DNA Distortion Caused by Uracil-Containing Intrastrand Cross-Links

Four uracil-containing intrastrand cross-links have been detected in human cells upon UV irradiation of 5-bromouracil-containing DNA, namely 5′-G­[8-5]­U-3′, 5′-U­[5-8]­G-3′, 5′-A­[8-5]­U-3′, and 5′-A­[2-5]­U-3′. These lesions feature unique composition and connectivity compared with other intrastrand cross-links reported in the literature. For the first time, structural information obtained using molecular dynamics (MD) simulations reveal that all four lesions distort the DNA helix, which can involve an extrahelical location of the cross-link, changes in the helical interactions of the complementary nucleotides, or disruption of hydrogen bonding in the flanking base pairs up to two positions from the cross-linked site; however, the degree of distortion varies between the cross-links, being affected by the sequence, nucleobase–nucleobase connectivity, and the purine involved. Most importantly, the relative distortion of the damaged DNA provides the first structural explanation for the observed abundances of the four uracil-containing cross-links. Furthermore, the highly distorted conformations suggest that these lesions will likely have severe implications for DNA replication and repair processes in cells

Inactivation of Protein Tyrosine Phosphatases by Peracids Correlates with the Hydrocarbon Chain Length

Background/Aims: Protein tyrosine phosphatases are crucial enzymes controlling numerous physiological and pathophysiological events and can be regulated by oxidation of the catalytic domain cysteine residue. Peracids are highly oxidizing compounds, and thus may induce inactivation of PTPs. The aim of the present study was to evaluate the inhibitory effect of peracids with different length of hydrocarbon chain on the activity of selected PTPs. Methods: The enzymatic activity of human CD45, PTP1B, LAR, bacterial YopH was assayed under the cell-free conditions, and activity of cellular CD45 in human Jurkat cell lysates. The molecular docking and molecular dynamics were performed to evaluate the peracids binding to the CD45 active site. Results: Here we demonstrate that peracids reduce enzymatic activity of recombinant CD45, PTP1B, LAR, YopH and cellular CD45. Our studies indicate that peracids are more potent inhibitors of CD45 than hydrogen peroxide (with an IC50 value equal to 25 nM for peroctanoic acid and 8 µM for hydrogen peroxide). The experimental data show that the inactivation caused by peracids is dependent on hydrocarbon chain length of peracids with maximum inhibitory effect of medium-chain peracids (C8-C12 acyl chain), which correlates with calculated binding affinities to the CD45 active site. Conclusion: Peracids are potent inhibitors of PTPs with the strongest inhibitory effect observed for medium-chain peracids

Designing and Testing of Novel Taxanes to Probe the Highly Complex Mechanisms by Which Taxanes Bind to Microtubules and Cause Cytotoxicity to Cancer Cells.

Our previous work identified an intermediate binding site for taxanes in the microtubule nanopore. The goal of this study was to test derivatives of paclitaxel designed to bind to this intermediate site differentially depending on the isotype of β-tubulin. Since β-tubulin isotypes have tissue-dependent expression--specifically, the βIII isotype is very abundant in aggressive tumors and much less common in normal tissues--this is expected to lead to tubulin targeted drugs that are more efficacious and have less side effects. Seven derivatives of paclitaxel were designed and four of these were amenable for synthesis in sufficient purity and yield for further testing in breast cancer model cell lines. None of the derivatives studied were superior to currently used taxanes, however computer simulations provided insights into the activity of the derivatives. Our results suggest that neither binding to the intermediate binding site nor the final binding site is sufficient to explain the activities of the derivative taxanes studied. These findings highlight the need to iteratively improve on the design of taxanes based on their activity in model systems. Knowledge gained on the ability of the engineered drugs to bind to targets and bring about activity in a predictable manner is a step towards personalizing therapies

IC₅₀ Values for Paclitaxel and Analogs in Cytotoxicity Assays with Breast Cancer Cell Lines.

<p>Cell lines were treated with a range of drug concentrations as indicated (left plot in each panel) to assess the cytotoxic activity of paclitaxel and analogs. Cell lines were exposed to drugs for 72 h. 30 μl of MTS reagent was administered to each well, and absorbance measurements were taken at 490 nm. Cell lines studied were: (A) SK-BR-3 (HER2⁺), (B) MDA-MB-231 (triple negative) and (C) T-47D (luminal A). All values are averages of replicates expressed relative to cell viability values in untreated cells normalized to 100%. Cytotoxicity curves represent n = 3 experiments with 6 replicates per drug concentration for each experiment. Bar graphs (right plots in each panel) show the IC₅₀ value of n = 3 independent experiments. Standard deviations, SD, are shown for each drug concentration (left plots) and for each IC₅₀ determined (right plots). * indicates P < 0.05 determined for IC₅₀ for an analog relative to paclitaxel.</p

Microtubule Polymerization in the Presence of Paclitaxel and Derivatives with αβII and αβIII.

<p>Isotypically pure bovine brain tubulin (αβII or αβIII) at 1.4 mg/ml was incubated in the presence of drugs and the absorbance at 350 nm was measured every 8 seconds for at least 11 minutes. The concentration of each drug was 10 μM. PTX is paclitaxel.</p

Western Blot Analysis of Expression of β-Tubulin Isotypes and P-gp for Breast Cancer Cell Lines.

<p>Breast cancer cell lines were used to determine expression of the βIII tubulin isotype and the P-gp (MDR1) efflux pump. Experiments were carried out under normal media conditions (−) or after 24 h paclitaxel exposure with concentration at one-half IC₅₀ (+). The image is representative of the results from n = 3 independent experiments. Actin expression on the blot was used as a loading control.</p

Western Blot Analysis of Expression of β-Tubulin Isotypes and P-gp for the P-gp^± Cell Lines.

<p>Two pairs of cell lines were used to determine expression of βIII tubulin isotype and P-gp efflux pump. Parental cell lines MES-SA (uterine sarcoma) and K-562 (chronic myelogenous leukemia) are P-gp negative, whereas daughter cell lines MES-SA/Dx5 and K-562/R7 express P-gp, and have been characterized in the literature as multidrug-resistant lines [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129168#pone.0129168.ref028" target="_blank">28</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129168#pone.0129168.ref030" target="_blank">30</a>]. Experiments were carried out under normal media conditions (−) and after 24 h paclitaxel exposure with concentration at one-half IC₅₀ (+). The image is representative of the results from n = 3 independent experiments. Actin protein was used as a loading control.</p

Summary of Drug Titrations Showing 50% Inhibitory Concentration, IC₅₀, in Breast Cancer Cell Lines Treated with Paclitaxel and Analogs.

<p>The IC₅₀ values are given in nM. Values and errors (SE) shown are representative of n = 3 independent experiments.</p><p>* P < 0.05 determined for IC₅₀ for each analog relative to paclitaxel.</p><p>Summary of Drug Titrations Showing 50% Inhibitory Concentration, IC₅₀, in Breast Cancer Cell Lines Treated with Paclitaxel and Analogs.</p