Conjugation of Cholesterol to HIV-1 Fusion Inhibitor C34 Synergistically Potentiates Peptide-Membrane Interaction

MATOS, PM; HOLLMANN, A; CASTANHO, MA; SANTOS NC

Congreso; Biophysical Society 56th Annual Meeting; 2012

Biophysical Society

HIV-1 fusion inhibitor peptides bind to gp41 and prevent its fusogenic conformation, inhibiting the initial step of viral entry into host cells. This early action suggests that these inhibitors can become new microbicide candidates. Enfuvirtide remains the only fusion inhibitor approved for clinical use. We studied the membrane interaction of a recent, highly potent, fusion inhibitor conjugate, C34-cholesterol (L’644), and compared it with other unconjugated peptides in order to better understand their mode of action. Large unilamellar vesicles were used to quantify the partition of the peptides using its intrinsic tryptophan fluorescence as a reporter. We confirmed that C34 has residual partition towards lipid membranes, except for DPPC, and observed that the conjugated peptide presents high partition. For cell membranes studies, isolated human peripheral blood mononuclear cells (PBMC) and erythrocytes were label with di-8-ANEPPS. This fluorescent probe is sensitive to dipole potential changes at the membrane level and can report the association of drugs to the cell membrane. C34 and cholesterol itself did not change membrane dipole potential significantly, in sharp contrast with the conjugated peptide that presented membrane affinity 115-fold higher than enfuvirtide and 14-fold higher than T-1249 for PBMC. Similar behavior was observed for erythrocytes. The high membranotropism of C34-cholesterol compared to the almost non existing binding of C34 and no effect of cholesterol itself in cells, suggests a synergistic potentiating effect when the two components are present in the conjugated molecule. Membrane binding can be a more efficient route than simple aqueous diffusion to facilitate the binding of the inhibitor to the pre-fusion conformation of gp41. Moreover, such a high binding to cell surface is an advantageous factor for the drug to be used as a topical microbicide.