-aryl-1-cyano-1-nitroethenes. As they exhibit strong bacteriostatic and bactericidal activities against reference bacteria and yeast species with no detectable cytotoxicity towards cultured human HepG2 and HaCaT cells, they could be promising candidates for the replacement of traditional nitrofurane-containing antibacterial drugs. Nevertheless, validation of the obtained data in an in vivo model and additional safety studies on mutagenicity are still required.

Homo- and hetero-oligomerization of β2-adrenergic receptor in receptor trafficking, signaling pathways and receptor pharmacology.

The β2-adrenergic receptor (β2AR) is the prototypic member of G protein-coupled receptors (GPCRs) involved in the production of physiological responses to adrenaline and noradrenaline. Research done in the past few years vastly demonstrated that β2AR can form homo- and hetero-oligomers. Despite the fact that currently this phenomenon is widely accepted, the spread and relevance of β2AR oligomerization are still a matter of debate. This review considers the progress achieved in the field of β2AR oligomerization with focus on the implications of the receptor-receptor interactions to β2AR trafficking, pharmacology and downstream signal transduction pathways.

The positive allosteric modulator of α7 nicotinic acetylcholine receptors, 3-furan-2-yl-N-p-tolyl-acrylamide, enhances memory processes and stimulates ERK1/2 phosphorylation in mice.

To determine whether 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), a positive allosteric modulator of α7 nicotinic acetylcholine receptors (nAChRs), improves memory processes, passive avoidance tests were conducted in male mice after acute and chronic treatments. To determine the neuronal mechanisms underlying the promnesic activity elicited by PAM-2, the effect of this ligand on α7 nAChR up-regulation and ERK1/2 phosphorylation was assessed in the hippocampus and prefrontal cortex. The results indicate that: (1) PAM-2 improves memory acquisition/consolidation after acute treatment (Day 2) and memory consolidation after chronic treatment (Day 22). Although no effect was observed on α7 nAChR up-regulation, the chronic, but not acute, PAM-2 treatment increases ERK1/2 kinase phosphorylation, (2) the promnesic activity of PAM-2 was inhibited by methyllycaconitine, a selective α7-antagonist, confirming the role of α7 nAChRs, (3) a synergistic (acute) effect was observed between inactive doses of PAM-2 (0.1 mg/kg) and DMXBA (0.3 mg/kg), a selective α7-agonist, and (4) PAM-2 reversed the memory impairment elicited by scopolamine, a muscarinic antagonist. The results demonstrate that PAM-2 presents promnesic activity mediated by α7 nAChRs, and is able to trigger ERK1/2 phosphorylation only after chronic treatment.

Antitumor activity of (R,R’)‐4‐methoxy‐1‐naphthylfenoterol in a rat C6 glioma xenograft model in the mouse

(R,R’)‐4‐methoxy‐1‐naphthylfenoterol (MNF) inhibits cancer cell proliferation in vitro through cell‐type specific modulation of β2‐adrenergic receptor and/or cannabinoid receptor function. Here, we report an investigation into antitumor activity of MNF in rat C6 glioma cells. The potent antiproliferative action of MNF in these cells (IC50 of ~1 nmol/L) was refractory to pharmacological inhibition of β2‐adrenergic receptor while a synthetic inverse agonist of cannabinoid receptor 1 significantly blocked MNF activity. The antitumor activity of MNF was then assessed in a C6 glioblastoma xenograft model in mice. Three days after subcutaneous implantation of C6 cells into the lower flank of nude mice, these animals were subjected to i.p. injections of saline or MNF (2 mg/kg) for 19 days and tumor volumes were measured over the course of the experiment. Gene expression analysis, quantitative RT‐PCR and immunoblot assays were performed on the tumors after treatment. Significant reduction in mean tumor volumes was observed in mice receiving MNF when compared with the saline‐treated group. We identified clusters in expression of genes involved in cellular proliferation, as well as molecular markers for glioblastoma that were significantly downregulated in tumors of MNF‐treated mice as compared to saline‐injected controls. The efficacy of MNF against C6 glioma cell proliferation in vivo and in vitro was accompanied by marked reduction in the expression of cell cycle regulator proteins. This study is the first demonstration of MNF‐dependent chemoprevention of a glioblastoma xenograft model and may offer a potential mechanism for its anticancer action in vivo.

The Stimulatory Effect of Strontium Ions on Phytoestrogens Content in Glycine max (L.) Merr

The amount of secondary metabolites in plants can be enhanced or reduced by various external factors. In this study, the effect of strontium ions on the production of phytoestrogens in soybeans was investigated. The plants were treated with Hoagland’s solution, modified with Sr2+ with concentrations ranging from 0.5 to 3.0 mM, and were grown for 14 days in hydroponic cultivation. After harvest, soybean plants were separated into roots and shoots, dried, and pulverized. The plant material was extracted with methanol and hydrolyzed. Phytoestrogens were quantified by HPLC. The significant increase in the concentration of the compounds of interest was observed for all tested concentrations of strontium ions when compared to control. Sr2+ at a concentration of 2 mM was the strongest elicitor, and the amount of phytoestrogens in plant increased ca. 2.70, 1.92, 3.77 and 2.88-fold, for daidzein, coumestrol, genistein and formononetin, respectively. Moreover, no cytotoxic effects were observed in HepG2 liver cell models after treatment with extracts from 2 mM Sr2+-stressed soybean plants when compared to extracts from non-stressed plants. Our results indicate that the addition of strontium ions to the culture media may be used to functionalize soybean plants with enhanced phytoestrogen content.

Angiotensin II Type 1 Receptor Blockers Inhibit KAT II Activity in the Brain—Its Possible Clinical Applications

Angiotensin II receptor blockers (ARBs) are one of the most frequently recommended antihypertensive drugs. Apart from their activity towards the circulatory system, ARBs also penetrate the blood-brain barrier and display neuroprotective effects. Kynurenic acid (KYNA) is an endogenous metabolite of tryptophan produced by kynurenine aminotransferase II (KAT II) in the brain. Antagonism towards all ionotropic glutamate (GLU) receptors is the main mechanism of KYNA action. An elevated brain level of KYNA is linked with memory impairment and psychotic symptoms. The aim of this study was to examine the influence of three ARBs: irbesartan, losartan, and telmisartan on KYNA production and KAT II activity in rat brain. The effect of ARBs on KYNA production was analyzed in rat brain cortical slices and on isolated KAT II enzyme. Irbesartan, losartan, and telmisartan decreased KYNA production and KAT II activity in a dose-dependent manner in rat brain cortex in vitro. Molecular docking suggested that the examined ARBs could bind to an active site of KAT II. In conclusion, ARBs decrease KYNA production in rat brain by direct inhibition of KAT II enzymatic activity. This novel mechanism of ARBs action may be advantageous in the treatment of cognitive impairment or the management of schizophrenia.

GPR55 receptor antagonist decreases glycolytic activity in PANC-1 pancreatic cancer cell line and tumor xenografts

The Warburg effect is a predominant metabolic pathway in cancer cells characterized by enhanced glucose uptake and its conversion to l‐lactate and is associated with upregulated expression of HIF‐1α and activation of the EGFR‐MEK‐ERK, Wnt‐β‐catenin, and PI3K‐AKT signaling pathways. (R,R′)‐4′‐methoxy‐1‐naphthylfenoterol ((R,R′)‐MNF) significantly reduces proliferation, survival, and motility of PANC‐1 pancreatic cancer cells through inhibition of the GPR55 receptor. We examined (R,R′)‐MNFs effect on glycolysis in PANC‐1 cells and tumors. Global NMR metabolomics was used to elucidate differences in the metabolome between untreated and (R,R′)‐MNF‐treated cells. LC/MS analysis was used to quantify intracellular concentrations of β‐hydroxybutyrate, carnitine, and l‐lactate. Changes in target protein expression were determined by Western blot analysis. Data was also obtained from mouse PANC‐1 tumor xenografts after administration of (R,R′)‐MNF. Metabolomics data indicate that (R,R′)‐MNF altered fatty acid metabolism, energy metabolism, and amino acid metabolism and increased intracellular concentrations of β‐hydroxybutyrate and carnitine while reducing l‐lactate content. The cellular content of phosphoinositide‐dependent kinase‐1 and hexokinase 2 was reduced consistent with diminished PI3K‐AKT signaling and glucose metabolism. The presence of the GLUT8 transporter was established and found to be attenuated by (R,R′)‐MNF. Mice treated with (R,R′)‐MNF had significant accumulation of l‐lactate in tumor tissue relative to vehicle‐treated mice, together with reduced levels of the selective l‐lactate transporter MCT4. Lower intratumoral levels of EGFR, pyruvate kinase M2, β‐catenin, hexokinase 2, and p‐glycoprotein were also observed. The data suggest that (R,R′)‐MNF reduces glycolysis in PANC‐1 cells and tumors through reduced expression and function at multiple controlling sites in the glycolytic pathway.

Influence of Cyclooxygenase-2 Inhibitors on Kynurenic Acid Production in Rat Brain in Vitro

Significant body of evidence suggests that abnormal kynurenic acid (KYNA) level is involved in the pathophysiology of central nervous system disorders. In the brain, KYNA is synthesized from kynurenine (KYN) by kynurenine aminotransferases (KATs), predominantly by KAT II isoenzyme. Blockage of ionotropic glutamate (GLU) receptors is a main cellular effect of KYNA. High KYNA levels have been linked with psychotic symptoms and cognitive dysfunction in animals and humans. As immunological imbalance and impaired glutamatergic neurotransmission are one of the crucial processes in neurological pathologies, we aimed to analyze the effect of anti-inflammatory agents, inhibitors of cyclooxygenase-2 (COX-2): celecoxib, niflumic acid, and parecoxib, on KYNA synthesis and KAT II activity in rat brain in vitro. The influence of COX-2 inhibitors was examined in rat brain cortical slices and on isolated KAT II enzyme. Niflumic acid and parecoxib decreased in a dose-dependent manner KYNA production and KAT II activity in rat brain cortex in vitro, whereas celecoxib was ineffective. Molecular docking results suggested that niflumic acid and parecoxib interact with an active site of KAT II. In conclusion, niflumic acid and parecoxib are dual COX-2 and KAT II inhibitors.

Effect of Kynurenic Acid on Pupae Viability of Drosophila melanogaster cinnabar and cardinal Eye Color Mutants with Altered Tryptophan-Kynurenine Metabolism

Kynurenic acid (KYNA) is one of the metabolites of evolutionary conserved tryptophan (Trp)/kynurenine (Kyn) metabolic pathway. Elevation of KYNA contributes to development of psychosis in schizophrenia but attenuates neurodegeneration in Drosophila model of Huntington’s disease. We have reported that KYNA increased lethality of pupae of wild-type flies, but not of vermilion (v) mutants with impaired formation of Kyn from Trp, suggesting that KYNA toxicity depends on its interaction with downstream Kyn metabolites [i.e., 3-hydroxykynurenine (3-HK) and/or xanthurenic acid (XA)]. The present study aimed to further explore the mechanisms of KYNA-induced lethality by the assessment of KYNA effect on pupae of two Drosophila mutants: cinnabar (cn), characterized by higher KYNA and lower 3-HK production, and cardinal (cd), characterized by higher 3-HK and XA levels compared to wild-type flies. Our microarray datamining revealed that the gene expression pattern of enzymes forming Trp/Kyn pathway stands in line with previously reported developmental changes in KYNA, 3-HK, and XA concentrations in wild-type and mutant flies. Administration of KYNA increased pupae lethality in cd, but not in cn mutants. Present data suggest that toxic effect of exogenous KYNA depends on the presence of 3-HK and/or XA. This is further supported by our finding that early stages of Drosophila development are associated with a positive expression pattern of genes encoding sulfotransferases, enzymes that are inhibited by KYNA and are involved in detoxification of XA. Alternatively, the toxic effect of KYNA might depend on anti-proliferative effects of KYNA.

Acute MDMA and nicotine co-administration: behavioral effects and oxidative stress processes in mice

3,4-Methylenedioxy-methylamphetamine (MDMA), a synthetic substance commonly known as ecstasy, is a worldwide recreational drug of abuse. As MDMA and nicotine activate the same neuronal pathways, we examined the influence of co-administration of nicotine (0.05 mg/kg) and MDMA (1 mg/kg) on cognitive processes, nicotine-induced behavioral sensitization and on processes linked with oxidative stress and α7 nicotinic acetylcholine receptors (nAChRs) expression in the brain of male Swiss mice. For behavioral study the passive avoidance (PA) test and locomotor sensitization paradigm were used. Also, the oxidative stress parameters as well as expression levels of α7 nAChRs in prefrontal cortex and hippocampus of mice treated with MDMA alone or in combination with nicotine were assessed. The results revealed that MDMA injections as well as co-administrations of MDMA and nicotine improved memory consolidation in male Swiss mice tested in PA task. Furthermore, one of the main findings of the present study is that MDMA increased locomotor activity in nicotine-sensitized mice. Our study showed for the first time strong behavioral and biochemical interactions between nicotine and MDMA. Both drugs are very often used in combination, especially by young people, thus these results may help explaining why psychoactive substances are being co-abused and why this polydrug administration is still a social problem.