The effects of SN Ps in the regions of positioning RNA polymerase II on the TBP/promoter affinity in the genes of human circadian clock

Genetic variability in the genes of circadian clock is manifested as the phenotypic variability of physiological functions and behavior as well as disorders of the function of not only the clock but also other systems, leading to the development of a pathologies. We analyzed the influence of SNPs localized in the [–70, –20] region from the transcription start site of the gene on TBP / promoter affinity in two groups of genes that are components of the system of human circadian clock. The first group comprises the genes of the circadian oscillator core (11 genes); the second, the genes of the nearest regulatory environment of the circadian oscillator (21 genes). A group for comparison included genes with another function (31 genes). The SNP_TATA_Comparator web service was used for prediction of the effect of SNPs in the regions of positioning of RNA polymerase II on the dissociation constant for TBP / promoter. It was shown that the number of SNP markers reducing the TBP / promoter affinity in the first group of genes significantly lower than the number of SNP markers increasing affinity (α < 10–3). The reverse was true of the comparison group: SNP markers reduced TBP / promoter affinity to a significantly greater extent than the SNP marker increased affinity (α < 10–6). This property may be a characteristic feature of genes  of the circadian oscillator. These predictions are important for identification of candidate SNP markers of various pathologies associated with the dysfunction of circadian clock genes for further testing them in experimental and clinical studies, as well as for verification of mathematical models of the circadian oscillator

Biomedical and candidate SN P markers of chronopathologies can significantly change affinity of ТАТА -binding protein for human gene promoters

Computational analysis of millions of unannotated SNPs from the 1000 Genomes Project may speed up the search for biomedical SNP markers. We combined the analysis of SNPs in the binding sites of ТАТА - binding protein (ТВР) using a previously described W eb service (http://beehive.bionet.nsc.ru/cgi-bin/mgs/ tatascan/start.pl) with a keyword search for biochemicalmarkers of chronopathologies, which correspond to clinical manifestations of these SNPs. In the [–70; –20] region of promoters of 14 human genes (location of proven binding sites of ТВР), we found 32 known and candidate SNP markers of circadian- rhythm disturbances, including rs17231520 and rs569033466 (both: risk of chronopathologies in liver); rs35036378 (behavioral chronoaberrations); rs549858786 (rheumatoid arthritis with a chronoaberration of IL1B expression); rs563207167, rs11557611, and rs5505 (all three: chronopathologies of the tumor – host balance, blood pressure, and the reproductive system); rs1143627 (bipolar disorder with circadian dependence of diagnosis and treatment); rs16887226 and rs544850971 (both: lowered resistance to endotoxins because of the imbalance between the circadian and immune systems); rs367732974 and rs549591993 (both: circadian dependence of heart attacks); rs563763767 (circadian dependence of myocardial infarction); rs2276109 and rs572527200 (both: circadian dependence of asthma attacks); rs34223104, rs563558831, and rs10168 (circadian optima of treatment with methotrexate and cyclophosphamide); and rs397509430, rs33980857,rs34598529, rs33931746, rs33981098, rs34500389, rs63750953, rs281864525, rs35518301, and rs34166473 (all: neurosensory hearing loss and restless legs syndrome). For these SNPs, we evaluated α (significance) of changes in the affinity of ТВР for promoters, where increased affinity corresponds to overexpression of the genes, and decreased affinity to deficient expression (Z-test). Verification of these 32 SNP markers according to clinical standards and protocols may advance the field of predictive preventive personalized medicine

Prediction and verification of the influence of the rs367781716 SN P on the interaction of ТАТА -binding protein with the promoter of the human АВСА9 gene

The high-throughput sequencing project “1 000 Genomes” made it possible to catalog and utilize genetic loci and single nucleotide polymorphisms (SNPs) in medicine. Analysis of SNP markers (significantly frequent differences of individual genomes of patients from the reference human genome) allows physicians to optimize treatment. On the other hand, tens of millions of unannotated SNPs correspond to a gigantic number of false positive (false negative) candidate SNP markers that are selected by computer methods for comparison of their frequency in patients with that in healthy people. This approach contributes to undervaluation of clinically relevant SNPs and to unnecessary computational expenses (on verification of neutral SNPs). Preclinical empirical verification of possible candidate SNP markers may eliminate neutral SNPs from the dataset. In the present study, we found, using the SNP_TATA_Comparator web service, the unannotated SNP rs367781716: the substitution of ancestral T (health) with minor C at position –37 before the transcription initiation site of the АВСА9 gene. This SNP significantly reduces affinity of TATAbinding protein (TBP) for this gene’s promoter and corresponds to a deficiency (low protein level) of the АВСА9 gene product (the transporter ATP-binding cassette A9) in patients with the –37C allele. For preclinical empirical verification of rs367781716, we used an electrophoretic mobility shift assay (EMSA) to measure the rates of formation (ka) and decay (kd) of the complexes of TBP with an oligonucleotide matching either allele –37C or –37T of the АВСА9 gene. We found that the rate of formation (ka) of the TBP/TATA complex for the minor allele is 2.4-fold lower than that for the ancestral allele. We calculated the empirical value of the change in the equilibrium constant of dissociation (KD = kd /ka), which characterizes binding affinity of TBP for a promoter containing the ТАТА box. This empirical value matched the value predicted by SNP_ТАТА _Comparator within the margin of error of the measurements and calculations. We also determined the half-life and Gibbs free energy of the complex of TBP with the АВСА9 promoter. Possible phenotypic manifestations of the candidate SNP marker rs367781716 are discussed

ВАРИАНТЫ И ПЕРСПЕКТИВЫ ПЕРЕПРОФИЛИРОВАНИЯ ЛЕКАРСТВЕННЫХ ПРЕПАРАТОВ ДЛЯ ИСПОЛЬЗОВАНИЯ В ТЕРАПИИ ОНКОЛОГИЧЕСКИХ ЗАБОЛЕВАНИЙ

Recently many new approaches for repurposing or repositioning of the clinically used drugs have been developed. Drug repurposing  allows not only to use known schemes for the synthesis of  biologically active compounds, but also to avoid multiple studies that  are necessary for drug approval process – analysis of  pharmacokinetics, carcinogenicity, acute and chronic toxicity,  including cardiotoxicity, nephrotoxicity, allergenicity etc. It makes  possible to reduce the number of experimental studies as well as  costs of investigations. In cancer research drug repurposing includes screening for medicines used nowadays for the treatment of patients with non-cancer diseases which possess anticancer activity or able to enhance the effects of the standard anticancer chemotherapy, and  search for new applications of known anticancer drugs for the  treatment of different cancer types. Scientific rationale for the search of the compounds with potential anticancer properties among drugs  with different applications is based on the multiple cross-talks of  signaling pathways, which can inhibit cell proliferation. Modern  advances in genomics, proteomics and bioinformatics, development  of permanently improving databases of drug molecular effects and  high throughput analytical systems allow researchers to analyze  simultaneously a large bulk of existing drugs and specific molecular targets. This review describes the main approaches and  resources currently used for the drug repurposing, as well as a  number of examples.В настоящее время появились новые программы по перепрофилированию или перепозиционированию лекарственных средств, используемых в медицинской практике.  Перепрофилирование препаратов позволяет не только использовать отработанные схемы  синтеза биологически активных соединений, но и избежать проведения исследований,  необходимых для внедрения новых лекарственных препаратов в медицинскую практику, по  фармакокинетике, канцерогенности, острой и хронической токсичности, в том числе  кардиотоксичности, нефротоксичности, аллергенности и т.д. Это создает возможность сократить объем необходимых исследований и снизить затраты на них. В онкологии  программы перепрофилирования лекарственных средств включают как поиск препаратов,  обладающих противоопухолевой активностью или потенцирующих действие  противоопухолевых препаратов, среди известных и широко применяемых лекарственных  средств, используемых для лечения неонкологических заболеваний, так и анализ  возможности использования уже известных противоопухолевых препаратов для лечения  каких-либо новых нозологических форм заболевания. Основанием для поиска  противоопухолевых свойств среди препаратов иного назначения является тот факт, что  сигнальные пути в клетке характеризуются большим количеством перекрестных  взаимодействий и некоторые из них могут ингибировать пролиферацию опухолевых клеток.  Современные достижения геномики, протеомики, биоинформатики, появление объемных баз данных по молекулярным эффектам лекарственных препаратов, мощных аналитических систем и их постоянное совершенствование уже позволяет исследователям  одновременно проанализировать большое количество существующих препаратов в  применении к конкретной молекулярной мишени. В обзоре рассмотрены основные подходы  и ресурсы, использующиеся в настоящее время для перепрофилирования лекарственных  препаратов, а также приведен ряд примеров

Correlation of physical development indicators with speed-strength performance in 11-12-year-old boys

© 2019 International Journal of Instruction. The paper is devoted to the issues of improving the methods of physical training. Currently, the method of teaching and training schoolchildren is developed mainly with a focus on a child's age, which does not always correspond to his or her biological maturity. These reasons together with the growing phenomenon of child acceleration reinforce the relevance of the research topic. The purpose of the study is to evaluate the impact of speed-strength exercises on the physical development and speed-strength qualities of schoolchildren aged 11-12. The authors used the measurements of anthropometric and physiometric indicators, as well as the tests that determine the level of development of speed and speed-strength qualities. The results of the research show that the physical indicators of the test group tend to increase, and during the school year, many indicators significantly change

Correlation of physical development indicators with speed-strength performance in 11-12-year-old boys

© 2019 International Journal of Instruction. The paper is devoted to the issues of improving the methods of physical training. Currently, the method of teaching and training schoolchildren is developed mainly with a focus on a child's age, which does not always correspond to his or her biological maturity. These reasons together with the growing phenomenon of child acceleration reinforce the relevance of the research topic. The purpose of the study is to evaluate the impact of speed-strength exercises on the physical development and speed-strength qualities of schoolchildren aged 11-12. The authors used the measurements of anthropometric and physiometric indicators, as well as the tests that determine the level of development of speed and speed-strength qualities. The results of the research show that the physical indicators of the test group tend to increase, and during the school year, many indicators significantly change

P85

Glucocorticoids (GCs) are the most important component of therapy for a number of diseases including combined chemotherapy of hematological malignancies. However, their application is strongly restricted by the development of serious side effects as well as glucocorticoid resistance. Both side effects and resistance affect child cancer patients more intensively. Adverse side-effects of glucocorticoid treatment are the result of glucocorticoid receptor (GR)-mediated gene activation, while the beneficial anti-inflammatory effects result from GR-mediated ‘transrepression’. Difference in mechanisms of therapeutic and side effects of GCs became obvious and selective glucocorticoid agonists (SEGRA) were developed aiming to separate transactivation from transrepression. Recently we have demonstrated that one of the modern SEGRAs, 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride or CpdA, selectively induces transrepression in lymphoma cells and reveals GR-dependent anti-lymphoma activity in vitro and in vivo. However, CpdA is a chiral molecule and exists as a racemic mixture of two enantiomers. Enantiomers often possess the same physical and chemical properties but their biological effects may differ drastically. They differently interact with cell receptors and it can lead to the essential diversity in pharmacokinetics and pharmacodynamics. The notorious example of such a molecule is Thalidomide which was originally designed and used as a sedative drug during the pregnancy, but it was withdrawn chiefly because of its severe teratogenicity. As it was demonstrated, only (R)-thalidomide exhibited significant sedative effects, while (S)-thalidomide revealed the teratogenic effects. The application of enantiopure compounds may lead to reduction of metabolism variability in patients and to decrease of drug effective dose. Thereby, the synthesis of enantiopure isomers of CpdA and the study of their anti-cancer activity are of immediate interest in cancer research. We synthesized for the first time enantiomers of the CpdA based on literature precedent Sharpless asymmetric dihydroxylation with AD-mix-alpha or AD-mix-beta. (S)- and (R)-enantiomers with enantiopure excess of 98% were obtained. Then we evaluated the cytotoxic effects on acute lymphoblastic leukemia cells CEM and mantle cell lymphoma cells Granta by direct cell counting and found that cytotoxic activities of both enantiomers were comparable with the effect of racemic mixture on cell growth and survival. Glucocorticoids modulate the expression of genes through transrepression and transactivation mechanisms realized in equal measure. In present work we estimated the potential ability of newly synthesized enantiomers to activate these mechanisms. Transactivation realizes through direct binding of receptor homodimers to specific sequences (glucocorticoid responsive elements (GREs)) in promoter or enhancer regions of GR target genes. Therefore, we studied the level of transactivation as the expression of GR-regulated genes, immunophiline FKBP51 and glucocorticoid-induced leucine zipper GILZ. As shown by Q-RT-PCR, the expression of FKBP51 and GILZ was unaffected after cell treatment by both CpdA enantiomers; hence, these compounds did not induce GR transactivation. GR-transrepression is chiefly mediated by protein–protein interaction between GR and other transcription factors like NF-kB and AP1, followed by inhibition of their transcriptional activity. We evaluated the transrepression by expression of NF-kB-depended genes, cyclines D1 and D2 (CCND and CCND2). (S)-enantiomer of CpdA down-regulated the expression of CCND1 and CCND2 to the level compared with Dex while (R)-enantiomer surprisingly increased level of CCND1 and CCND2 expression. This fact demonstrated that (S)-CpdA is perspective as antiproliferative and cell growth inhibiting agent. Overall, our data provide the potential for further studies of CpdA optical isomers, especially (S)-enantiomer, as GR-dependent anti-cancer agents

T25

Glucocorticoids (GCs) are widely used in treatment of many cancer types due to its ability to induce apoptosis in malignant cells in blood cancer therapy, and to prevent nausea, emesis and chemotherapy-associated hepatotoxicity in case of solid tumors. However, severe dose-limiting side effects occur, including osteoporosis, diabetes and other metabolic complications. Moreover, in therapy of solid tumors GCs strongly affect microenvironment which could be associated with poor prognosis, risk of metastasis and high frequency of relapses. Biological response to GCs is mediated by glucocorticoid receptor (GR), a well-characterized transcription factor. GR controls gene expression via (1) transactivation, which requires binding of GR homodimers to glucocorticoid-responsive elements (GRE) in gene promoters and enhancers, and (2) dimerization-independent transrepression mediated via negative interaction between GR and other transcription factors including major effectors of inflammation and proliferation. Transrepression plays an important role in anti-inflammatory and anti-cancer effects of GR, including normalizing influence on microenvironment, while side effects are associated with GR transactivation. In particular, GCs induce insulin resistance in adipocytes, a major component of the mammary microenvironment, which secrete pro-inflammatory cytokines and growth factors, implicated in tumor progression. Selective GR agonists (SEGRA) that preferentially activate GR transrepression could be a better option for treatment of cancer. Dozens of candidate SEGRAs were identified, synthesized and tested by industry and academia, with some having reached clinical trials. One of the novel GR modulators is 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium-chloride, or CpdA, synthetic analogue of aziridine precursor isolated from Namibian shrub Salsola tuberculatiformis Botschantzev. It was shown that CpdA acts as “dissociated” GR ligand: it competes with GCs for GR binding and efficiently induces GR transrepression but not transactivation. We and other authors reported recently that CpdA inhibits survival of prostate cancer cells as well as blood cancer cells in GR-dependent fashion. Furthermore, primary leukemia cells from T-ALL patients appeared to be equally sensitive to GCs and CpdA. Our further studies were concentrated on three directions: (1) GC/SEGRA-based chemotherapy. We screened biological effect of CpdA in combination with traditional agents (doxorubicin, vincristine) and newer therapeutics (Bortezomib, Carfilzomib, MLN-4924, Rapamycin). Pretreatment of lymphoma cells with proteasome inhibitor Bortezomib resulted in GR accumulation and enhanced ligand properties of CpdA. We also revealed remarkable GR-dependent cooperation between CpdA and Bortezomib in suppressing survival of lymphoma cells in vitro and in vivo. Also surprising findings were substantial cooperation in anti-cancer effect of immunosuppressant Rapamycin and CpdA in vitro, and unexpected “dissociated” effect of Rapamycin on GR signaling realized through down -regulation of REDD1, mTORC1 inhibitor. These data suggested high clinical potential of Rapamycin/GC combination in cancer treatment. (2) SEGRA list extention We used two approaches to extend SEGRA list: (1) synthesis of CpdA enantiomers and (2) its chemical derivatives. Chemical analogues of CpdA were designed by appending of bulky substituent into benzene ring, alkylation of carbon atom adjacent to chlorine atom or appending of substituents to nitrogen atom. Evaluation of biological properties of enantiomers revealed higher GR-dependent anti-cancer potential of S-CpdA. Cytotoxic and proapoptotic effects of CpdA analogues were comparable with precursor. (3) Selection of tumor types acceptable for SEGRA treatment. CpdA was selected for NCI-60 in Vitro Cell Line Screening Project providing direct support to anticancer drug discovery program. It was shown that CpdA affect viability of some adherent cancer cell lines. We demonstrated that CpdA unlike GCs did not modify microenvironment and disintegrate tight junctions between cells decreasing risk of metastasis in case of solid tumors. It demonstrates reasonability of further investigations. Overall, our data provide the rationale for novel therapy of cancer based on combination of non-steroidal GR modulators with classic and modern chemotherapeutics. Approaches to obtain more SEGRAs were elaborated

COMPARATIVE ANALYSIS OF BIOLOGICAL EFFECTS OF SELECTIVE ACTIVATOR OF THE GLUCOCORTICOID RECEPTOR CPDA ON DIFFERENT SUBTYPES OF BREAST CANCER CELL LINES

Glucocorticoids (GCs) are often used as an adjuvant therapy to reduce the adverse effects of chemotherapy in breast cancer patients. Moreover, GCs can display pro-proliferative or anti-proliferative effects on BC cells depending on their molecular subtype. In addition, long-term use of GCs can induce drug resistance and tumor progression. The biological activity of GCs is mediated by glucocorticoid receptor (GR) via either transrepression or transactivation. The anti-inflammatory effects of GCs are thought to be due to transrepression, while side effects, drug resistance and tumor progression/metastasis are associated with transactivation. We have previously demonstrated that Compound A, a selective GR agonist (SEGRA), has a GR-dependent antitumor effect on blood cancer cells in vitro, not triggering the GR transactivation. This study was focused on the analysis of the CpdA activity in BC models in vitro. We demonstrated the antiproliferative effect of CpdA on BC cells and its ability to induce transrepression of GR-depended genes such as CCND1-3, COX-2, iNOS without the induction of transactivation. A comparative analysis showed that CpdA was an effective and safe alternative to dexamethasone in adjuvant chemotherapy for breast cancer