Natural Guanine Derivatives Exert PARP-Inhibitory and Cytoprotective Effects in a Model of Cardiomyocyte Damage under Oxidative Stress.

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.

HLDF-6 peptides exhibit neuroprotective effects in the experimental model of preclinical Parkinson's disease.

The mechanisms of the neuroprotective action of the hexapeptides HLDF-6 encoded by the amino acid sequence 41-46 of Human Leukemia Differentiation Factor and its homoserine derivative HLDF-6H were studied in an experimental 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced model of Parkinson's disease (PD). C57Bl/6 mice received two intraperitoneal injections of 18 mg/kg MPTP-HCl, with an interval of 2 hours. MPTP-induced motor dysfunction was assessed using horizontal grid test. Our data show that chronic intranasal administration of peptides (3 weeks, 300 µg/kg/day) restored normal levels of dopamine and improved its turnover rates in the striatum. Furthermore, peptide administration increased serum estradiol levels and led to a significant improvement in motor functions in MPTP-treated mice. Additionally, peptide treatment increased the levels of mRNA encoding neurotrophin BDNF, but normalized the levels of mRNA encoding the inflammatory mediators TGFß1, IL1ß and IFNγ in the brain. Collectively, our behavioral and biochemical studies demonstrate that HLDF-6 peptides have a therapeutic potential for treating PD. We propose that HLDF-6 peptides may exert their neuroprotective mechanism, at least in part, by normalizing estradiol levels and modulating the expression of key factors involved in neurotrophic support and neuroinflammation.

Inhibitory Effects of 7-Methylguanine and Its Metabolite 8-Hydroxy-7-Methylguanine on Human Poly(ADP-Ribose) Polymerase 1.

Previously, we have found that a nucleic acid metabolite, 7-methylguanine (7mGua), produced in the body can have an inhibitory effect on the poly(ADP-ribose) polymerase 1 (PARP1) enzyme, an important pharmacological target in anticancer therapy. In this work, using an original method of analysis of PARP1 activity based on monitoring fluorescence anisotropy, we studied inhibitory properties of 7mGua and its metabolite, 8-hydroxy-7-methylguanine (8h7mGua). Both compounds inhibited PARP1 enzymatic activity in a dose-dependent manner, however, 8h7mGua was shown to be a stronger inhibitor. The IC50 values for 8h7mGua at different concentrations of the NAD+ substrate were found to be 4 times lower, on average, than those for 7mGua. The more efficient binding of 8h7mGua in the PARP1 active site is explained by the presence of an additional hydrogen bond with the Glu988 catalytic residue. Experimental and computational studies did not reveal the effect of 7mGua and 8h7mGua on the activity of other DNA repair enzymes, indicating selectivity of their inhibitory action.

Pharmacokinetics and Molecular Modeling Indicate nAChRα4-Derived Peptide HAEE Goes through the Blood-Brain Barrier.

One of the treatment strategies for Alzheimer's disease (AD) is based on the use of pharmacological agents capable of binding to beta-amyloid (Aß) and blocking its aggregation in the brain. Previously, we found that intravenous administration of the synthetic tetrapeptide Acetyl-His-Ala-Glu-Glu-Amide (HAEE), which is an analogue of the 35-38 region of the α4 subunit of α4ß2 nicotinic acetylcholine receptor and specifically binds to the 11-14 site of Aß, reduced the development of cerebral amyloidogenesis in a mouse model of AD. In the current study on three types of laboratory animals, we determined the biodistribution and tissue localization patterns of HAEE peptide after single intravenous bolus administration. The pharmacokinetic parameters of HAEE were established using uniformly tritium-labeled HAEE. Pharmacokinetic data provided evidence that HAEE goes through the blood-brain barrier. Based on molecular modeling, a role of LRP1 in receptor-mediated transcytosis of HAEE was proposed. Altogether, the results obtained indicate that the anti-amyloid effect of HAEE, previously found in a mouse model of AD, most likely occurs due to its interaction with Aß species directly in the brain.

Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose.

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.

Disaccharide pyrimidine nucleosides and their derivatives: a novel group of cell-penetrating inhibitors of poly(ADP-ribose) polymerase 1.

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.

Isolation and characterization of high affinity aptamers against DNA polymerase iota.

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.

5'-aminocarbonyl phosphonates as new zidovudine depot forms: antiviral properties, intracellular transformations, and pharmacokinetic parameters.

The main disadvantages of 3'-azido-3'-deoxythymidine (zidovudine, AZT), the most common anti-HIV drug, are toxicity and a short half-life in the organism. The introduction of an H-phosphonate group into the AZT 5' position resulted in significant improvement of its therapeutic properties and allowed a new anti-HIV drug, Nikavir (AZT H-phosphonate). In this work, we described a new group of AZT derivatives, namely, AZT 5'-aminocarbonylphosphonates. The synthesized compounds displayed antiviral properties in cell cultures infected with HIV-1 and the capacity to release the active nucleoside in animals (rabbits and dogs) in a dose-dependent manner. The compounds were less toxic in MT-4 and HL-60 cell cultures and experimental animals compared with AZT. Major metabolites found in MT-4 cells after their incubation with AZT 5'-aminocarbonylphosphonate 1 were AZT and AZT 5'-phosphate (25 and 55%, respectively). Among the tested compounds, phosphonate 1 was the most effective AZT donor, and its longest t(1/2) and T(max) values in the line phosphonate 1--AZT H-phosphonate--AZT imply that compound 1 is an extended depot form of AZT. Although bioavailability of AZT after oral administration of phosphonate 1 was lower than those of AZT H-phosphonate and AZT (8 against 14 and 49%), we expect that this reduction would not cause essential decrease of antiviral activity but noticeably decrease toxicity as a result of gradual accumulation of AZT in blood and the absence of sharp difference between C(max) and C(min). Such a combination of properties makes the compounds of this group promising for further studies as extended-release forms of AZT.