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Recently, the covalent binding of a cholesterol moiety to a classical HIV-1 fusion inhibitor peptide, C34, was shown to potentiate its antiviral activity. Our purpose was to evaluate the interaction of cholesterol-conjugated and native C34 with membrane model systems and human blood cells to understand the effects of this derivatization. Lipid vesicles and monolayers with defined compositions were used as model membranes. C34-cholesterol partitions more to fluid phase membranes that mimic biological membranes. Importantly, there is a preference of the conjugate for liquid ordered membranes, rich in cholesterol and/or sphingomyelin, as observed both from partition and surface pressure studies. In human erythrocytes and peripheral blood mononuclear cells (PBMC), C34-cholesterol significantly decreases the membrane dipole potential. In PBMC, the conjugate was 14- and 115-fold more membranotropic than T-1249 and enfuvirtide, respectively. C34 or cholesterol alone did not show significant membrane activity. The enhanced interaction of C34-cholesterol with biological membranes correlates with its higher antiviral potency. Higher partitions for lipid-raft like compositions direct the drug to the receptor-rich domains where membrane fusion is likely to occur. This intermediary membrane binding step may facilitate the drug delivery to gp41 in its pre-fusion state.

Human paramyxoviruses include global causes of lower respiratory disease like the parainfluenza viruses, as well as agents of lethal encephalitis like Nipah virus. Infection is initiated by viral glycoprotein-mediated fusion between viral and host cell membranes. Paramyxovirus viral fusion proteins (F) insert into the target cell membrane, and form a transient intermediate that pulls the viral and cell membranes together as two heptad-repeat regions refold to form a six-helix bundle structure that can be specifically targeted by fusion-inhibitory peptides. Antiviral potency can be improved by sequence modification and lipid conjugation, and by adding linkers between the protein and lipid components. We exploit the uniquely broad spectrum antiviral activity of a parainfluenza F-derived peptide sequence that inhibits both parainfluenza and Nipah viruses, to investigate the influence of peptide orientation and intervening linker length on the peptides’ interaction with transitional states of F, solubility, membrane insertion kinetics, and protease sensitivity. We assessed the impact of these features on biodistribution and antiviral efficacy in vitro and in vivo. The engineering approach based on biophysical parameters resulted in a peptide that is a highly effective inhibitor of both paramyxoviruses and a set of criteria to be used for engineering broad spectrum antivirals for emerging paramyxoviruses.

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.