Relevant Interactions of Antimicrobial Iron Chelators and Membrane Models Revealed by Nuclear Magnetic Resonance and Molecular Dynamics Simulations

The dynamics and interaction of 3-hydroxy-4-pyridinone fluorescent iron chelators, exhibiting antimicrobial properties, with biological membranes were evaluated through NMR and molecular dynamics simulations. Both NMR and MD simulation results support a strong interaction of the chelators with the lipid bilayers that seems to be strengthened for the rhodamine containing compounds, in particular for compounds that include ethyl groups and a thiourea link. For the latter type of compounds the interaction reaches the hydrophobic core of the lipid bilayer. The molecular docking and MD simulations performed for the potential interaction of the chelators with DC-SIGN receptors provide valuable information regarding the cellular uptake of these compounds since the results show that the fluorophore fragment of the molecular framework is essential for an efficient binding. Putting together our previous and present results, we put forward the hypothesis that all the studied fluorescent chelators have access to the cell, their uptake occurs through different pathways and their permeation properties correlate with a better access to the cell and its compartments and, consequently, with the chelators antimicrobial properties

600 MHz ¹H-NMR spectra of: A. DMEM; B. 24 h DMEM; C. 48 H <i>Com. Medium</i>; D. 24 h <i>Com. Medium</i>¹; E. 24 h <i>Com. Medium</i>²; F. <i>Com. Medium</i>; G. <i>Plasma1</i>; H. <i>Plasma2</i>; I. <i>Plasma3</i>; J. <i>Plasma4</i>; K. <i>Plasma5</i>.

<p>The codes (1-28) represent metabolites from <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113769#pone.0113769.s006" target="_blank">Table S5</a></b>. *D and E correspond to two different samples with the same basal medium (<i>Com. Medium</i>) and time of conditioning (24 h). <i>Com. Medium is</i> Commercial medium from PromoCell (LabClinics, Promocell, C-28010).</p

600 MHz ¹H/¹H 2D COSY NMR spectra of <i>48 h Com. Medium</i> sample.

<p><i>Com. Medium is</i> Commercial medium from PromoCell (LabClinics, Promocell, C-28010).</p

drug

drug n[on slide]Used I and SupUsed I and SupNot use

Total nucleated cells per liter of umbilical cord blood collected measured (TNC cell x 10⁹/l) obtained by using the hematology autoanalyser (Ac T diff2, Beckman Coulter, Inc.), number of viable CD34⁺ cells per µl of UCB collected (cells/µl), viability of CD34⁺ cells (%), number of viable CD45⁺ cells per liter of UCB collected (cells ×10⁹/l) and viability of CD45⁺ cells (%) measured by flow citometry of the UCB samples used for ¹H NMR and Multiplexing LASER Bead analysis.

<p>The average and standard deviation (SD) of N  =  11 is also presented. * Count using the hematology autoanalyser.</p><p>Total nucleated cells per liter of umbilical cord blood collected measured (TNC cell x 10⁹/l) obtained by using the hematology autoanalyser (Ac T diff2, Beckman Coulter, Inc.), number of viable CD34⁺ cells per µl of UCB collected (cells/µl), viability of CD34⁺ cells (%), number of viable CD45⁺ cells per liter of UCB collected (cells ×10⁹/l) and viability of CD45⁺ cells (%) measured by flow citometry of the UCB samples used for ¹H NMR and Multiplexing LASER Bead analysis.</p

600 MHz ¹H/¹H 2D TOCSY NMR spectra of <i>48 h Com. Medium</i> sample.

<p><i>Com. Medium is</i> Commercial medium from PromoCell (LabClinics, Promocell, C-28010).</p

600/150 MHz ¹H/¹³C 2D HSQC NMR spectra of <i>48 h Com. Medium</i> sample.

<p><i>Com. Medium is</i> Commercial medium from PromoCell (LabClinics, Promocell, C-28010).</p

Graphical representation of the relative metabolite concentration of the main aminoacids after normalization of the integral signal intensity obtained in the ¹H-NMR spectra of <i>DMEM</i>, <i>24 h DMEM</i>, <i>Com. Medium</i>, <i>24 h</i> and <i>48 h Com.</i>

<p><i>Medium</i> and <i>UCB Plasma</i> (average) samples. Chemical structures of the main metabolites are represented. <i>Com. Medium is</i> Commercial medium from PromoCell (LabClinics, Promocell, C-28010).</p