Identification of new promising plant-based lead compounds for inhibition of prokaryotic replicative DNA polymerases: combination of in silico and in vitro studies.

Emerging widespread bacterial resistance to current antibiotics with traditional targets is one of the major global concerns. Therefore, so many investigations are exploring the potential of other druggable macromolecules of bacteria such as replication machinery components that are not addressed by previous antibiotics. DNA polymerase is the major part of this machine. However, a few studies have been done on it so far. In this respect, we report the discovery of four new plant-based leads against DNA polymerase (pol) IIIC (three leads) and pol IIIE (one lead) of Gram-positive and negative bacteria by combining a sequentially constrained high-throughput virtual screenings on Traditional Chinese Medicine Database with in vitro assays. The compounds displayed relatively good levels of inhibitory effect. They were active against their designated targets at micromolar concentrations. The IC50 values for them are ranged from 25 to 111 µM. In addition, they showed minimum inhibitory concentrations in the range of 8-128 µg/mL against five representatives of pathogenic bacteria species. However, they were inactive against Pseudomonas aeruginosa. Given these results, these leads hold promise for future modification and optimization to be more effective in lower concentrations and also against most of the important bacterial species. Communicated by Ramaswamy H. Sarma.

Targeting the ß-clamp in Helicobacter pylori with FDA-approved drugs reveals micromolar inhibition by diflunisal.

The ß-clamp is the processivity-promoting factor for most of the enzymes in prokaryotic DNA replication; hence, it is a crucial drug target. In the present study, we investigated the ß-clamp from Helicobacter pylori, aiming to seek potential drug molecules against this gastric-cancer-causing bacterium. An in silico screening of Food and Drug Administration (FDA) approved drugs against the H. pylori ß-clamp, followed by its in vitro inhibition using a surface competition approach, yielded the drug diflunisal as a positive initial hit. Diflunisal inhibits the growth of H. pylori in the micromolar range. We determined the structure of diflunisal in complex with the ß-clamp to show that the drug binds at subsite I, which is a protein-protein interaction site. Successful identification of FDA-approved molecules against H. pylori may lead to better and faster drug development.

Quinazolin-2-ylamino-quinazolin-4-ols as novel non-nucleoside inhibitors of bacterial DNA polymerase III.

High throughput screening led to the discovery of a novel series of quinazolin-2-ylamino-quinazolin-4-ols as a new class of DNA polymerase III inhibitors. The inhibition of chromosomal DNA replication results in bacterial cell death. The synthesis, structure-activity relationships and functional activity are described.

PolC-type polymerase III of Streptococcus pyogenes and its use in screening for chemical inhibitors.

The polC gene from Streptococcus pyogenes (S. pyogenes, strain SF370) has been cloned and expressed in Escherichia coli (E. coli) as a fusion protein containing an N-terminal histidine tag. The purified recombinant enzyme showed an apparent molecular mass of 160 kDa on SDS-PAGE and a specific activity of 3.5 nmol/min/mg when assayed in the presence of calf thymus DNA and the four deoxyribonucleoside triphosphates. This activity was inhibited by TMAU, a specific inhibitor of PolC. To facilitate kinetic studies, and high-throughput assays, a double-stranded oligo DNA primer/template was used as a substrate. The minimum requirement for the length of the substrate was a 20-base oligo primer annealed to a 35-base template. PolC activity was detected either by a filter-binding format or by a novel homogeneous scintillation proximity assay (SPA). Sensitivity to inhibition by anilinouracil analogs was improved by incorporating three deoxycytidines in the template strand as the first 3 bases to be copied by the polymerase. Inhibition of PolC activity by trimethyleneanilinouracil by the filtration and SPA methods gave comparable results, but the SPA assay uses less radioactive label, is less time-consuming, and is amenable to high-throughput formatting.

Effect of protracted mild hyperthermia on polymerase activity in a human melanoma cell line.

Radioresistant human melanoma SkMel-3 was evaluated with its sensitivity to thermal cell killing and polymerase inactivation. Cells were heated from 40 to 45 degrees C and demonstrated no thermal tolerance development for any of the temperatures tested. In addition, at 45 degrees C the heat survival curve showed a large shoulder indicating capacity for accumulation of sublethal heat damage. Also at 45 degrees C heating polymerase beta was more sensitive than polymerase alpha + delta + epsilon. At 42 degrees C, the polymerase sensitivities were nearly the same but at the lower temperatures (41 and 40 degrees C) polymerase beta became progressively more resistant than the polymerase alpha + delta + epsilon. Thus, mild hyperthermia effects may be different than high temperature hyperthermia and may be related to polymerase alpha + delta + epsilon activity.

Elucidation of the mechanism of selective inhibition of mammalian DNA polymerase alpha by 2-butylanilinopurines: development and characterization of 2-(p-n-butylanilino)adenine and its deoxyribonucleotides.

2-(p-n-Butylanilino)adenine (BuAA), an homolog of the DNA polymerase alpha (pol alpha)-specific inhibitor, N2-(p-n-butylphenyl)guanine (BuPG), was transformed to its 2'-deoxyribonucleoside, BuAdA, and the corresponding 2'-deoxyribonucleoside 5'-phosphates, BuAdAMP, BuAdADP, and BuAdATP. All five forms of BuAA are highly selective inhibitors of mammalian pol alpha, and the action of each is subject to specific competitive antagonism by dATP. BuAdADP, and BuAdATP, like the corresponding forms of BuPG, are very potent pol alpha inhibitors, displaying apparent Ki's of less than 3 nanomolar on natural activated templates. BuAdATP, like BuPdGTP, also inhibits pol alpha-catalysed reactions directed by non-complementary, thymine-deficient templates, and it does so via a mechanism subject to specific antagonism by its natural homolog, dATP. The results of the BuAdATP-homopolymer experiments complement those of analogous experiments with BuPdGTP and the dCTP-specific pol alpha inhibitor, aphidicolin, and strengthen the suggestion that mammalian pol alpha contains dNDP and dNTP binding sites which can recognize specific bases without direction by templates.