Relevant Interactions of Antimicrobial Iron Chelators and Membrane Models Revealed by Nuclear Magnetic Resonance and Molecular Dynamics Simulations Joa ̃ o T. S. Coimbra, †,§ Ta ̂ nia Moniz, †,§ Nate ́ rcia F. Bra ́ s, †,§ Galya Ivanova, †,§ Pedro A. Fernandes, † Maria J. Ramos,* ,† and Maria Rangel* ,‡ † REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciê ncias, Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal ‡ REQUIMTE, Instituto de Ciê ncias Biome ́ dicas de Abel Salazar, Universidade do Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313, Porto, Portugal * S Supporting Information ABSTRACT: 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. ■ INTRODUCTION Our group has been working on the design of iron chelators that may be of use in new strategies to fight infection based on the concept of iron deprivation. Chelators were prepared by conjugating 3-hydroxy-4-pyridinone chelating units with xanthene fluorophores in order to provide compounds with distinct fluorescence emission spectrum, hydrophilic/lipophilic balance and charge at physiological pH (Figure 1). Evaluation of the effect of chelators in a Mycobacterium avium infection model proved that the compounds limit the access of iron to bacteria and have a significant inhibitory effect in the intramacrophagic growth of M. avium bacteria. 1-3 The results showed that (i) the activity of the chelators is strongly dependent on the presence of the fluorophore on the molecular framework and (ii) the inhibitory effect of the rhodamine B isothiocyanate derivatives (MRH7=CP777=4 and MRB7) is superior to those observed for carboxytetramethylrhodamine chelators (MRH8 and MRB8) and fluorescein chelators (MRH5=CP852). Results of comparative studies of the partition of the compounds and distribution of the compounds, obtained from fluorescence spectroscopy and confocal microscopy, provide evidence that the biologically active compounds strongly interact with lipid phases and remain largely membrane bound while the less-active do not. 3 Moreover, the results suggest that a surface effect is quite important for the interaction with the membrane as reported for rhodamine B conjugated peptides. 4,5 Considering the latter results, we hypothesized that rhodamine B potentiates the effect of the iron chelator by targeting the phagosomal membrane and by tethering the chelator to it, thus allowing successful competition with mycobacterial siderophores. Considering that the permeation process of drugs across a lipid bilayer is crucial to understand the mechanism of drug action and a significant contribution to the development of new bioactive molecules we further explore, in the present work, the dynamics and interaction of the synthesized iron chelators with lipid bilayers by Nuclear Magnetic Resonance (NMR) and Molecular Dynamics simulations in order to substantiate our former premise. Moreover, our previous confocal microscopy data show that the chelators go into macrophages but no detailed information regarding their pathways or mechanism of entrance has been obtained yet. Considering the chelators large size and the fact Received: September 19, 2014 Revised: November 18, 2014 Published: December 1, 2014 Article pubs.acs.org/JPCB © 2014 American Chemical Society 14590 dx.doi.org/10.1021/jp509491p | J. Phys. Chem. B 2014, 118, 14590-14601