Abstract: Enfuvirtide and T-1249 are two HIV-1 fusion inhibitor peptides that bind to gp41 and prevent its fusogenic conformation, inhibiting viral entry into host cells. Previous studies established the relative preferences of these peptides for membrane model systems of defined lipid compositions. We aimed to understand the interaction of these peptides with the membranes of erythrocytes and peripheral blood mononuclear cells. The peptide behavior toward cell membranes was followed by di-8-ANEPPS fluorescence, a lipophilic probe sensitive to the changes in membrane dipole potential. We observed a fusion inhibitor concentration-dependent decrease on the membrane dipole potential. Quantitative analysis showed that T-1249 has an approximately eight-fold higher affinity towards cells, when compared with enfuvirtide. We also compared the binding towards di-8-ANEPPS labeled lipid vesicles that model cell membranes and obtained concordant results. We demonstrated the distinct enfuvirtide and T-1249 membranotropism for circulating blood cells, which can be translated to a feasible in vivo scenario. The enhanced interaction of T-1249 with cell membranes correlates with its higher efficacy, as it can increase and accelerate the drug binding to gp41 in its pre-fusion state.
Notes: Recommended by F1000 at http://f1000.com/2953959
Abstract: Peptide-membrane interactions have been gaining increased relevance, mainly in biomedical investigation, as the potential of the natural, nature-based and synthetic peptides as new drugs or drug candidates also expands. These peptides must face the cell membrane when they interfere with or participate in intracellular processes. Additionally, several peptide drugs and drug leads actions occur at the membrane level (e.g., antimicrobial peptides, cell-penetrating peptides and enveloped viruses membrane fusion inhibitors). Here we explore fluorescence spectroscopy methods that can be used to monitor such interactions. Two main approaches are considered, centered either on the peptide or on the membrane. On the first, we consider mainly the methodologies based on the intrinsic fluorescence of the aminoacid residues tryptophan and tyrosine. Regarding membrane-centric approaches, we review methods based on lipophilic probes sensitive to membrane potentials. The use of fluorescence constitutes a simple and sensitive method to measure these events. Unraveling the molecular mechanisms that govern these interactions can unlock the key to understand specific biological processes involving natural peptides or to optimize the action of a peptide drug.
Abstract: Sifuvirtide is a gp41 based peptide that inhibits HIV-1 fusion with the host cells and is currently under clinical trials. Previous studies showed that sifuvirtide partitions preferably to saturated phosphatidylcholine lipid membranes, instead of fluid-phase lipid vesicles. We extended the study to the interaction of the peptide with circulating blood cells, by using the dipole potential sensitive probe di-8-ANEPPS. Sifuvirtide decreased the dipole potential of erythrocyte and lymphocyte membranes in a concentration dependent manner, demonstrating its interaction. Also, the lipid selectivity of the peptide towards more rigid phosphatidylcholines was confirmed based on the dipole potential variations. Overall, the interaction of the peptide with the cell membranes is a contribution of different lipid preferences that presumably directs the peptide towards raft-like domains where the receptors are located, facilitating the reach of the peptide to its molecular target, the gp41 in its pre-fusion conformation.
Abstract: Peptide-membrane interaction is an important step to be evaluated in a study of the activity and mode of action of several bioactive peptides. A variety of methods are available; however, few of them satisfy the criteria of being sensitive, biocompatible, versatile, easy to perform, and allowing real-time monitoring as the use of potential-sensitive fluorescent probes. Here we review methods for detecting the effects of membrane-active peptides, even those that are not intrinsically fluorescent, on the different types of membrane potentials, with a special emphasis on studies conducted with living cells. FPE is a probe sensitive to surface potential and detects electrostatic interactions at the water-lipid interface. Di-8-ANEPPS is sensitive to dipole potential and detects membrane incorporations. Transmembrane potential changes reveal major membrane destabilizations, such as in pore formation. The combination of the information obtained from the three potential variations can lead to a more elucidative picture of the mechanisms of the interaction of relevant peptides with biomembranes.
