Design, Synthesis and Assay of Novel Methylxanthine-Alkynylmethylamine Derivatives as Acetylcholinesterase Inhibitors.

Xanthine derivatives have been a great area of interest for the development of potent bioactive agents. Thirty-eight methylxanthine derivatives as acetylcholinesterase inhibitors (AChE) were designed and synthesized. Suzuki-Miyaura cross-coupling reactions of 8-chlorocaffeine with aryl(hetaryl)boronic acids, the CuAAC reaction of 8-ethynylcaffeine with several azides, and the copper(I) catalyzed one-pot three-component reaction (A3-coupling) of 8-ethynylcaffeine, 1-(prop-2-ynyl)-, or 7-(prop-2-ynyl)-dimethylxanthines with formaldehyde and secondary amines were the main approaches for the synthesis of substituted methylxanthine derivatives (yield 53-96%). The bioactivity of all new compounds was evaluated by Ellman's method, and the results showed that most of the synthesized compounds displayed good and moderate acetylcholinesterase (AChE) inhibitory activities in vitro. The structure-activity relationships were also discussed. The data revealed that compounds 53, 59, 65, 66, and 69 exhibited the most potent inhibitory activity against AChE with IC50 of 0.25, 0.552, 0.089, 0.746, and 0.121 µM, respectively. The binding conformation and simultaneous interaction modes were further clarified by molecular docking studies.

Trehalose Activates Hepatic and Myocardial Autophagy and Has Anti-Inflammatory Effects in db/db Diabetic Mice.

Db/db mice (carrying a mutation in the gene encoding leptin receptor) show autophagy suppression. Our aim was to evaluate the effect of autophagy inducer trehalose on liver and heart autophagy in db/db mice and to study inflammation dysregulation and the suitability of chitinases' expression levels as diabetes markers. Thirty-eight male db/db mice and C57/BL mice (control) were used. The db/db model manifested inflammation symptoms: overexpression of TNF-α in the spleen and underexpression of IL-10 in the liver and spleen (cytokine imbalance). Simultaneously, we revealed decreased expression of chitotriosidase (CHIT1) and acid mammalian chitinase (CHIA) in the liver of db/db mice. CHIA expression in db/db mice is significantly lower only in the spleen. Trehalose treatment significantly reduced blood glucose concentration and glycated hemoglobin. Treatment of db/db mice by trehalose was followed by increased autophagy induction in the heart and liver (increased autolysosomes volume density studied by morphometric electron-microscopic method). Trehalose exerted beneficial cardiac effects possibly via increased lipophagy (uptake of lipid droplets). The autophagy activation by trehalose had several positive effects on the heart and liver of db/db mice; therefore, lipophagy activation seems to be a promising therapy for diabetes.

Analyzing the relationship between the cytokine profile of invasive breast carcinoma, its histopathological characteristics and metastasis to regional lymph nodes.

This study was aimed at analyzing the relations of metastasis to regional lymph nodes (RLNs) with histopathological indicators of invasive breast carcinoma of no special type (IC-NST) and its cytokine profile. Enzyme-linked immunosorbent assays were performed to determine concentrations of IL-2, IL-6, IL-8, IL-10, IL-17, IL-18, IL-1ß, IL-1Ra, TNF-α, IFN-γ, G-CSF, GM-CSF, VEGF-A, and MCP-1 in the culture supernatant of IC-NST samples from 48 female patients. Histopathological indicators (degree of tumor cell differentiation, mitoses, and others) and ER, PR, Her2/neu, Ki-67, and CD34 expression levels were determined. By means of three types of neural network models, it was shown that for different parameters of the output layer, different groups of parameters are involved that have predictive value regarding metastasis to RLNs. As a result of multi-dimensional cluster analysis, three clusters were formed with different cytokines profiles of IC-NST. Different correlations between indicators of cytokine production by IC-NST and its histopathological parameters were revealed in groups with different cytokine profiles. It was shown that at simultaneous evaluation of the production of even two cytokines, the importance of which relationship with metastasis was revealed by neural network modeling, can increase the probability of determining the presence of metastasis in the RLNs.

Hybrides of Alkaloid Lappaconitine with Pyrimidine Motif on the Anthranilic Acid Moiety: Design, Synthesis, and Investigation of Antinociceptive Potency.

Convenient and efficient routes to construct hybrid molecules containing diterpene alkaloid lappaconitine and pyrimidine fragments are reported. One route takes place via first converting of lappaconitine to 1-ethynyl-lappaconitine, followed by the Sonogashira cross-coupling-cyclocondensation sequences. The other involves the palladium-catalyzed carbonylative Sonogashira reaction of 5'-iodolappaconitine with aryl acetylene and Mo (CO)6 as the CO source in acetonitrile and subsequent cyclocondensation reaction of the generated alkynone with amidines. The reaction proceeded cleanly in the presence of the PdCl2-(1-Ad)2PBn∙HBr catalytic system. The protocol provides mild reaction conditions, high yields, and high atom and step-economy. Pharmacological screening of lappaconitine-pyrimidine hybrids for antinociceptive activity in vivo revealed that these compounds possessed high activity in experimental pain models, which was dependent on the nature of the substituent in the 2 and 6 positions of the pyrimidine nucleus. Docking studies were undertaken to gain insight into the possible binding mode of these compounds with the voltage-gated sodium channel 1.7. The moderate toxicity of the leading compound 12 (50% lethal dose (LD50) value was more than 600 mg/kg in vivo) and cytotoxicity to cancer cell lines in vitro encouraged the further design of therapeutically relevant analogues based on this novel type of lappaconitine-pyrimidine hybrids.

Effects of polyclonal activators on cell differentiation and cytokine production of cultured invasive breast carcinoma of no special type, their association with tumour histopathological parameters and lymph node metastasis.

Currently, a number of promising strategies and approaches to cancer treatment include differentiation therapy. However, theoretical and methodological foundations of this field are not yet well developed. The objective of this study was to determine the effects of a mixture of polyclonal activators (PAs; phytohaemagglutinin, concanavalin A and lipopolysaccharide) on cytokine production by biopsy samples of invasive breast carcinoma of no special type (IBC-NST) having various differentiation abilities and metastatic potentials as well as on differentiation status of the IBC-NST biopsy samples. We used ELISAs to investigate spontaneous and PA-stimulated cytokine production in the IBC-NST biopsy samples; from these data, we calculated a cytokine production stimulation index (SIPA). The effect of PAs on tumour cell differentiation was determined via a differentiation stimulation index (DSI). DSI was found to vary within the range 1.0-5.0. After treatment with PAs, in the IBC-NST biopsy samples of group I (DSI <1.25), the production of IL-2, IL-6, IL-8, IL-17, IL-18, IL-1ß, IL-1Ra, TNF-α and GM-CSF increased; in the biopsy samples of group II (DSI >1.25), the production of IL-6, IL-1ß, IL-1Ra, TNF-α, G-CSF and GM-CSF significantly increased, while the production of VEGF-A decreased. Receiver operating characteristic (ROC) analysis of SIPA revealed that increased production of IL-18 in the IBC-NST biopsy samples after exposure to PAs may block the PA-driven, cytokine-mediated differentiation of moderately differentiated into highly differentiated tumour cells. The ROC analysis also uncovered an association between the responses of tumour cells to PAs and lymph node metastasis observed in the patients. The findings suggest that there is a need for research aimed at finding new drugs for differentiating cancer therapy and at searching for targeted inducers of cytokine production or specific suppressors of their induction.

Mitochondrial Dysfunction as a Predictor and Driver of Alzheimer's Disease-Like Pathology in OXYS Rats.

Growing evidence suggests that mitochondrial dysfunction is an early event in sporadic Alzheimer's disease (AD), but the impact of mitochondrial dysfunction on the transition from healthy aging to AD remains elusive. Here we estimated the influence of mitochondrial dysfunction on the initiation of AD signs in OXYS rats, which simulate key characteristics of sporadic AD. We assessed the mitochondrial ultrastructure of pyramidal neurons of the hippocampus at the age preceding the development (age 20 days), during manifestation (4-5 months), and at the well-pronounced stages (18-24 months) of the AD-like pathology in OXYS rats. Ultrastructural alterations were collated with the amounts of proteins mediating mitochondrial dynamics [mitofusins (MFN1 and MFN2) and dynamin-1-like protein (DRP1)]; with activity of respiratory chain complexes I, IV, and V in the hippocampal mitochondria; with reactive oxygen species (ROS) production; and with expression of uncoupling protein 2 (UCP2) regulating ROS production. Already at the preclinical stage, OXYS rats showed some characteristic changes in hippocampal mitochondria, which increased in size with the manifestation and progression of AD-like pathology, including decreased activity of respiratory complexes against the background of greater fusion and formation of larger mitochondria. Signs of AD developed simultaneously with increasing dysfunction of mitochondria, with a dramatic decrease in their number, and with increased fission but without upregulation of ROS production (observed only in 20-day-old OXYS rats). Summarizing the data from our present and previous studies, we conclude that mitochondrial dysfunction appears to mediate or possibly even initiate pathological molecular cascades of AD-like pathology in OXYS rats and can be considered a predictor of the early development of the late-onset form of AD in humans.

The gene expression profile of a drug metabolism system and signal transduction pathways in the liver of mice treated with tert-butylhydroquinone or 3-(3'-tert-butyl-4'-hydroxyphenyl)propylthiosulfonate of sodium.

Tert-butylhydroquinone (tBHQ) is a highly effective phenolic antioxidant used in edible oils and fats in foods as well as in medicines and cosmetics. TBHQ has been shown to have both chemoprotective and carcinogenic effects. Furthermore, it has potential anti-inflammatory, antiatherogenic, and neuroprotective activities. TBHQ induces phase II detoxification enzymes via the Keap1/Nrf2/ARE mechanism, which contributes to its chemopreventive functions. Nonetheless, there is growing evidence that biological effects of tBHQ may be mediated by Nrf2-independent mechanisms related to various signaling cascades. Here, we studied changes in gene expression of phase I, II, and III drug metabolizing enzymes/transporters as well as protein levels and activities of cytochromes P450 (CYPs) elicited by tBHQ and its structural homolog TS-13 in the mouse liver. Next, we carried out gene expression analysis to identify signal transduction pathways modulated by the antioxidants. Mice received 100 mg/kg tBHQ or TS-13 per day or only vehicle. The liver was collected at 12 hours and after 7 days of the treatment. Protein and total RNA were extracted. Gene expression was analyzed using Mouse Drug Metabolism and Signal Transduction PathwayFinder RT2Profiler™PCR Arrays. A western blot analysis was used to measure protein levels and a fluorometric assay was employed to study activities of CYPs. Genes that were affected more than 1.5-fold by tBHQ or TS-13 treatment compared with vehicle were identified. Analysis of the gene expression data revealed changes in various genes that are important for drug metabolism, cellular defense mechanisms, inflammation, apoptosis, and cell cycle regulation. Novel target genes were identified, including xenobiotic metabolism genes encoding CYPs, phase II/III drug metabolizing enzymes/transporters. For Cyp1a2 and Cyp2b, we observed an increase in protein levels and activities during tBHQ or TS-13 treatment. Changes were found in the gene expression regulated by NFκB, androgen, retinoic acid, PI3K/AKT, Wnt, Hedgehog and other pathways.

Functional Analysis of the Unique Cytochrome P450 of the Liver Fluke Opisthorchis felineus.

The basic metabolic cytochrome P450 (CYP) system is essential for biotransformation of sterols and xenobiotics including drugs, for synthesis and degradation of signaling molecules in all living organisms. Most eukaryotes including free-living flatworms have numerous paralogues of the CYP gene encoding heme monooxygenases with specific substrate range. Notably, by contrast, the parasitic flatworms have only one CYP gene. The role of this enzyme in the physiology and biochemistry of helminths is not known. The flukes and tapeworms are the etiologic agents of major neglected tropical diseases of humanity. Three helminth infections (Opisthorchis viverrini, Clonorchis sinensis and Schistosoma haematobium) are considered by the International Agency for Research on Cancer (IARC) as definite causes of cancer. We focused our research on the human liver fluke Opisthorchis felineus, an emerging source of biliary tract disease including bile duct cancer in Russia and central Europe. The aims of this study were (i) to determine the significance of the CYP activity for the morphology and survival of the liver fluke, (ii) to assess CYP ability to metabolize xenobiotics, and (iii) to localize the CYP activity in O. felineus tissues. We observed high constitutive expression of CYP mRNA (Real-time PCR) in O. felineus. This enzyme metabolized xenobiotics selective for mammalian CYP2E1, CYP2B, CYP3A, but not CYP1A, as determined by liquid chromatography and imaging analyses. Tissue localization studies revealed the CYP activity in excretory channels, while suppression of CYP mRNA by RNA interference was accompanied by morphological changes of the excretory system and increased mortality rates of the worms. These results suggest that the CYP function is linked to worm metabolism and detoxification. The findings also suggest that the CYP enzyme is involved in vitally important processes in the organism of parasites and is a potential drug target.

Filaggrin mutations in a Western siberian population and their association with atopic dermatitis in children.

We determined the frequencies of null mutations of the FLG gene--2282del4, R501X, R2447X, 3702delG, S3247X, and the 12-repeat allele (rs12730241)--among 460 Caucasians of the city of Novosibirsk, Russia. The frequency was 17.7% for rs12730241, 2.73% for 2282del4, 0.22% for R501X, 0.33% for R2447X, and 0% for 3702delG and S3247X in a western Siberian population. A case-control study showed that the deletion 2282del4 was associated with atopic dermatitis in children (odds ratio 7.01; p<0.001). The other mutations were not.

Water-soluble phenol TS-13 combats acute but not chronic inflammation.

OBJECTIVE: This study was conducted to evaluate the effect of the synthetic water-soluble phenolic antioxidant TS-13 (sodium 3-(4'-methoxyphenyl)propyl thiosulfonate), an inducer of the redox-dependent Keap1/Nrf2/ARE signaling system, in experimental models of acute and chronic inflammation. METHODS: Acute local inflammation was induced by intraplantar carrageenan injection into rat hind paws, and acute systemic inflammation was modeled by intravenous zymosan injection (in rats) or LPS-induced endotoxic shock (in mice). Chronic inflammation was investigated in rat models of air pouch and collagen-induced arthritis. The effects of TS-13 treatment were estimated by changes in the intensity of inflammation (paw edema, liver infiltration, animal survival, exudation, and clinical score of arthritis) and by the effects on reactive oxygen species (ROS) generation by leukocytes from peripheral blood and inflammatory exudates. RESULTS: We found the significant increase in expression of mRNA, content of protein and activity of a well-characterized Nrf2 target enzyme glutathione S-transferase P1, as well as nuclear extract protein binding to the ARE consensus sequence in liver of mice fed with diet containing TS-13. TS-13 markedly attenuated carrageenan-induced paw edema, reduced blood granulocyte number and volume density of liver infiltrates in the systemic zymosan-induced inflammation model, and increased mice survival after lipopolysaccharide-induced septic shock. However, TS-13 administration did not influence cell and protein exudation into air pouches and suppressed clinical manifestation of collagen-induced polyarthritis only at early stages. Nevertheless, TS-13 inhibited the generation of ROS by leukocytes in all inflammation models. CONCLUSION: The data suggest that the anti-inflammatory effects of Keap1/Nrf2/ARE system are more prominent against acute innate-mediated inflammation than chronic immune inflammation. This narrows the potential therapeutic efficacy of ARE inducers in inflammation treatment.

Fatty acids in energy metabolism of the central nervous system.

In this review, we analyze the current hypotheses regarding energy metabolism in the neurons and astroglia. Recently, it was shown that up to 20% of the total brain's energy is provided by mitochondrial oxidation of fatty acids. However, the existing hypotheses consider glucose, or its derivative lactate, as the only main energy substrate for the brain. Astroglia metabolically supports the neurons by providing lactate as a substrate for neuronal mitochondria. In addition, a significant amount of neuromediators, glutamate and GABA, is transported into neurons and also serves as substrates for mitochondria. Thus, neuronal mitochondria may simultaneously oxidize several substrates. Astrocytes have to replenish the pool of neuromediators by synthesis de novo, which requires large amounts of energy. In this review, we made an attempt to reconcile ß-oxidation of fatty acids by astrocytic mitochondria with the existing hypothesis on regulation of aerobic glycolysis. We suggest that, under condition of neuronal excitation, both metabolic pathways may exist simultaneously. We provide experimental evidence that isolated neuronal mitochondria may oxidize palmitoyl carnitine in the presence of other mitochondrial substrates. We also suggest that variations in the brain mitochondrial metabolic phenotype may be associated with different mtDNA haplogroups.