A New Approach for Studying Poly(ADP-Ribose) Polymerase Inhibitors Using Permeabilized Adherent Cells

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Stanislav I. Shram^1,a*, Tatyana A. Shcherbakova², Tatyana V. Abramova³, Maria S. Smirnovskaya⁴, Anastasia I. Balandina^1,5, Andrey V. Kulikov⁶, Vytas K. Švedas^2,7, Vladimir N. Silnikov³, Nikolay F. Myasoedov¹, Dmitry K. Nilov²

¹National Research Centre “Kurchatov Institute”, 123182 Moscow, Russia

²Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia

³Institute of Chemical Biology and Fundamental Medicine, Russian Academy of Sciences, Siberian Branch, 630090 Novosibirsk, Russia

⁴Faculty of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia

⁵Faculty of Biotechnology and Industrial Ecology, Mendeleev University of Chemical Technology of Russia, 125047 Moscow, Russia

⁶Medical Institute, Peoples’ Friendship University of Russia, 117198 Moscow, Russia

⁷Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119234 Moscow, Russia

^* To whom correspondence should be addressed.

Received: January 29, 2024; Revised: May 3, 2024; Accepted: June 10, 2024

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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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KEY WORDS: poly(ADP-ribose) polymerase, inhibitor, 7-methylguanine, 7,8-dimethylguanine, chemical synthesis of purines, adherent cells, cell permeabilization, digitonin, cardiomyoblasts, molecular docking

DOI: 10.1134/S0006297924090086

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