A new approach for studying poly(ADP-ribose) polymerase inhibitors using permeabilized adherent cells

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Abstract

Poly(ADP-ribose) polymerase (PARP) inhibitors have been proposed as pharmacological agents in the treatment of various diseases. Recently, factors and mechanisms responsible for regulating PARP catalytic activity have been identified, some of which can significantly influence the effectiveness of inhibitors of this enzyme. In this regard, it is important to develop new models and methods that would reflect the cellular context in which PARP functions. We proposed to use digitonin-permeabilized adherent cells to study poly(ADP-ribosyl)ation reaction (PARylation) in order to maintain the nuclear localization of PARP and to control the concentrations of its substrate (NAD+) and tested compounds in the cell. A specific feature of the approach is that before permeabilization, cellular PARP is converted to the DNA-bound state under conditions preventing premature initiation of the PARylation reaction. Experiments were carried out in rat H9c2 cardiomyoblasts. The activity of PARP in permeabilized cells was analyzed by measuring the immunofluorescence of the reaction product poly(ADP-ribose). The method was verified in the studies of PARP inhibition by the classic inhibitor 3-aminobenzamide and a number of new 7-methylguanine derivatives. One of them, 7,8-dimethylguanine, was found to be a stronger inhibitor compared to 7-methylguanine, due to a formation of additional hydrophobic contact with the protein. The proposed approach opens up new prospects for studying the mechanisms of PARP activity regulation in cells and can be used in high-throughput screening of PARP inhibitors.

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About the authors

S. I. Shram

National Research Centre “Kurchatov Institute”

Author for correspondence.
Email: shram.img@yandex.ru
Russian Federation, 123182 Moscow

T. A. Shcherbakova

Lomonosov Moscow State University, Belozersky Institute of Physico-Chemical Biology

Email: shram.img@yandex.ru
Russian Federation, 119992 Moscow

T. V. Abramova

Institute of Chemical Biology and Fundamental Medicine, Russian Academy of Sciences, Siberian Branch

Email: shram.img@yandex.ru
Russian Federation, 630090 Novosibirsk

M. S. Smirnovskaya

Lomonosov Moscow State University

Email: shram.img@yandex.ru

Faculty of Chemistry

Russian Federation, 119991 Moscow

A. I. Balandina

National Research Centre “Kurchatov Institute”; Mendeleev University of Chemical Technology of Russia

Email: shram.img@yandex.ru

Faculty of Biotechnology and Industrial Ecology

Russian Federation, 123182 Moscow; 125047 Moscow

A. V. Kulikov

RUDN University

Email: shram.img@yandex.ru

Medical Institute

Russian Federation, 117198 Moscow

V. K. Švedas

Lomonosov Moscow State University, Belozersky Institute of Physico-Chemical Biology; Lomonosov Moscow State University

Email: shram.img@yandex.ru

Faculty of Bioengineering and Bioinformatics

Russian Federation, 119992 Moscow; 119234 Moscow

V. N. Silnikov

Institute of Chemical Biology and Fundamental Medicine, Russian Academy of Sciences, Siberian Branch

Email: shram.img@yandex.ru
Russian Federation, 630090 Novosibirsk

N. F. Myasoedov

National Research Centre “Kurchatov Institute”

Email: shram.img@yandex.ru
Russian Federation, 123182 Moscow

D. K. Nilov

Lomonosov Moscow State University, Belozersky Institute of Physico-Chemical Biology

Email: shram.img@yandex.ru
Russian Federation, 119992 Moscow

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2. 1. Permeabilization of H9c2 cells by digitonin. a–b is a hypotonic solution without digitonin. g–e is a hypotonic solution with digitonin (35 microns). The nuclei of permeabilized cells are stained with ethidium III homodimer (a, b, g, e). The length of the scale strip corresponds to 100 microns. 10× Lens

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3. Fig. 2. Effect of pretreatment of PJ34 and H2O2 cells on PARP activity in digitonin permeabilized H9c2 cells. a–z – Microfluorescence analysis of PAR content in cell nuclei (1-4 – various variants of the analyzed preparations). The length of the scale bar corresponds to 50 microns. The 20×. i lens is the results of the quantitative analysis of PAR in individual cores. Number of cores analyzed: 198 (1), 236 (2), 229 (3), 144 (4). The average and median values are reflected by the blue and red lines, respectively. k is the variation in the values of the PAR level when analyzing individual microfluorescence images of different drugs (mean ± standard deviation; n = 6). Reagent concentrations: PJ34 – 5 microns, H2O2 – 1 mM, digitonin – 35 microns, oligo-DNA – 50 mcg/ml, NAD+ – 10 microns; incubation time with H2O2 – 5 min; incubation time with NAD+ – 5 min. *** p < 0.001 between all pairs of samples (ANOVA analysis of variance/Bonferroni a posteriori analysis)

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4. Fig. 3. Optimized scheme for the analysis of PARP activity in permeabilized adhered cells. 1 – Adhered intact cells; 2 – preincubation with PJ34 (5 microns, 1 hour, 37 °C, 5% CO2); 3 – treatment with H2O2 (1 mM, 5 min, 25 °C); 4 – formation of a PARP–DNA complex; 5 – permeabilization with digitonin (35 microns, 5 min, 0 °C); 6 – washing from digitonin, PJ34 and endogenous NAD+; 7 – incubation of permeabilized cells with exogenous NAD+ (10 microns) and the test compound (2-10 min, 25 °C); 8 – Protein methylation

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5. 4. Chemical structures of 7-methylguanine and its derivatives

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6. 5. Concentration dependences of PARP inhibition by the tested compounds in permeabilized H9c2 cells. Designations: 1 – 3-aminobenzamide; 2 – 7-methylguanine; 3 – 7,8-dimethylguanine; 4 – N2,7-dimethylguanine. Conditions: NAD+ – 10 microns; incubation time – 10 min; concentrations of 3-aminobenzamide and 7,8-dimethylguanine – 0,2, 1, 2,5, 5, 10, 25, 50, 100 microns; concentrations of 7-methylguanine and N2,7-dimethylguanine – 10, 25, 75, 100, 150, 200, 250, 400 microns

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7. 6. Modeling of inhibitor binding in the active center of PARP1. a – 3-Aminobenzamide; b – 7-methylguanine; c – 7,8-dimethylguanine; d – N2,7-dimethylguanine. Nonpolar hydrogen atoms of amino acid residues are not shown. The dotted lines indicate the key hydrogen bonds with Gly863

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