Yu. V. Vakhitova

Synthesis and neuropharmacological activity of N -1-adamantylcytisine-12-carbamide and its 12-thiocarbonyl analog

The neuropharmacological activity of two (–)-cytisine derivatives with adamantyl fragments was studied. It was shown that N-1-adamantylcytisine-12-thiocarbamide exhibited in tests in vivo a pronounced mnestic effect, increased the lifespan of laboratory animals under hypoxic conditions, and also enhanced in vitro binding of transcription factors NFAT and NF-κB to the DNA sequences corresponding to them.

NMDA receptors as a possible component of store-operated Ca2+ entry in human T-lymphocytes

Elevation of intracellular Ca2+ in T-lymphocytes as a consequence of T cell antigen receptor activation triggers transcriptional programs resulting in effector cytokine secretion and immune response coordination. Increase of Ca2+ concentration in T-lymphocytes follows both the Ins(1,4,5)P3-dependent release from an intracellular store and subsequent influx from extracellular milieu. Flow cytometry and the fluorescent dye Fluo-4AM have been used to demonstrate that noncompetitive NMDA receptor antagonist (+)-MK801 inhibits Ca2+ influx in T cells induced by thapsigargin. Combination of thapsigargin and (+)-MK801 with following incubation does not affect Ca2+ mobilization from intracellular stores, while decreased Ca2+ entry was observed. Overall data indicate that the ion channel blocker (+)-MK801 is able to inhibit the Ca2+ influx and confirm our suggestion about involvement of NMDA receptor in the store-operated Ca2+ entry mechanisms in human T-lymphocytes. To identify the signal transduction pathways associated with NMDA receptors in mitogen-stimulated T-lymphocytes, the cells were incubated with (+)-MK801, then activity of key phosphorylated protein kinases of MAP-activated (pERK1/2, pSAPK/JNK, p-p38), Ca2+-dependent (pCaMKII), PI3/Akt-dependent (pGSK-3β), and PKC-activated (pPKCθ) pathways were detected. The data we obtained demonstrate that (+)-MK801 treatment leads to more prominent decrease in Ras-activated protein kinases pERK1/2 and Rac-activated proteins p-p38 and pSAPK/JNK, as compared to DAG-dependent pPKCθ and Ca2+-dependent pCaMKII. These results show that NMDA receptors are mainly involved in regulation of Ras/Rac-dependent signaling in T-lymphocytes.

A study of the mechanism of the antiarrhythmic action of Allapinin

Allapinin (lappaconitine hydrobromide) is a drug used for the treatment of cardiac rhythm disturbances; its properties are characteristic of class IC antiarrhythmics. The mechanism of its electrophysiological action involves the blockade of Na+ channels with a subsequent decrease of depolarization rate leading to a slowing of impulse propagation and a decrease of excitability in the conductive system of the heart. Factors underlying the side effects of Allapinin (tachycardia, arterial hypertension, impaired coordination, etc.) are currently unknown, and therefore a study of the molecular mechanisms of its action seems relevant. The target genes of the drug were identified in rats with induced aconitine arrhythmia using the commercially available Rat Neuroscience Ion Channels & Transporters RT2 Profiler™ PCR Array kit (SA Biosciences). A comparison of expression levels of 84 genes in rats treated with Allapinin, after the induction of arrhythmia by aconitine (experiment) and in physiological saline-treated arrhythmic rats (control), revealed 18 mRNAs which were up- or downregulated twofold or more in the experiment relative to the control. Allapinin was shown to stimulate the expression of genes coding for various types of K+ channels (kcna6, kcnj1, kcnj4, kcnq2, and kcnq4), Ca2+ channel (cacna1g), and vesicular acetylcholine transporter (slc18a3). A decrease in mRNA levels was detected for genes coding for K+ channels (kcne1, kcns1), a Na+ channel (scn8a), and membrane transporter genes (atp4a, slc6a9). Our data shows that Allapinin administered to animals with aconitine arrhythmia modulates the expression of genes accounting for ion current conductances involved in the formation of various phases of action potential (INa, Ito, IKs, IK1, ICaT). The effect of the drug on the levels of mRNAs coding for acetylcholine and glycine transporters suggests the involvement of these neuromediators in the mechanisms underlying the antiarrhythmic effect of Allapinin.

Cytosine demethylation in the tyrosine hydroxylase gene promoter in hypothalamus cells of rat brain under the action of 2-aminoadamantane compound Ladasten

In our previous study of mechanisms of pharmacological activity of a derivative of 2-aminoadamantane, Ladasten, we have shown more than a twofold increase in the mRNA level of the tyrosine hydroxylase (TH) gene in the rat brain hypothalamus 1.5–2.0 h after a single administration of this substance. In the present study, we employed the bisulfate sequencing technique to detect changes of GpG islands methylation status in the TH gene 5′-flanking region (−650)−(+84) in hypothalamus cells of control and experimental rats after a single exposure to Ladasten. Ladasten was shown to increase frequency of cytosine demethylation in binding sites of some transcription factors or in neighboring motifs. It is suggested that the Ladasten-induced increase of TH gene transcriptional activity in rat hypothalamus is associated with cytosine demethylation in CpG islands in some regulatory gene elements.

Conjugates of natural chlorins and isobornylphenols with a different length of the spacer between the chlorin and terpenephenolic fragments: synthesis and antioxidant activity

Conjugates containing chlorine and isobornylphenolic fragments, which are connected by spacers of different lengths, were synthesized from methyl pheophorbide a. The toxicity and antioxidant activity of the obtained compounds were studied. Derivatives of pyropheophorbide a containing the most distant from the macrocycle terpenephenolic fragment combine a low toxicity with the high antioxidant activity, and are the most promising for further designing of new medicinal agents for the treatment of diseases associated with a disorder in oxidation-reduction processes in the body.

Search for (-)-cytisine derivatives as potential inhibitors of NF-κB and STAT1

Design and synthesis of new derivatives of (-)-cytisine with a wide spectrum of pharmacological activity, represents a potential therapeutic interest for development of drug candidates for neurodegenerative disorders, inflammatory diseases, and treatment of nicotine addiction. We used HEK293 cell line, transiently transfected with NF-κB and STAT1 luciferase reporter constructs, to select (-)-cytisine derivatives for their potency to modulate basal and induced NF-κB and STAT1 activity. Currently, NF-κB, STAT1 and components of their signaling pathways, are considered as attractive targets for pharmacological intervention, primarily in chronic inflammation, cancer, autoimmune, neurodegenerative and infectious diseases. Library of tested compounds included derivatives of (-)-cytisine with amino, amide, thionyl and carboxamide groups at the 3rd, 5th and 12th position in the original molecule, as well as other bimolecular derivatives. Our experimental results revealed compounds with moderate inducing, as well as inhibitory, effects on basal NF-κB and STAT1 activity (IC50 or EC50 values are mainly in the micromolar range). The structure-activity relationship analysis demonstrated that mode of activity (activation or inhibition of NF-κB and STAT1) is determined by the topology of substituents in (-)-cytisine molecule, whereas the nature of substitutions determines the severity of the effect (introduction of aromatic and adamantyl substitutions, as well as thionyl or ketone groups are of principal importance). Assessments of effects of (-)-cytisine derivatives on activity of NF-κB and STAT1, induced by specific agents (TNFα and IFNγ, respectively), revealed that certain compounds inhibited both basal and stimulated activity of NF-κB and STAT1, whereas other compounds showed a dual effect (increase in basal and decrease in stimulated NF-κB activity), in turn, several compounds increased both basal and induced activity of NF-κB and STAT1. In summary, obtained results indicate that one possible mechanism of biological action of (-)-cytisine derivatives lies is their ability to influence components of NF-κB and STAT1 signaling pathways.

Synthesis and Nootropic Activity of new 3-Amino-12- N -Methylcytisine Derivatives

Reductive alkylation of 3-amino-12-N-methylcytisine by aromatic aldehydes synthesized a series of secondary amines. The nootropic activity of the synthesized compounds was studied in vivo (mnestic and antihypoxic properties) and in vitro (antiradical properties and ability to affect transcription factor HIF-1 DNA-binding activity). The cytotoxicity of the synthesized compounds was assessed. The lead compound was identified.

Amines, Amides, and Thio- and Carboxamides of (–)-Cytisine as Nfat Transcription Factor Modulators

A library of derivatives of the quinolizidine alkaloid (–)-cytisine with amine, amide, and thio- and carboxamides in the 3-, 5-, and 12-positions were synthesized. Their activity with respect to NFAT transcription factor was studied. It was shown that NFAT modulation activity was a function of the nature of the substituent.

Cellular test systems for the search of transcription factors activity modulators

Test systems for monitoring activities and the search for substances activating or inhibiting transcription factors as biological targets have been designed on the basis of luciferase constructs containing binding sites for transcription factors CREB, NFAT, NF-κB, p53, STAT1, GAS, VDR, HSF1, and HIF1α. An assessment of the functional activity of reporter constructs has been carried out using their transient transfection into HEK293 cells followed by treatment with specific inducers. The functional activity of all reporter constructs was observed based on the increased luciferase expression. In order to evaluate the efficiency of the suggested test systems, aspirin was used. Incubation of cells transfected with the above-mentioned constructs treated with aspirin was accompanied by the suppression of NF-κB, HIF1α, GAS, VDR, and HSF binding activity. The findings revealed for NF-κB, NFAT, and STAT1 confirm the published data concerning the mechanisms of aspirin action. The detected effects of this drug on the HIF1α, GAS, VDR, and CREB activity have been demonstrated for the first time.

Genes targets identification of 5-amino-exo-3-azatricyclo[5.2.1.02,6]decane-4-one in an in vivo model of arrhythmia

The molecular mechanisms of action of 5-amino-exo-3-azatricyclo[5.2.1.02,6]decane-4-one (P11), a compound possessing strong antiarrhythmic, nootropic, anti-inflammatory and analgesic activity, have been studied. Cardiac rhythm disturbances were modeled by administering the arrhythmogenic compound aconitin in a dose of 50 μg/kg to the animals from the control group. P-11 in a dose of 0.3 mg/kg was injected intravenously in the experimental group of animals 2 min before aconitin administration. P-11 target genes were identified using the Atlas™ Rat cDNA Expression macroarray (#7738-1, BD Biosciences, United States). Reproducible changes in the expression levels of 16 genes in the heart of rats treated with P-11 concommitantly to arrhythmia modeling in vivo were detected. The genes regulated by the substance coded for proteins of the extracellular matrix are (glypican 1, Gpc1; tissue inhibitor of metalloproteinase 2, 3, Timp2, Timp3), intracellular signaling proteins (rho GTPase activating protein 7, Dlc1; protein tyrosine phosphatase 4a1, Ptp4a1; phosphodiesterase 4D, PDE4D; PI3-kinase regulatory subunit alpha, PIK3R1; guanine nucleotide binding protein alpha 12, Gna12), proteins involved in glycolysis (phosphofructokinase 1, Pfk1), intercellular interactions (junction plakoglobin, Jup), and hemostasis (tissue plasminogen activator, Plat), membranebound pumps and transporters (solute carrier family 16, member 1, Slc16a1; ATPase, Na+/K+ transporting, Atp1a), and others (c-fos proto-oncogene, c-fos; telomerase protein component 1, tlp; Annexin 1, anxa1). Therefore, the data concerning the selective effect of P-11 on genes coding for proteins involved in arrhythmogenesis allow for considering this compound as a promising medication for pathogenetically oriented therapy of arrhythmias.