ABSTRACT
Inhibitors of human poly(ADP-ribose) polymerase (PARP) are considered as promising agents for treatment of cardiovascular, neurological, and other diseases accompanied by inflammation and oxidative stress. Previously, the ability of natural compounds 7-methylguanine (7mGua) and 8-hydroxy-7-methylguanine (8h7mGua) to suppress activity of the recombinant PARP protein was demonstrated. In the present work, we have investigated the possibility of PARP-inhibitory and cytoprotective action of 7mGua and 8h7mGua against the rat cardiomyoblast cultures (undifferentiated and differentiated H9c2). It was found that 7mGua and 8h7mGua rapidly penetrate into the cells and effectively suppress the H2O2-stimulated PARP activation (IC50 = 270 and 55 µM, respectively). The pronounced cytoprotective effects of 7mGua and 8h7mGua were shown in a cellular model of oxidative stress, and effectiveness of 8h7mGua exceeded the classic PARP inhibitor 3-aminobenzamide. The obtained data indicate promise for the development of PARP inhibitors based on guanine derivatives and their testing using the models of ischemia-reperfusion tissue damage.
Subject(s)
Myocytes, Cardiac , Poly(ADP-ribose) Polymerase Inhibitors , Humans , Animals , Rats , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Hydrogen Peroxide/pharmacology , Oxidative Stress , Guanine/pharmacologyABSTRACT
Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.
Subject(s)
Bacterial Proteins/analysis , Fluorescence Resonance Energy Transfer/methods , Fluorescent Dyes/analysis , Luminescent Proteins/analysis , Poly Adenosine Diphosphate Ribose/analysis , Bacterial Proteins/genetics , Biosensing Techniques/methods , Cell Line , Fluorescent Dyes/metabolism , Humans , Luminescent Proteins/genetics , Open Reading Frames , Protein Domains , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics , Ubiquitin-Protein Ligases/analysis , Ubiquitin-Protein Ligases/geneticsABSTRACT
Nearly 30 synthetic nucleosides were tested with human recombinant poly(ADP-ribose) polymerase 1 as potential inhibitors of this enzyme. The most active compounds were some disaccharide analogues of thymidine: 3'-O-ß-D-ribofuranosyl-5-iodo-dUrd (2d; IC50 = 45 µM), 3'-O-ß-D-ribofuranosyl-2'-deoxythymidine (2e; IC50 = 38 µM), and 3'-O-ß-D-ribofuranosyl-2'-deoxythymidine oxidized (4; IC50 = 25 µM). These compounds also reduced H2O2-induced synthesis of poly(ADP-ribose) in cultured human ovarian carcinoma (SKOV-3) cells in a dose-dependent manner. Furthermore, compounds 2d or 2e until a concentration of 1 mM did not affect growth of SKOV-3 cells, whereas dialdehyde compound 4, as well as thymidine, exhibited a significant cytotoxicity.
Subject(s)
Disaccharides/chemical synthesis , Poly(ADP-ribose) Polymerase Inhibitors , Pyrimidine Nucleosides/chemical synthesis , Thymidine/chemical synthesis , Cell Line, Tumor/drug effects , Disaccharides/chemistry , Disaccharides/pharmacology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Hydrogen Peroxide/metabolism , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/metabolism , Pyrimidine Nucleosides/chemistry , Pyrimidine Nucleosides/pharmacology , Structure-Activity Relationship , Thymidine/analogs & derivatives , Thymidine/chemistryABSTRACT
Human DNA-polymerase iota (Pol ι) is an extremely error-prone enzyme and the fidelity depends on the sequence context of the template. Using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) procedure, we obtained an oligoribonucleotide with a high affinity to human Pol ι, named aptamer IKL5. We determined its dissociation constant with homogenous preparation of Pol ι and predicted its putative secondary structure. The aptamer IKL5 specifically inhibits DNA-polymerase activity of the purified enzyme Pol ι, but did not inhibit the DNA-polymerase activities of human DNA polymerases beta and kappa. IKL5 suppressed the error-prone DNA-polymerase activity of Pol ι also in cellular extracts of the tumor cell line SKOV-3. The aptamer IKL5 is useful for studies of the biological role of Pol ι and as a potential drug to suppress the increase of the activity of this enzyme in malignant cells.