Design of a Novel Peptide Inhibitor of HIV Fusion That Disrupts the Internal Trimeric Coiled-coil of gp41*

The pre-hairpin intermediate of gp41 from the human immunodeficiency virus (HIV) is the target for two classes of fusion inhibitors that bind to the C-terminal region or the trimeric coiled-coil of N-terminal helices, thereby preventing formation of the fusogenic trimer of hairpins. Using rational design, two 36-residue peptides, N36Mut(e,g) and N36Mut(a,d), were derived from the parent N36 peptide comprising the N-terminal helix of the gp41 ectodomain (residues 546–581 of HIV-1 envelope), characterized by analytical ultracentrifugation and CD, and assessed for their ability to inhibit HIV fusion using a quantitative vaccinia virus-based fusion assay. N36Mut(e,g) contains nine amino acid substitutions designed to disrupt interactions with the C-terminal region of gp41 while preserving contacts governing the formation of the trimeric coiled-coil. N36Mut(a,d) contains nine substitutions designed to block formation of the trimeric coiled-coil but retains residues that interact with the C-terminal region of gp41. N36Mut(a,d) is monomeric, is largely random coil, does not interact with the C34 peptide derived from the C-terminal region of gp41 (residues 628–661), and does not inhibit fusion. The trimeric coiled-coil structure is therefore a prerequisite for interaction with the C-terminal region of gp41. N36Mut(e,g) forms a monodisperse, helical trimer in solution, does not interact with C34, and yet inhibits fusion about 50-fold more effectively than the parent N36 peptide (IC50∼ 308 nm versus ∼16 μm). These results indicate that N36Mut(e,g) acts by disrupting the homotrimeric coiled-coil of N-terminal helices in the pre-hairpin intermediate to form heterotrimers. Thus N36Mut(e,g)represents a novel third class of gp41-targeted HIV fusion inhibitor. A quantitative model describing the interaction of N36Mut(e,g) with the pre-hairpin intermediate is presented.