Sergey V. Leonov

Efficient Synthesis of Glaziovianin A Isoflavone Series from Dill and Parsley Extracts and Their in Vitro/in Vivo Antimitotic Activity

A concise six-step protocol for the synthesis of isoflavone glaziovianin A (GVA) and its alkoxyphenyl derivatives 9 starting with readily available plant metabolites from dill and parsley seeds was developed. The reaction sequence involved an efficient conversion of the key intermediate epoxides 7 into the respective β-ketoaldehydes 8 followed by their Cu(I)-mediated cyclization into the target series 9. The biological activity of GVA and its derivatives was evaluated using a panel of seven human cancer cell lines and an in vivo sea urchin embryo assay. Both screening platforms confirmed the antimitotic effect of the parent GVA (9cg) and its alkoxy derivatives. Structure-activity relationship studies suggested that compounds 9cd and 9cf substituted with trimethoxy- and dillapiol-derived B-rings, respectively, were less active than the parent 9cg. Of the evaluated human cancer cell lines, the A375 melanoma cell line was the most sensitive to the tested molecules. Notably, the target compounds were not cytotoxic against human peripheral blood mononuclear cells up to 10 μM concentration. Phenotypic readouts from the sea urchin assay unequivocally suggest a direct microtubule-destabilizing effect of isoflavones 9cg, 9cd, and 9cf.

A Comprehensive Insight into the Chemical Space and ADME Features of Small Molecule NS5A Inhibitors

Non-structural 5A (NS5A) protein plays a crucial role in the replication of hepatitis C virus (HCV) and during the past decade has attracted increasing attention as a promising biological target for the treatment of viral infections and related disorders. Small-molecule NS5A inhibitors have shown significant antiviral activity in vitro and in vivo. Several lead molecules are reasonably regarded as novel highly potent drug candidates with favorable ADME features and tolerable side effects. The first-in-class daclatasvir has recently been launched into the market and 14 novel molecules are currently under evaluation in clinical trials. From this perspective, we provide an overview of the available chemical space of small-molecule NS5A inhibitors and their PK properties, mainly focusing on the diversity in structure and scaffold representation.

In Silico Approaches to the Design of NS5A Inhibitors

In recent years, nonstructural protein 5A (NS5A) has rapidly emerged as a promising therapeutic target for Hepatitis C (HCV) virus therapy. It is involved in both viral RNA replication and virus assembly and NS5A plays a critical role in the regulation of HCV life cycle. NS5A replication complex inhibitors (NS5A RCIs) have demonstrated strong antiviral activity in vitro and in vivo. However, wild-type resistance mutations and a wide range of genotypes significantly reduce their clinical efficacy. The exact mechanism of NS5A action still remains elusive, therefore several in silico models have been constructed to gain insight into the drug binding and subsequent structural optimization to overcome resistance. This paper provides a comprehensive overview of the computational studies towards NS5A mechanism of action and the design of novel small-molecule inhibitors.

Discovery of a Novel Series of Tankyrase Inhibitors by a Hybridization Approach

A structure-guided hybridization approach using two privileged substructures gave instant access to a new series of tankyrase inhibitors. The identified inhibitor 16 displays high target affinity on tankyrase 1 and 2 with biochemical and cellular IC50 values of 29 nM, 6.3 nM and 19 nM, respectively, and high selectivity toward other poly (ADP-ribose) polymerase enzymes. The identified inhibitor shows a favorable in vitro ADME profile as well as good oral bioavailability in mice, rats, and dogs. Critical for the approach was the utilization of an appropriate linker between 1,2,4-triazole and benzimidazolone moieties, whereby a cyclobutyl linker displayed superior affinity compared to a cyclohexane and phenyl linker.

Diffuse colonies of human skin fibroblasts in relation to cellular senescence and proliferation

Development of personalized skin treatment in medicine and skin care may benefit from simple and accurate evaluation of the fraction of senescent skin fibroblasts that lost their proliferative capacity. We examined whether enriched analysis of colonies formed by primary human skin fibroblasts, a simple and widely available cellular assay, could reveal correlations with the fraction of senescent cells in heterogenic cell population. We measured fractions of senescence associated β-galactosidase (SA-βgal) positive cells in either mass cultures or colonies of various morphological types (dense, mixed and diffuse) formed by skin fibroblasts from 10 human donors. Although the donors were chosen to be within the same age group (33-54 years), the colony forming efficiency of their fibroblasts (ECO-f) and the percentage of dense, mixed and diffuse colonies varied greatly among the donors. We showed, for the first time, that the SA-βgal positive fraction was the largest in diffuse colonies, confirming that they originated from cells with the least proliferative capacity. The percentage of diffuse colonies was also found to correlate with the SA-βgal positive cells in mass culture. Using Ki67 as a cell proliferation marker, we further demonstrated a strong inverse correlation (r=−0.85, p=0.02) between the percentage of diffuse colonies and the fraction of Ki67+ cells. Moreover, a significant inverse correlation (r=−0.94, p=0.0001) between the percentage of diffuse colonies and ECO-f was found. Our data indicate that quantification of a fraction of diffuse colonies may provide a simple and useful method to evaluate the extent of cellular senescence in human skin fibroblasts.

Computational insight into the chemical space of plant growth regulators.

An enormous technological progress has resulted in an explosive growth in the amount of biological and chemical data that is typically multivariate and tangled in structure. Therefore, several computational approaches have mainly focused on dimensionality reduction and convenient representation of high-dimensional datasets to elucidate the relationships between the observed activity (or effect) and calculated parameters commonly expressed in terms of molecular descriptors. We have collected the experimental data available in patent and scientific publications as well as specific databases for various agrochemicals. The resulting dataset was then thoroughly analyzed using Kohonen-based self-organizing technique. The overall aim of the presented study is to investigate whether the developed in silico model can be applied to predict the agrochemical activity of small molecule compounds and, at the same time, to offer further insights into the distinctive features of different agrochemical categories. The preliminary external validation with several plant growth regulators demonstrated a relatively high prediction power (67%) of the constructed model. This study is, actually, the first example of a large-scale modeling in the field of agrochemistry.

OP VIII – 4 Type of physical activity, diet, bmi and tobacco/alcohol consumption relationship: which of them affect more our health?

Background/aim Many studies related to health include such parameters as physical activity, diet, BMI and substances consumption. The purpose of this article was to check the magnitude of their effects on health together with other parameters as age, sex and level of education. Methods Design The study was based on epidemiological data (9617 participants from 19 to 81 y.o. for whom we have health indicators over a period of 11 years) together with questionnaires about diet, alcohol, tobacco and the frequency of different types of physical activities together with socio-demographical parameters. Methods Descriptive statistics and non-parametric correlational analysis of epidemiological data was performed with use of SPSS v.19. Results The magnitude of association of factors with days of hospitalisation were the following ones (from greater to lesser): physical activity type 3 (gardening); sport activity type 1 (aerobic, swimming, running); level of studies; age; BMI; menu variability; smoking; weight maintenance; alcohol consumption; sport activity type 2 (Pilates, yoga) and limitation of fats consumption (1–9 were statistically significant at p Conclusion Our findings confirm that leading a healthy life style is an important issue since it has a significant relationship with physical and mental health indicators. Moreover, our results show different associations by ranking the studied factors with different health indicators (number of days spent in hospital, visits of doctors and psychiatrists, drug consumption, etc.).

γH2AX, 53BP1 and Rad51 protein foci changes in mesenchymal stem cells during prolonged X-ray irradiation

At high exposure levels ionizing radiation is a carcinogen. Little is known about how human stem cells, which are known to contribute to tumorigenesis, respond to prolonged radiation exposures. We studied formation of DNA double strand breaks, accessed as γH2AX and 53BP1 foci, in human mesenchymal stem cells (MSCs) exposed to either acute (5400 mGy/h) or prolonged (270 mGy/h) X-irradiation. We show a linear γH2AX and 53BP1 dose response for acute exposures. In contrast, prolonged exposure resulted in a dose-response curve that had an initial linear portion followed by a plateau. Analysis of Rad51 foci, as a marker of homologous recombination, in cells exposed to prolonged irradiation revealed a threshold in a dose response. Using Ki67 as a marker of proliferating cells, we show no difference in the γH2AX distribution in proliferating vs. quiescent cells. However, Rad51 foci were found almost exclusively in proliferating cells. Concurrent increases in the fraction of S/G2 cells were detected in cells exposed to prolonged irradiation by scoring CENPF-positive cells. Our data suggest that prolonged exposure of MSCs to ionizing radiation leads to cell cycle redistribution and associated activation of homologous recombination. Also, proliferation status may significantly affect the biological outcome, since homologous repair is not activated in resting MSCs.At high exposure levels ionizing radiation is a carcinogen. Little is known about how human stem cells, which are known to contribute to tumorigenesis, respond to prolonged radiation exposures. We studied formation of DNA double strand breaks, accessed as γH2AX and 53BP1 foci, in human mesenchymal stem cells (MSCs) exposed to either acute (5400 mGy/h) or prolonged (270 mGy/h) X-irradiation. We show a linear γH2AX and 53BP1 dose response for acute exposures. In contrast, prolonged exposure resulted in a dose-response curve that had an initial linear portion followed by a plateau. Analysis of Rad51 foci, as a marker of homologous recombination, in cells exposed to prolonged irradiation revealed a threshold in a dose response. Using Ki67 as a marker of proliferating cells, we show no difference in the γH2AX distribution in proliferating vs. quiescent cells. However, Rad51 foci were found almost exclusively in proliferating cells. Concurrent increases in the fraction of S/G2 cells were detected in cells exposed to prolonged irradiation by scoring CENPF-positive cells. Our data suggest that prolonged exposure of MSCs to ionizing radiation leads to cell cycle redistribution and associated activation of homologous recombination. Also, proliferation status may significantly affect the biological outcome, since homologous repair is not activated in resting MSCs.

The Evaluation of Geroprotective Effects of Selected Flavonoids in Drosophila melanogaster and Caenorhabditis elegans

Flavonoids is an intensively studied group of natural compounds with antioxidant, antineoplastic, antihyperglycemic, cardioprotective, and neuroprotective properties. The present study intends to investigate the geroprotective action of three selected flavonoids (naringin, luteolin, chrysin) in two model organisms, Caenorhabditis elegans and Drosophila melanogaster. Luteolin and chrysin were shown to improve lifespan parameters when administered to both model organisms. The observed positive effects of these flavonoids in D. melanogaster were limited to females and were not associated with reduced fecundity or locomotor impairment. The life-extending effects of flavonoids were observed in N2 wild-type worms but absent in aak-2(gt33) mutants implying that these effects can be associated with AMP-activated protein kinase activity. Naringin improved lifespan parameters of C. elegans, but had no effect on D. melanogaster females; in some cases, naringin was found to decrease the lifespan of males. Compared to chrysin and luteolin, however, naringin more effectively activates Nrf2 target genes (particularly, GstD1) under oxidative stress. Then we compared molecular mechanisms of studied compounds and a well-known geroprotector rapamycin, using software tool GeroScope. There are no transcriptomic data on luteolin or chrysin provided by LINCS Project database. The bioinformatics comparison of transcriptomics data for A549 and MCF7 human cell lines treated with rapamycin or naringin revealed that these compounds share just a few common signaling pathways and quite distinct in their geroprotective action. Thus, based on C. elegans effects of naringin, luteolin, chrysin on lifespan we have revealed new potential geroprotectors.

Residual γH2AX foci induced by low dose x-ray radiation in bone marrow mesenchymal stem cells do not cause accelerated senescence in the progeny of irradiated cells

Mechanisms underlying the effects of low-dose ionizing radiation (IR) exposure (10-100 mGy) remain unknown. Here we present a comparative study of early (less than 24h) and delayed (up to 11 post-irradiation passages) radiation effects caused by low (80 mGy) vs intermediate (1000 mGy) dose X-ray exposure in cultured human bone marrow mesenchymal stem cells (MSCs). We show that γН2АХ foci induced by an intermediate dose returned back to the control value by 24 h post-irradiation. In contrast, low-dose irradiation resulted in residual γН2АХ foci still present at 24 h. Notably, these low dose induced residual γН2АХ foci were not co-localized with рАТМ foci and were observed predominantly in the proliferating Кi67 positive (Кi67+) cells. The number of γН2АХ foci and the fraction of nonproliferating (Кi67-) and senescent (SA-β-gal+) cells measured at passage 11 were increased in cultures exposed to an intermediate dose compared to unirradiated controls. These delayed effects were not seen in the progeny of cells that were irradiated with low-dose X-rays, although such exposure resulted in residual γН2АХ foci in directly irradiated cells. Taken together, our results support the hypothesis that the low-dose IR induced residual γH2AХ foci do not play a role in delayed irradiation consequences, associated with cellular senescence in cultured MSCs.