Analysis of nuclear glucocorticoid receptor-DNA interaction in aged rat liver

Abstract: In order to contribute to the understanding of mechanisms by which regulatory proteins recognize genetic information stored in DNA, analyses of their interaction with specific nucleotides are usually performed. In this study, the electrophoretic mobility shift assay (EMSA) was applied to analyze the interaction of nuclear proteins from the liver of rats of different age i.e., young (3-month-old), middle- aged (12-month-old) and aged (24-month-old), with radioactively labelled synthetic oligonucleotide analogues, corresponding to GRE. The levels of GRE binding activity were assessed by quantitative densitometric scanning of the autoradiograms. The results showed statistically significant decreasing values of up to 78% and 49% in middle aged and old animals, respectively, compared to young animals (p < 0.05). The specificity of the nuclear proteins-GRE interaction was demonstrated by competition experiments with unlabelled GRE. In a supershift assay, using the antibody BuGR2, it was shown that the GR proteins present in nuclear extracts have a high affinity for the GRE probe. The stabilities of the protein-DNA complexes were analysed and it was concluded that they changed during ageing.U cilju doprinosa razumevanju mehanizama pomoću kojih regulatorni proteini prepoznaju genetičku informaciju koju nosi DNK, analiziraju se njihove interakcije sa specifičnim nukleotidima. U ovom radu je metodom EMSA analizirana interakcija jedarnih proteina iz jetri pacova iz tri starosne grupe (mladi - 3 meseca, srednje doba – 12 meseci i stari – 24 meseca) sa sintetičkim, radioaktivno obeleženim, oligonukleotidnim analogom GRE. Nivo vezujuće aktivnosti GRE je određivan kvantitativno denzitometrijskom autoradiografijom. Rezultati su pokazali da postoji statistički značajan pad vrednosti GRE-vezujuće aktivnosti do 78% kod životinja srednjeg starosnog doba i do 49 % kod starih životinja, u poređenju sa vrednostima dobijenim za mlade životinje (p < 0.05). Specifičnost interakcije jedarnih proteina i GRE je određena eksperimentima kompeticije sa neobeleženim GRE. Korišćenjem antitela BuGR2 pokazano je da je glukokortikoidni receptor protein koji u jedarnom ekstraktu ima najveći afinitet za GRE probu. Analizirana je stabilnost kompleksa protein-DNK i zaključeno je da se menja tokom starenja.nul

Supplementary data for the article: Marković, O. S.; Cvijetić, I. N.; Zlatović, M. V.; Opsenica, I. M.; Konstantinović, J. M.; Terzić Jovanović, N. V.; Šolaja, B. A.; Verbić, T. Ž. Human Serum Albumin Binding of Certain Antimalarials. Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy 2018, 192, 128–139. https://doi.org/10.1016/j.saa.2017.10.061

Supplementary material for: [https://doi.org/10.1016/j.saa.2017.10.061]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2085

Iron salt-promoted oxidation of steroidal phenols by m -chloroperbenzoic acid: a route to possible antitumor agents

Iron salt-promoted reaction of estrone and its derivatives with meta -chloroperoxybenzoic acid was developed and epoxyquinols were further transformed. Most compounds showed in vitro antiproliferative activity. , A new oxidant, containing m -chloroperoxybenzoic acid (MCPBA) and an iron salt, was developed and used for oxidation of steroidal phenols to a quinol/epoxyquinol mixture. Reaction was optimized for estrone, by varying initiators (Fe-salts), reaction temperature, time and mode of MCPBA application. A series of five more substrates (17β-estradiol and its hydrophobized derivatives) was subjected to the optimized oxidation, providing corresponding p -quinols and 4β,5β-epoxyquinols in good to moderate yields. The obtained epoxyquinols were additionally transformed by oxidation, as well as the acid-catalyzed oxirane opening. In a preliminary study of the antiproliferative activity against human cancer cell lines, all newly synthesized compounds expressed moderate to high activity

Supplementary data for the article: Konstantinović, J.; Videnović, M.; Orsini, S.; Bogojević, K.; D’Alessandro, S.; Scaccabarozzi, D.; Terzić Jovanović, N.; Gradoni, L.; Basilico, N.; Šolaja, B. A. Novel Aminoquinoline Derivatives Significantly Reduce Parasite Load in Leishmania Infantum Infected Mice. ACS Medicinal Chemistry Letters 2018, 9 (7), 629–634. https://doi.org/10.1021/acsmedchemlett.8b00053

Supplementary material for: [https://pubs.acs.org/doi/10.1021/acsmedchemlett.8b00053]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2209]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/2948

Analysis of nuclear glucocorticoid receptor-DNA interaction in aged rat liver

Abstract: In order to contribute to the understanding of mechanisms by which regulatory proteins recognize genetic information stored in DNA, analyses of their interaction with specific nucleotides are usually performed. In this study, the electrophoretic mobility shift assay (EMSA) was applied to analyze the interaction of nuclear proteins from the liver of rats of different age i.e., young (3-month-old), middle- aged (12-month-old) and aged (24-month-old), with radioactively labelled synthetic oligonucleotide analogues, corresponding to GRE. The levels of GRE binding activity were assessed by quantitative densitometric scanning of the autoradiograms. The results showed statistically significant decreasing values of up to 78% and 49% in middle aged and old animals, respectively, compared to young animals (p < 0.05). The specificity of the nuclear proteins-GRE interaction was demonstrated by competition experiments with unlabelled GRE. In a supershift assay, using the antibody BuGR2, it was shown that the GR proteins present in nuclear extracts have a high affinity for the GRE probe. The stabilities of the protein-DNA complexes were analysed and it was concluded that they changed during ageing.U cilju doprinosa razumevanju mehanizama pomoću kojih regulatorni proteini prepoznaju genetičku informaciju koju nosi DNK, analiziraju se njihove interakcije sa specifičnim nukleotidima. U ovom radu je metodom EMSA analizirana interakcija jedarnih proteina iz jetri pacova iz tri starosne grupe (mladi - 3 meseca, srednje doba – 12 meseci i stari – 24 meseca) sa sintetičkim, radioaktivno obeleženim, oligonukleotidnim analogom GRE. Nivo vezujuće aktivnosti GRE je određivan kvantitativno denzitometrijskom autoradiografijom. Rezultati su pokazali da postoji statistički značajan pad vrednosti GRE-vezujuće aktivnosti do 78% kod životinja srednjeg starosnog doba i do 49 % kod starih životinja, u poređenju sa vrednostima dobijenim za mlade životinje (p < 0.05). Specifičnost interakcije jedarnih proteina i GRE je određena eksperimentima kompeticije sa neobeleženim GRE. Korišćenjem antitela BuGR2 pokazano je da je glukokortikoidni receptor protein koji u jedarnom ekstraktu ima najveći afinitet za GRE probu. Analizirana je stabilnost kompleksa protein-DNK i zaključeno je da se menja tokom starenja.nul

Supplementary data for the article: Marković, O. S.; Cvijetić, I. N.; Zlatović, M. V.; Opsenica, I. M.; Konstantinović, J. M.; Terzić Jovanović, N. V.; Šolaja, B. A.; Verbić, T. Ž. Human Serum Albumin Binding of Certain Antimalarials. Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy 2018, 192, 128–139. https://doi.org/10.1016/j.saa.2017.10.061

Supplementary material for: [https://doi.org/10.1016/j.saa.2017.10.061]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2085

Human serum albumin binding of certain antimalarials

Tested compounds, previously synthesized, are derivatives of chloroquine, drug commonly used in the treatment and prevention of malaria. Human serum albumin (HSA) has the role in transport of endogenous (fatty acids, hormones, bile acids, amino acids) and exogenous compounds (drug molecules and nutrients). Interaction between tested compounds and HSA has been studied by fluorescence spectroscopy in phosphate buffered saline (1× PBS, pH 7.4) [1]. Results show that among tested compounds, all positively charged at pH 7.4, derivatives with thiophene substructure bind to HSA. Molecular docking studies were used to determine HSA–compound binding mode. Fluorescence quenching data were processed using Stern-Volmer (S-V) equation [2]. Almost linear S-V plot for binding of 1 to HSA (Fig. 1a) indicates single type of quenching mechanism. Results show that Ksv decreases (20C: (2.60±0.07)×105 M -1 ; 25C: (2.33±0.07)×105 M -1 and 37C: (2.18±0.08)×105 M -1 ) as temperature increases indicating static quenching mechanism. Downward curvature in S-V plots of 2 and 3 (Fig. 1b and 1c) indicates that tryptophan residues are not fully accessible to the drug and that dynamic quenching dominates over static. Fraction of tryptophan residues that are buried and inaccessible to the quencher and effective quenching constants can be determined by modified S-V equation. The effective quenching constant for 2 and 3 increases as temperature increases, this is another indication that dynamic quenching process is dominant in binding of 2 and 3 to HSA. Effective quenching constants of all three compounds are in the order of 10 5 M-1, meaning that these compounds can be effectively carried and stored by HSA in the human body

Supplementary data for the article: Konstantinović, J.; Videnović, M.; Orsini, S.; Bogojević, K.; D’Alessandro, S.; Scaccabarozzi, D.; Terzić Jovanović, N.; Gradoni, L.; Basilico, N.; Šolaja, B. A. Novel Aminoquinoline Derivatives Significantly Reduce Parasite Load in Leishmania Infantum Infected Mice. ACS Medicinal Chemistry Letters 2018, 9 (7), 629–634. https://doi.org/10.1021/acsmedchemlett.8b00053

Supplementary material for: [https://pubs.acs.org/doi/10.1021/acsmedchemlett.8b00053]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2209]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/2948

Supplementary data for the article: Konstantinović, J.; Kiris, E.; Kota, K. P.; Kugelman-Tonos, J.; Videnović, M.; Cazares, L. H.; Terzić Jovanović, N.; Verbić, T. Ž.; Andjelković, B.; Duplantier, A. J.; et al. New Steroidal 4-Aminoquinolines Antagonize Botulinum Neurotoxin Serotype A in Mouse Embryonic Stem Cell Derived Motor Neurons in Postintoxication Model. Journal of Medicinal Chemistry 2018, 61 (4), 1595–1608. https://doi.org/10.1021/acs.jmedchem.7b01710

Supplementary material for: [https://doi.org/10.1021/acs.jmedchem.7b01710]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2099

Supplementary data for the article: Terzić, N.; Konstantinović, J.; Tot, M.; Burojević, J.; Djurković-Djaković, O.; Srbljanović, J.; Štajner, T.; Verbić, T.; Zlatović, M.; Machado, M.; et al. Reinvestigating Old Pharmacophores: Are 4-Aminoquinolines and Tetraoxanes Potential Two-Stage Antimalarials? Journal of Medicinal Chemistry 2016, 59 (1), 264–281. https://doi.org/10.1021/acs.jmedchem.5b01374

Supplementary material for: [https://doi.org/10.1021/acs.jmedchem.5b01374]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2036