| Autor: |
Shram SI; National Research Centre 'Kurchatov Institute', Moscow, 123182, Russia. shram.img@yandex.ru., Shcherbakova TA; Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia., Abramova TV; Institute of Chemical Biology and Fundamental Medicine, Russian Academy of Sciences, Siberian Branch, Novosibirsk, 630090, Russia., Smirnovskaya MS; Faculty of Chemistry, Lomonosov Moscow State University, Moscow, 119991, Russia., Balandina AI; National Research Centre 'Kurchatov Institute', Moscow, 123182, Russia.; Faculty of Biotechnology and Industrial Ecology, Mendeleev University of Chemical Technology of Russia, Moscow, 125047, Russia., Kulikov AV; Medical Institute, Peoples' Friendship University of Russia, Moscow, 117198, Russia., Švedas VK; Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia.; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, 119234, Russia., Silnikov VN; Institute of Chemical Biology and Fundamental Medicine, Russian Academy of Sciences, Siberian Branch, Novosibirsk, 630090, Russia., Myasoedov NF; National Research Centre 'Kurchatov Institute', Moscow, 123182, Russia., Nilov DK; Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia. |
| Abstrakt: |
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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