Fusion peptides binding to different micelles as measured by acrylamide quenching.

<p>All measurements were performed in triplicate in the same experiment, and the results were obtained from at least six independent experiments. The Ksv values are expressed as the mean ± SEM. The significance coefficient was obtained using the paired Student’s t test. p values were calculated by buffer-SDS<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047596#pone.0047596-Weissenhorn1" target="_blank">[¹]</a> and buffer-n-OGP<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047596#pone.0047596-White1" target="_blank">[²]</a> pairs. ns – not significant;</p>*<p>0.01**</p><p>0.001***</p><p>p<0.001 (extremely significant).</p

Molecular dynamics simulation of the interaction of the fusion peptides with the POPE membrane.

<p>Representative snapshots from simulations of FLA_G (A) and FLA_H (B) interaction with a POPE membrane at 35°C. The peptides are shown in light green, the membranes are in gray and, in each peptide, the side chains of residues Trp101 and Phe 108 are highlighted in dark green and blue, respectively.</p

Molecular dynamics studies of the POPE membrane environment.

<p>(A) The RMSD of FLA_G (black) and FLA_H (red). (B) The distance between the peptide and membrane was determined as follows: the distance between the center of mass of the peptide and the membrane in the axis perpendicular to the membrane surface plane for both FLA_G (black) and FLA_H (red); and the minimal distance between Gly104 (green) or His104 (blue) atoms and the phosphorus atoms of the lipids. (C and D) The number of intramolecular hydrogen bonds (black) and those formed between the fusion peptides FLA_G (C) or FLA_H (D) and the water (green) or the POPE membrane (red). (E and F) The minimal distance between the Trp101 residue and the POPE membrane (red) and between the Phe108 residue and the POPE membrane (black) during MD simulation in the presence of the POPE membrane at 35°C. The intermolecular distance between Trp101 and Phe108 is also presented (green). The results for MD simulation of FLA_G and FLA_H are shown in E and F, respectively.</p

The secondary structures of FLA_G and FLA_H in the presence of membrane models.

<p>(A and B) The secondary structures of the fusion peptides FLA_G (A) and FLA_H (B) in the presence of POPE membranes at 35°C. (C and D) The circular dichroism spectra of the FLA_G (C) and FLA_H (D) FP in solution (solid line) and in the presence of SDS (dashed line) or n-OGP (dotted line) micelles. The experiments were performed at room temperature at pH 5.5.</p

Fluorescence spectroscopy data of FLA_G and FLA_H in the presence of micelles.

<p>All measurements were performed in triplicate in the same experiment, and the results were obtained from at least six independent experiments. ΔCM and S/S₀ are expressed as the mean ± SD.</p>[1]<p>Blue shift was determined by subtracting emission wavelength from control.</p

The secondary structure of peptides FLA_G and FLA_H in aqueous buffer.

<p>The secondary structure patterns of the fusion peptides FLA_G (A) and FLA_H (B), at 35°C and secondary structure patterns of the FP FLA_G (C) and FLA_H (D) at 85°C in solution. (E and F) The MD simulation of FLA_G (E) and FLA_H (F) in water at 35°C (red line) and at 85°C (black line). (G and H) The minimal distance between FLA_G (G) or FLA_H (H) Trp101 and Phe108 residues at 35°C (red line) and at 85°C (black line). (I and J) The circular dichroism spectra of the FLA_G (I) and FLA_H (J) fusion peptides in solution at 25°C (solid line), at 85°C (dashed line), and the return to 25°C (dotted line). The experiments were performed at room temperature at pH 5.5.</p

The flavivirus E glycoprotein fusion loop and hydrophobicity plots of the fusion peptides.

<p>(A) The crystallographic structure of West Nile virus E protein (PDB ID 2HG0) and the schematic representation and sequence of the two fusion peptides of flaviviruses studied in this work. FLA_G has a Gly residue (red) in position 104 of glycoprotein E, while FLA_H presents a His residue (blue). Trp101, Gly104 and His104 are indicated in bold, red and blue, respectively. The conserved amino acids are underlined. (B) Hydrophobicity plots for the fusion peptides FLA_G (solid line) and FLA_H (dashed line) were elaborated using the Wimley-White hydrophobicity scale.</p

Anti-Prion Activity of a Panel of Aromatic Chemical Compounds: <i>In Vitro</i> and <i>In Silico</i> Approaches

<div><p>The prion protein (PrP) is implicated in the Transmissible Spongiform Encephalopathies (TSEs), which comprise a group of fatal neurodegenerative diseases affecting humans and other mammals. Conversion of cellular PrP (PrP^C) into the scrapie form (PrP^Sc) is the hallmark of TSEs. Once formed, PrP^Sc aggregates and catalyzes PrP^C misfolding into new PrP^Sc molecules. Although many compounds have been shown to inhibit the conversion process, so far there is no effective therapy for TSEs. Besides, most of the previously evaluated compounds failed <i>in vivo</i> due to poor pharmacokinetic profiles. In this work we propose a combined <i>in vitro</i>/<i>in silico</i> approach to screen for active anti-prion compounds presenting acceptable drugability and pharmacokinetic parameters. A diverse panel of aromatic compounds was screened in neuroblastoma cells persistently infected with PrP^Sc (ScN2a) for their ability to inhibit PK-resistant PrP (PrP^Res) accumulation. From ∼200 compounds, 47 were effective in decreasing the accumulation of PrP^Res in ScN2a cells. Pharmacokinetic and physicochemical properties were predicted <i>in silico</i>, allowing us to obtain estimates of relative blood brain barrier permeation and mutagenicity. MTT reduction assays showed that most of the active compounds were non cytotoxic. Compounds that cleared PrP^Res from ScN2a cells, were non-toxic in the MTT assay, and presented a good pharmacokinetic profile were investigated for their ability to inhibit aggregation of an amyloidogenic PrP peptide fragment (PrP^109–149). Molecular docking results provided structural models and binding affinities for the interaction between PrP and the most promising compounds. In summary, using this combined <i>in vitro</i>/<i>in silico</i> approach we have identified new small organic anti-scrapie compounds that decrease the accumulation of PrP^Res in ScN2a cells, inhibit the aggregation of a PrP peptide, and possess pharmacokinetic characteristics that support their drugability. These compounds are attractive candidates for prion disease therapy.</p></div

Full ESI-LIT-MS spectra of <i>T. cruzi</i> phospholipids.

<p>(<b>A</b>) MS1 spectra of lipids obtained in the Folch lower phase prior to fractionation. Lipid samples from all <i>T. cruzi</i> stages were diluted in methanol containing 5 mM LiOH and analyzed by direct infusion in an LTQXL ESI-LIT-MS (positive-ion mode, MS+). Note that the region of spectrum corresponding to LPAF, LPC, and PAF species in Epi, Meta, and TCT has been magnified for better visualization. (<b>B</b>) MS1 spectra of phospholipids obtained by SPE followed by POROS R1 fractionation. Lipids eluted in 25% n-propanol were diluted in methanol containing 5 mM LiOH and analyzed as above. Since the same initial total number of cells (5×10⁸) was used for lipid fractionation from each parasite stage, all spectra were normalized. Magnification of the MS range where PAF and LPC species would be found is indicated (insets). Epi, epimastigote; Meta, metacyclic trypomastigote; ICA, intracellular amastigote; TCT, tissue culture-derived trypomastigote. <i>m/z</i>, mass to charge ratio.</p

Activity of C16:0-PAF and different <i>T. cruzi</i> LPC species on the aggregation of rabbit platelets.

<p>Platelet aggregation assays were performed as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003077#s2" target="_blank">Materials and Methods</a>, using synthetic 16:0-PAF and C16:0-, C18:0-, C18:1-LPC, and purified C18:2-LPC. Control platelets or platelets pre-treated for 30 min with 10 µM WEB 2086 were assayed in the absence or presence of 1 µM C16:0-PAF or the LPC species at 10 µM (C16:0-LPC, C18:0-LPC, C18:1-LPC, and C18:2-LPC) and 100 µM (C18:1-LPC). Each lipid was tested in duplicate as indicated by black and blue curves in each graph.</p