Synthesis of peroxytetradecanoic acid.

<p>The peroxytetradecanoic acid is formed in the reaction of tetradecanoic acid with hydrogen peroxide.</p

Oxidation steps of PTP catalytic cysteine residue.

<p>The cysteine residue exists in a thiolate anion form and may undergo oxidation to the inactive sulfenic acid residue form. This conversion is reversible, but highly oxidizing conditions can further induce oxidation to the sulfinic and sulfonic acid residues, which is considered irreversible under physiological conditions.</p

The calculated binding affinities to CD45 catalytic center.

<p>The binding affinity of the peroxytetradecanoic acid, hydrogen peroxide and phosphotyrosine (natural substrate as a control) calculated with docking software AutoDock Vina version 1.1.1. The receptor structure used for affinity calculations was based on the PDB structure 1YGU and ligands were drawn in ChemDraw and processed with Schrodinger LigPrep version 25111. The data present the means of 6 repetitions with different random seeds.</p

The enzymatic activity of PTP CD45.

<p>The effect of tetradecanoic acid, peroxytetradecanoic acid and H₂O₂ on enzymatic activity of PTP CD45 before and after treatment with DTT. The enzyme CD45 (130 nM) was incubated with 50 nM tetradecanoic acid, peroxytetradecanoic acid and H₂O₂ at 37°C for 15 minutes. The activity was measured after adding 1 mM pNPP in 50 mM Tris buffer, pH 7.4. After the following 5 min of incubation, PTP activity was measured using microplate reader at 405 nm. Subsequently 20 mM dithiothreitol (DTT) was added to each sample for 30 minutes and a potential recovery of the enzymatic activity was assessed using the same technique. The results were expressed as percent of activity of control sample in Tris buffer. The data from three independent experiments were present as mean ± S.E.M; * significantly different (P<0.05) from control, ** significantly different (P<0.001) from control. The data were analysed using the combination of ANOVA and Tukey’s test (GraphPad Prism Software v.4).</p

The best predicted binding poses of ligands in the CD45 active site.

<p>The best predicted binding poses for peroxy acid C14 (panel A) and hydrogen peroxide (panel B) in the CD45 active site. The receptor was based on the CD45 D1 domain from PDB structure 1YGU <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052495#pone.0052495-Nam1" target="_blank">[20]</a>, with residues mutated to correspond to a CD45 reference sequence (UniProtKB accession number P08575) using the SWISS-MODEL web server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052495#pone.0052495-Arnold1" target="_blank">[11]</a>. The docking was performed with the AutoDock Vina version 1.1.1 software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052495#pone.0052495-Trott1" target="_blank">[15]</a> using a rigid receptor and a binding box centered on the CD45 phosphatase active site. The best binding pose was defined as the pose with the strongest affinity in the largest cluster of poses, with poses clustered with a 1.5 Å RMSD thresholds. Also highlighted are four important residues involved in binding (Tyr683, His822, Arg859 and Gln897) and the catalytic cysteine (Cys853). Predicted hydrogen bonds with a 3.5 Å distance cutoff are shown as green dashed lines. The residues are numbered according the P08575 sequence.</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

Chemical Structures of Paclitaxel, Docetaxel, and Paclitaxel Derivatives.

<p>Chemical Structures of Paclitaxel, Docetaxel, and Paclitaxel Derivatives.</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

Summary of Drug Titrations Showing 50% Inhibitory Concentration, IC₅₀, for the P-gp^± Cell Lines Treated with Paclitaxel and Derivatives (and ± Verapamil).

<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 an analog relative to paclitaxel.</p><p>^‡ P < 0.05 determined for IC₅₀ for an analog with improved potency relative paclitaxel.</p><p>^§ VRP is verapamil.</p><p>^¶ P-gp⁺ cell line.</p><p>Summary of Drug Titrations Showing 50% Inhibitory Concentration, IC₅₀, for the P-gp^± Cell Lines Treated with Paclitaxel and Derivatives (and ± Verapamil).</p