Vol 45, No 5 (2019)

Nicking endonucleases (NE) are a special group of the restriction endonucleases family. These unique enzymes catalyze the hydrolysis of only one DNA strand in a predetermined position relative to the recognition site. In this review, we summarize the engineering methods for NE construction: inactivation of the catalytic center of restriction endonucleases, disruption of enzyme dimerization interface, or random mutagenesis of the genes оf restriction endonucleases. The main methods of NE application in biotechnology and gene engineering are described. NE-mediated amplification for the enhancement of analytical signal in the detection of nucleic acids, proteins, and small molecules is characterized.

The emerging development of new analytical mass spectrometry technologies resulted in the possibility of a detailed fractionation of biologically important compounds. As applied to lipids, it promoted the development of opportunities for identification of the whole set of lipid molecular species, or the lipidome of a biological object. The review summarizes approaches to and results of human blood plasma lipidome analysis in medical studies. The main principal approaches of MS lipid analysis are considered, including targeted lipidomics after preliminary HPLC or direct MS of the whole lipid extract, termed recently “shotgun lipidomics.” New methods reveal the diversity of individual species (more than 1000) of blood plasma lipids due to many variants of combinations of polar and fatty acid fragments of lipid molecules in each lipid class. Some of them change abundance in different ways in some diseases. Analytical and physiological factors influencing lipidome analysis are also shortly considered, including sample preparation, normalization, and effect of genes associated with lipid metabolism. The prospects of lipidome analysis in clinical studies are noted, as well as the need for standardization of used processes and conditions. Due to the involvement of lipids in many cellular and metabolic processes, their detailed analysis will contribute to unveiling of new biomarkers, as well as to increased understanding of pathogenetic mechanisms of a number of diseases.

HLDF-6 hexapeptide, which corresponds to the ₄₁TGENHR₄₆ fragment of human leukemia differentiation factor (HLDF), shows a wide range of neuroprotective, normalizing, anxiolytic, nootropic and antitumor activities. A promising drug with antitumor activity is being developed based on the N-acetyl, C‑amide form of HLDF-6 peptide, obtained by the solid phase method. Proteolytic hydrolysis of HLDF-6-AA peptide in blood plasma was studied using its derivatives labeled with hydrogen isotopes. The HLDF-6-AA peptide samples uniformly labeled with tritium and deuterium were obtained by the reaction of high-temperature solid-state catalytic isotope exchange at 170°С. [³H] HLDF-6-AA peptide was obtained with the molar radioactivity of 50 Ci/mmol and the average deuterium incorporation for [²H] HLDF-6-AA peptide was 2.90 atoms per peptide molecule. Proteolytic hydrolysis of HLDF-6-AA peptide in rat blood plasma was studied using radiochromatography. It was established that the main pathway of its proteolytic hydrolysis in rat blood plasma consists in cleavage of His-Arg-NH₂ (HR-NH₂) dipeptide from the C-end of the amide with the half-degradation period of 35 min.

The membrane receptor for advanced glycation endproducts (RAGE) is involved in the development of a number of pathological conditions, including Alzheimer’s disease (AD), in which the receptor overexpression in brain cells and its increasing activity is observed. We have previously shown that the synthetic fragment RAGE (60–76), administered intranasally, could prevent the disturbance of the spatial memory of olfactory bulbectomized mice, that develop features of AD. We suggested that N-terminal amino function of the peptide protected with acetic group and C-terminal carboxyl group replaced with amide, would increase the stability of this peptide in in vivo experiments, and this protected peptide would show higher activity compared to the original free one. In the current study the protected peptide analog Ac-(60–76)-NH₂ was synthesized. The activity of the peptide (60–76) and its protected analog has studied in transgenic 5xFAD mice, which represent a generally accepted model of AD. The memory testing was performed in the Morris water maze. It was shown that intranasal administration of these peptides to transgenic 5xFAD mice for two months preserved the spatial memory of animals, and both peptides exhibited the same ability to prevent the spatial memory. The difference in the activity of the tested peptides was revealed 7 days after drug administration had been over, and only animals, previously received the modified peptide Ac-(60–76)-NH₂, showed the ability to find the Morris water maze learning sector. The animals received the peptide (60–76) lost their ability to find the learning sector after 7 days and were completely disoriented. The data obtained allow us to conclude that the modified fragment (60–76) with protected N- and C-terminal functional groups has a more pronounced and long lasting protective effect compared to the free peptide (60–76). Thus, Ac-(60–76)-NH₂ is a promising candidate for development of the drug for the treatment of Alzheimer’s disease.

A series of amides based on (2S)-cyanopyrrolidine and α, β-unsaturated aryl- and hetarylcarboxylic acids have been synthesized. The dependence of the hypoglycemic activity of compounds on the structure of the aromatic fragment has been studied in the oral glucose tolerance test in mice. Amides based on (E)-3-phenylprop-2-enoic and (E)-3-(4-methoxyphenyl)prop-2-enoic acids and (2S)-cyanopyrrolidine have been shown to significantly reduce blood glucose levels in mice. The observed hypoglycemic effect at a dose of 10 mg/kg is comparable to the effect of hypoglycemic drug vildagliptin.

A series of new N-benzoyl-N'-triazine thiourea derivatives have been synthesized via the reaction of 4-amino-6-methyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one with benzoyl chloride derivatives and ammonium thiocyanate in acetone under reflux conditions. 4-Amino-6-methyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one was prepared from the reaction of two equivalents of hydrazine hydrate with carbon disulfide and sodium pyruvate. The chemical structure of thioureas was confirmed using FT-IR, ¹H NMR, ¹³C NMR, and high-resolution mass spectrometry, and elemental analysis. The synthesized thioureas were assayed for their antibacterial activity against both gram-positive (Micrococcusluteus and Bacilluscereus) and gram-negative (Pseudomonasaeruginosa and Escherichiacoli) bacteria using the agar well diffusion method.

Inhibitors of aldose reductase provide a feasible mode of action against diabetic complications. Based on the marketed aldose reductase inhibitor epalrestat containing rhodanine nucleus, rhodanine-3-hippuric acid and its 5-arylidene derivatives were synthesized. The structure of newly synthesized compounds was confirmed by IR, ¹H NMR, and ¹³C NMR spectrometry. In vitro aldose reductase inhibitory activity of the synthesized compounds was assayed and the results showed that most of the derivatives were potent against aldose reductase with IC₅₀ values ranging from 0.2 to 2.36 µM. Two of the compounds with the highest efficacies among the tested compounds (IC₅₀ values of 0.2 and 0.6 µM) were more potent than epalrestat. Molecular docking studies were undertaken to explore the binding modes of all compounds into the active site of aldose reductase in order to rationalize the inhibitory efficacy of these derivatives. Rhodanine-3-hippuric acid and all its arylidene derivatives fulfil Lipinski’s rule and show good drug-likeness property. In conclusion, novel compounds synthesized in the present study have proved to be potential drugs for diabetic complications.

The synthesis of two derivatives of lignan 4'-O-methylhonokiol (MH), i.e., 4'-methoxy-5-propyl-1,1'-biphenyl-2-ol (III) and 4'-(2-fluoroethoxy)-2-hydroxy-5-propyl-1,1'-biphenyl (VI) has been described. Derivative (VI) demonstrates the highest anti-inflammatory activity in the lipopolysaccharide-induced (LPS-induced) neuroinflammation mouse model. Its effect on the morphological changes in the brain is comparable to that of celecoxib, a well-known nonsteroid anti-inflammatory drug and a selective inhibitor of cyclooxygenase-2 (COX 2).