Cell membrane features in HIV entry and its inhibition

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Singlet oxygen effects on lipid membranes : implications for the mechanism of action of broad-spectrum viral fusion inhibitors

Publication . Hollmann, Axel; Castanho, Miguel A. R. B.; Lee, Benhur; Santos, Nuno C.

It was reported recently that a new aryl methyldiene rhodamine derivative, LJ001, and oxazolidine-2,4-dithione, JL103, act on the viral membrane, inhibiting its fusion with a target cell membrane. The aim of the present study was to investigate the interactions of these two active compounds and an inactive analogue used as a negative control, LJ025, with biological membrane models, in order to clarify the mechanism of action at the molecular level of these new broad-spectrum enveloped virus entry inhibitors. Fluorescence spectroscopy was used to quantify the partition and determine the location of the molecules on membranes. The ability of the compounds to produce reactive oxygen molecules in the membrane was tested using 9,10-dimethylanthracene, which reacts selectively with singlet oxygen (1O2). Changes in the lipid packing and fluidity of membranes were assessed by fluorescence anisotropy and generalized polarization measurements. Finally, the ability to inhibit membrane fusion was evaluated using FRET. Our results indicate that 1O2 production by LJ001 and JL103 is able to induce several changes on membrane properties, specially related to a decrease in its fluidity, concomitant with an increase in the order of the polar headgroup region, resulting in an inhibition of the membrane fusion necessary for cell infection.

2014Journal article

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Improvement of HIV fusion inhibitor C34 efficacy by membrane anchoring and enhanced exposure

Publication . Augusto, Marcelo T.; Hollmann, Axel; Castanho, Miguel A. R. B.; Porotto, Matteo; Pessi, Antonello; Santos, Nuno C.

Objectives: The aim of the present work was to evaluate the interaction of two new HIV fusion inhibitors {HIVP3 [C34–polyethylene glycol (PEG)4–cholesterol] and HIVP4 [(C34–PEG4)2–cholesterol]} with membrane model systems and human blood cells in order to clarify where and how the fusion inhibitors locate, allowing us to understand their mechanism of action at the molecular level, and which strategies may be followed to increase efficacy. Methods: Lipid vesicles with defined compositions were used for peptide partition and localization studies, based on the intrinsic fluorescence of HIVP3 and HIVP4. Lipid monolayers were employed in surface pressure studies. Finally, human erythrocytes and peripheral blood mononuclear cells (PBMCs) isolated from blood samples were used in dipole potential assays. Results: Membrane partition, dipole potential and surface pressure assays indicate that the new fusion inhibitors interact preferentially with cholesterol-rich liquid-ordered membranes, mimicking biological membrane microdomains known as lipid rafts. HIVP3 and HIVP4 are able to interact with human erythrocytes and PBMCs to a similar degree as a previously described simpler drug with monomeric C34 and lacking the PEG spacer, C34–cholesterol. However, the pocket-binding domain (PBD) of both HIVP3 and HIVP4 is more exposed to the aqueous environment than in C34–cholesterol. Conclusions: The present data allow us to conclude that more efficient blocking of HIV entry results from the synergism between the membranotropic behaviour and the enhanced exposure of the PBD.

2014Journal article

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Structural requirements of glycosaminoglycans for their interaction with HIV-1 envelope glycoprotein gp120

Publication . Matos, Pedro M.; Andreu, David; Santos, Nuno C.; Gutiérrez-Gallego, Ricardo

Heparan sulfate proteoglycans are known to assist HIV-1 entry into host cells, mediated by the viral envelope glycoprotein gp120. We aimed to determine the general structural features of glycosaminoglycans that enable their binding to gp120, by surface plasmon resonance. Binding was found to be dependent on sequence type, size and sulfation patterns. HIV-1 gp120 prefers heparin and heparan sulfate (with at least 16 monomers in length) over chondroitin and dermatan. Sulfate groups were essential to promote this interaction. These results advance the understanding of the molecular-level requirements for virus attachment and cell entry.

2014Journal article

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