[Design of an Efficient Inhibitor for the Influenza A Virus M2 Ion Channel].

Influenza A virus is capable of rapidly infecting large human populations, warranting the development of novel drugs to efficiently inhibit virus replication. A transmembrane ion channel formed by the M2 protein plays an important role in influenza virus replication. A reasonable approach to designing an effective antivirus drug is constructing a molecule that binds in the M2 transmembrane proton channel, blocks H^(+) proton diffusion through the channel, and thus the influenza A virus cycle. The known anti-influenza drugs amantadine and rimantadine have a weak effect on influenza A virus replication. A new class of positively charged molecules, diazabicyclooctane derivatives with a constant charge of +2, was proposed to block proton diffusion through the M2 ion channel. Molecular dynamics simulations were performed to study the temperature fluctuations in the M2 structure, and ionization states of histidine residues were established at physiological pH values. Two types of diazabicyclooctane derivatives were analyzed for binding with the M2 ion channel. An optimal structure was determined for a blocker to most efficiently bind with the M2 ion channel and block proton diffusion. The new molecule is advantageous over amantadine and rimantadine in having a positive charge of +2, which creates a positive electrostatic potential barrier to proton transport through the M2 ion channel in addition to a steric barrier.

Catalytically Competent Conformation of the Active Site of Human 8-Oxoguanine-DNA Glycosylase.

8-Oxoguanine-DNA N-glycosylase (OGG1) is a eukaryotic DNA repair enzyme responsible for the removal of 8-oxoguanine (oxoG), one of the most abundant oxidative DNA lesions. OGG1 catalyzes two successive reactions - N-glycosidic bond hydrolysis (glycosylase activity) and DNA strand cleavage on the 3'-side of the lesion by ß-elimination (lyase activity). The enzyme also exhibits lyase activity with substrates containing apurinic/apyrimidinic (AP) sites (deoxyribose moieties lacking the nucleobase). OGG1 is highly specific for the base opposite the lesion, efficiently excising oxoG and cleaving AP sites located opposite to C, but not opposite to A. The activity is also profoundly decreased by amino acid changes that sterically interfere with oxoG binding in the active site of the enzyme after the lesion is everted from the DNA duplex. Earlier, the molecular dynamics approach was used to study the conformational dynamics of such human OGG1 mutants in complexes with the oxoG:C-containing substrate DNA, and the population density of certain conformers of two OGG1 catalytic residues, Lys249 and Asp268, was suggested to determine the enzyme activity. Here, we report the study of molecular dynamics of human OGG1 bound to the oxoG:A-containing DNA and OGG1 mutants bound to the AP:C-containing DNA. We showed that the enzyme low activity is associated with a decrease in the populations of Lys249 and Asp268 properly configured for catalysis. The experimentally measured rate constants for the OGG1 mutants show a good agreement with the models. We conclude that the enzymatic activity of OGG1 is determined majorly by the population density of the catalytically competent conformations of the active site residues Lys249 and Asp268.

PELDOR analysis of enzyme-induced structural changes in damaged DNA duplexes.

PELDOR (pulsed electron-electron double resonance) spectroscopy was applied to determine spin-spin distances in spin-labeled DNA duplexes (13-mer and 17-mer) containing the damaged sites 8-oxoguanine or uncleavable abasic site analogue tetrahydrofuran. The lesions were located in one strand of the DNA, and two nitroxyl spin labels were attached at the 5'- and 3'-ends of the complementary strand. PELDOR data allow us to obtain distances between the two spin labels in DNAs, which turned out to be around 5 nm for the 13-mer DNA and around 6 nm for 17-mer DNA. Results of PELDOR measurements were supported by molecular dynamics calculations. Study of the interaction of DNA fragments with DNA repair enzyme 8-oxoguanine-DNA glycosylase from E. coli (Fpg protein) showed that this interaction leads to a noticeable decrease of the distance between spin labels, which indicates the enzyme-induced bending of the DNA duplex. This bending may be important for the mechanisms of recognition of damaged sites by DNA repair enzymes.