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Olivomycin A (OA) exerts its cytotoxic potency due to binding to the minor groove of the G/C-rich DNA and interfering with replication and transcription. Screening of the complete set of tetranucleotide G/C sites by electrophoretic mobility gel shift assay (EMSA) revealed that the sites containing central GC or GG dinucleotides were able to bind OA, whereas the sites with the central CG dinucleotide were not. However, studies of equilibrium OA binding in solution by fluorescence, circular dichroism and isothermal titration calorimetry failed to confirm the sequence preference of OA, indicating instead a similar type of complex and comparable affinity of OA to all G/C binding sites. This discrepancy was resolved by kinetics analysis of the drug&ndash, DNA interaction: the dissociation rate significantly differed between SGCS, SGGS and SCGS sites (S stands for G or C), thereby explaining the disintegration of the complexes during EMSA. The functional relevance of the revealed differential kinetics of OA&ndash, DNA interaction was demonstrated in an in vitro transcription assay. These findings emphasize the crucial role of kinetics in the mechanism of OA action and provide an important approach to the screening of new drug candidates.

Novel generations of antitumor anthraquinones are expected to be advantageous over the conventional chemotherapeutic agents. Previous structure–activity relationship studies demonstrated an importance of the positively charged side chains conjugated to anthra[2,3-b]thiophene-5,10-dione scaffolds. Exploring a role of individual side chain moieties in binding to the duplex and G-quadruplex DNA, modulation of telomerase and topoisomerase I activities, intracellular accumulation and cytostatic potency, we herein analyzed a series of reported and newly synthesized guanidine-containing derivatives of anthra[2,3-b]thiophene-5,10-dione. We found that the number of cationic side chains (namely, two) is critical for a tight interaction with human telomeric G-quadruplex (TelQ). Along with a larger drug–TelQ association constant, the telomerase attenuation by anthrathiophenediones with two basic groups in the side chains was more pronounced than by the analogs bearing one basic group. For mono-guanidinated compounds the substituent with the amino group in the side chain provided better TelQ affinity than the methylamine residue. The intracellular uptake of the mono-guanidino derivative with two side chains was >2-fold higher than the respective value for the bis(guanidino) derivative. This difference can explain a lower antiproliferative potency of bis(guanidine) containing compounds. Thus, the modifications of side chains of anthra[2,3-b]thiophene-5,10-dione differently modulated drug–target interactions and cellular effects. Nevertheless, the selected compound 11-(3-aminopropylamino)-4-(2-guanidinoethylamino)anthra[2,3-b]thiophene-5,10-dione 13 demonstrated a high affinity to TelQ and the ability to stabilize the quadruplex structure. These properties were paralleled by reasonable potency of 13 as a telomerase/topoisomerase I inhibitor and an antiproliferative agent. These results indicate that the structural elements of anthra[2,3-b]thiophene-5,10-dione derivatives can be balanced to yield a candidate for further preclinical study.

The porphyrin-based photosensitizers capable of binding to DNA are perspective drug candidates. Here we report the interactions with calf thymus DNA of 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium)porphyrin (P1) and its derivatives containing Zn(II) or Ni(II) in the coordination sphere. These interactions were studied with absorption and circular dichroism spectroscopy. NiP1 and ZnP1 formed different types of complexes with DNA. NiP1 intercalated into the double helix, whereas ZnP1 bound the DNA groove. Compound P1 displayed both binding modes. The ZnP1-DNA binding constant was approximately three times smaller than the respective values for P1-DNA and NiP1-DNA complexes. Light induced degradation of the reactive oxygen species (ROS) trap 1,3-diphenylisobenzofuran in the presence of P1 and its metal derivatives revealed that NiP1 was a weaker photooxidative agent, whereas P1 and ZnP1 generated ROS to similar extents. Nevertheless, the DNA photodamaging effect of ZnP1 was the most pronounced. Illumination of the supercoiled plasmid caused single-strand DNA photocleavage in the presence of P1 and ZnP1; double strand breaks were detectable with micromolar concentrations of ZnP1. The concentration of ZnP1 required for plasmid photonicking was two times smaller than that of P1 and ~20 times lower than that for NiP1. Thus, the modes of P1, NiP1 and ZnP1 binding to DNA determine the differential photodamaging potency of these porphyrins. A greater accessibility to the solvent of the groove binder ZnP1, compared to the shielded intercalator NiP1 and the intercalated P1 molecules, allows for an efficient local generation of ROS followed by DNA photocleavage.

The homology of subunit primary sequence of 40 glutamate decarboxylases (GAD) of different origin was analyzed by multiple alignment. A phylogenetic tree was designed on the basis of the resulting data. The following groups are distinguished in the consensus tree: archeans, bacteria, plant eukaryotes, and animal eukaryotes. The latter are clearly divided into two branches according to two enzyme isoforms. Borders of PLP domains in each enzyme were detected. The consensus phylogenetic tree for PLP domains is structurally rather similar to that obtained for subunits. Twenty homologous motifs of from 15 to 87 amino acid residues were revealed in all GAD studied. The results revealed the division of all of the enzymes into groups with characteristic sets of motifs in each and a fixed order of their arrangement along the sequence. Thus, we can show the divergent evolution of the enzyme. The results of multiple alignments during structural analysis of the 40 GAD confirmed and extended our previous data on conserved residues that arrange the position of the coenzyme (PLP) in the enzyme active center. The following residues should be noted: lysine forming a Schiff base with the PLP aldehyde group, an adjacent histidine, and aspartic acid that establishes a link with nitrogen of the PLP pyridine ring. The homology of the primary sequence fragments was also found in the residues in contact with the PLP phosphate group. Comparison of the GAD amino acid sequence with that of another PLP enzyme, aspartate aminotransferase, revealed a binding site for carboxylic group of the substrate—glutamic acid. The structures carrying out a particular catalytic function of all GAD studied were detected, i.e., convergent evolution of the enzyme was revealed.

Amino acid sequences of E. coli glutamate decarboxylase (GADa) and those of 36 GAD of different origin were compared by pairwise alignment using computer program CLUSTAL. GADalpha and plant enzymes showed 59.8-67.8% subunit homology, GADalpha and other bacterial GAD--49.8-77.6%, whereas GADalpha and animal enzymes--13.9-58.8%. Two PLP domains exhibited higher homology comparing to that of the whole subunit in the case of GAD67, plant (68.4-73.9%), and bacterial (46.7-83.2%) enzymes. The alignment of PLP-domains of 37 GAD, three group II decarboxylases, and two pyridoxal enzymes with known 3D structures (bacterial ORD and mAAT from chicken heart) allowed us to reveal conserved residues of the active sites. Their functional role is discussed. Modelling of the PLP-binding sites in active centers for GADalpha and human brain GAD67 was done using the Swiss-PdbViewer homology modelling program. Although the homology between GADalpha and GAD67 is rather low, structural similarity of their active sites allows us to consider here a functional convergence. Thus, glutamate decarboxylation by GADalpha may be helpful for understanding general mechanism of this reaction.

In this study, various proviral HIV-1 DNA forms were analyzed in a chronically infected culture of human monocytes. To elucidate the role of viral RT and cellular DNA polymerases in the formation of the circular forms, we used azidothymidine (AZT) as a specific terminator of viral RT. We previously developed a method for testing the circular proviral DNA forms by means of polymerase chain reaction (PCR) [5] and specific pairs of primers located at the 3' and 5' ends of the HIV-1 DNA (in the env and gag gene regions, respectively). The primers were so oriented that both one-LTR and two-LTR circular proviral DNAs could be determined simultaneously [5]. The total HIV-1 DNA concentration was measured to prepare diluted samples with an equal amount of proviral DNA, in which the relative content of circular nonintegrated forms of viral DNA was detected by means of PCR. The primers nov1‐nov2 were used. An additional amplification with primers V1‐V2 or nov3‐nov4 was performed in samples with low two-LTR circular DNA concentrations.

The paper presents results obtained in conformational analysis of homopolymeric four- stranded poly(dT) · poly(dA) · poly(dA) · poly(dT) DNA helices in which the pairs of strands with identical bases are parallel and have a two-fold symmetry axis. All possible models of base binding to yield a symmetric complex have been considered. The dihedral angles of sugar-phosphate backbones and helix parameters, which are consistent with the minima of conformational energy for four-stranded DNAs, have been determined using the results of optimization of conformational energy calculated at atom-atom approximation. Potential energy is shown to depend on the structure of base complexes and on the mutual orientation of unlike strands. Possible biological functions of four-stranded helices are discussed.

The use of the o-orienting effect of the sulfonyl group during metallation with BuLi made possible the synthesis of o-sulfonylbenzaldehydes. Oxidation of their oximes with hypochlorite gave o-sulfonylbenzonitrile oxides, stable at 20°C, which are compounds not obtained previously. They form normal 1,3-dipolar cycloaddition products with styrene, and on heating, they dimerize into furoxanes. o-Sulfonyl-bisbenzonitrile oxide cyclizes into dibenzo[b,f]furazano[3,4-d]thiepin 1(3),8,8-trioxide. 2- and 2,2′-Bistrimethylsilyldiphenyl sulfones were also synthesized, and their metallation was investigated.