l‑α-Phosphatidylglycerol Chlorohydrins as Potential Biomarkers for Chlorine Gas Exposure

Chlorine is a widely available toxic chemical that has been repeatedly used in armed conflict globally. The Organization for the Prohibition of Chemical Weapons (OPCW) have on numerous occasions found “compelling confirmation” that chlorine gas has been used against civilians in northern Syria. However, currently, there are no analytical methods available to unambiguously prove chlorine gas exposure. In this study, we describe the screening for chlorinated biomolecules by the use of mass isotope ratio filters followed by the identification of two biomarkers present in bronchoalveolar lavage fluid (BALF) from chlorine gas exposed mice. The relevance of these markers for human exposure was verified by their presence in in vitro chlorinated human BALF. The biomarkers were detectable for 72 h after exposure and were absent in nonexposed control animals. Furthermore, the biomarkers were not detected in humans diagnosed with chronic respiratory diseases. The potential chlorine specific markers were all chlorohydrins of unsaturated pulmonary surfactant phospholipids; phosphatidylglycerols, and phosphatidylcholines. Mass spectrometry fragmentation characteristics were favorable for the phosphatidylglycerol chlorohydrins, and they were therefore proposed as the best biomarker candidates

Distinguishing orthosteric and allosteric effects in Hsp90.

<p><b>(A)</b> The absolute difference in numbers of deuterons (y-axis) between the free and ligand bound state is plotted for each pepsin digest fragment listed from the N to C terminus (x-axis) of Hsp90 for each deuterium exchange time point (t = 0.5, 2, 5, 10 min) in a ‘difference plot’. Shifts in the positive scale represent decreases in deuterium exchange and shifts in the negative scale represent increases in deuterium exchange when compared to apo-Hsp90. Regions showing significant differences above a threshold of 0.5 Da (red dashed line) are compared with orthosteric sites (blue boxes) to predict allosteric regions. Peptides highlighted in red show regions showing differences in distal allosteric regions, not involved in orthosteric binding. Peptides spanning these regions are marked in red boxes and divided into four allosteric regions A1 to A4. Radicicol and 17-AAG shows differences in A1 and A2, while only radicicol showed changes in A3 and A4. Time points are colored according to key. <b>(B)</b> Predicted allosteric regions are mapped on to the structure of Hsp90 (red), together with the orthosteric regions, in blue. Radicicol bound at the ligand binding pocket is shown as sticks (PDB ID: 4EGK).</p

Mapping protein-ligand interactions by HDXMS.

<p>Protein-ligand interactions can be analyzed by HDXMS by comparing deuterium exchange of the unliganded state of the protein with that bound to ligand (shown in yellow sticks). An ensemble view entails that the target protein <b>(E)</b> would exist in multiple conformations in the absence of ligand. Here a representative target protein is shown containing two sites- an orthosteric <b>(O)</b> site forming the ligand binding pocket (sites 1–4 are represented) and an allosteric <b>(A)</b> site. Deuterium exchange at the orthosteric site (O-site) (blue) is then governed by ligand binding kinetic parameters: kon, koff, concentration of ligand as well as the observed rate of HDX exchange, kex, which varies across different regions of the protein. The HDXMS output encompasses changes at the orthosteric O-site and long range conformational changes (red) at the allosteric A-site. Binding of ligand at the O-site <b>(E:L)</b> would result in decreased exchange while changes at the A-site <b>(E*:L)</b> could be reflected as decreases or increases in deuterium exchange.</p

Fragments 1 and 2 differ in the nature of the allosteric effect in Hsp90.

<p><b>(A)</b> The absolute difference in numbers of deuterons (inferred from difference in mass in Daltons (Da) (y-axis) between the free and ligand bound state is plotted for each pepsin digest fragment listed from the N to C terminus (x-axis) of Hsp90 for each deuterium exchange time point (t = 0.5, 2, 5, 10 min) in a ‘difference plot’. Shifts in the positive scale represent decreases in deuterium exchange and shifts in the negative scale represent increases in deuterium exchange when compared to the apo-Hsp90. Regions showing significant differences above a threshold of 0.5 Da (red dashed line) are compared with orthosteric sites (blue boxes) to establish allosteric regions (red boxed). Fragment <b>2</b> does not show any changes in region A4, similar to 17-AAG, while fragment <b>1</b> shows differences, similar to Radicicol. In addition, fragment <b>1</b> shows an allosteric response at the regions A5 (residues 201–213 shown in orange box), which is not observed in the other three ligands. Time points are colored according to key. <b>(B,C)</b> The identified orthosteric (blue) and allosteric regions (red) for fragments are mapped on to the structure of Hsp90 in blue. <b>(C)</b> The allosteric site A5 in Hsp90, which is observed only fragment <b>2</b> is highlighted in orange. Radicicol bound at the ligand binding pocket is shown as sticks (PDB ID: 4EGK).</p

Modifications of the 7-Hydroxyl Group of the Transthyretin Ligand Luteolin Provide Mechanistic Insights into Its Binding Properties and High Plasma Specificity - Fig 2

<p>Reagents and conditions: (a) 2.1 equivalents Ph₂CCl₂, 2 equivalents DIPEA, Dioxane/NMP, 2 x 30 min, microwave 180°C. (b) 1.2 equivalents MeI, 1.4 equivalents K₂CO₃, DMF, 18 h, RT. (c) AcOH/H₂O 4:1, 1 h, microwave 190°C. (d) 3.3 equivalents POCl₃, DMF, 5 h, 130°C.</p

Data collection and refinement statistics for the TTR-7-Cl-Lut and TTR-7-MeO-Lut-complexes.

<p>Data collection and refinement statistics for the TTR-7-Cl-Lut and TTR-7-MeO-Lut-complexes.</p

Modifications of the 7-Hydroxyl Group of the Transthyretin Ligand Luteolin Provide Mechanistic Insights into Its Binding Properties and High Plasma Specificity - Fig 4

<p>A and C) The TTR monomers in the tetrameric structure are shown as ribbons and are labeled A (gold) and B (ice-blue). The symmetry-related monomers are labeled A´ and B´, respectively. Two 7-Cl-Lut molecules (shown in A) and two 7-MeO-Lut molecules (shown in C) bind at the T4 hormone binding pocket and are shown as sticks. (B) The quality of the electron density map at the BB´ dimer-dimer interface of 7-Cl-Lut. The σA-weighted (m|Fo|-D|Fc|) electron density map contoured at 3 times the root-mean-square value of the map is shown in orange. The anomalous log-likelihood-gradient (LLG) map shown in dark blue shows unambiguously the positions of the two symmetry-related chlorine atoms and verifies the modeled orientation of the 7-Cl-Lut compound in the binding site. (D) The quality of the difference Fourier electron density map at the BB´ dimer-dimer interface of 7-MeO-Lut, calculated as described in (B). To reduce model bias, the 7-Cl-Lut and 7-MeO-Lut molecules were excluded from the coordinate files that were subjected to one round of simulated annealing before calculation of the electron density map.</p

Chemical structures showing luteolin, luteolin-7-O-glucuronide, and the modified variants of luteolin where the 7-OH has been substituted with a chlorine (7-Cl-Lut) or a methoxy group (7-MeO-Lut).

<p>Chemical structures showing luteolin, luteolin-7-O-glucuronide, and the modified variants of luteolin where the 7-OH has been substituted with a chlorine (7-Cl-Lut) or a methoxy group (7-MeO-Lut).</p

Depletion profile of 10 μM luteolin (triangle), 7-Cl-Lut (square) and 7-MeO-Lut (diamond) after incubation in the presence of 0.1 mg/mL human liver microsomes for various times.

<p>Each time point represents the average of three independent samples (mean ± SD) acquired through plotting the integral of the corresponding peaks from the UHPLC analysis.</p

Determination of IC₅₀ for luteolin, 7-Cl-Lut and 7-MeO-Lut in human plasma.

<p>The drug’s ability to selectively stabilize TTR in plasma was evaluated using a urea denaturation assay [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153112#pone.0153112.ref025" target="_blank">25</a>]. The dissociation of the TTR tetramer is indicated by the presence of a monomeric TTR band on a western blot. (A) Gel-shift assay of various concentrations of luteolin in human plasma. The IC₅₀ is ~5 μM. (B) Gel-shift assay of various concentrations of 7-Cl-Lut in human plasma. The IC₅₀ is ~65 μM. (C) Gel-shift assay of various concentrations of 7-MeO-Lut in human plasma. The IC₅₀ is ~105 μM.</p