Background:  HIV fusion is mediated by assembly of the gp41 6-helix bundle, a validated target for pharmacologic inhibition of HIV infection. Although the prototype therapeutic in this arena, enfuvirtide (Fuzeon, T20) suppresses HIV viral load in treatment-experienced patients, this peptidic drug suffers from the general drawbacks of traditional peptide therapeutics. These include loss of biologically relevant secondary structure, a resultant decrease in bioactivity, and susceptibility to proteolytic degradation. Here we apply a hydrocarbon stapling strategy to overcome these peptide shortcomings, yielding remarkably stable and protease resistant gp41-based compounds that potently inhibit HIV viral fusion.

Methods:  We inserted non-natural amino acids bearing olefin tethers into peptides derived from the C-terminal heptad repeat of gp41, followed by ruthenium-catalyzed olefin metathesis to generate stabilized alpha helices of gp41 (SAH-gp41) compounds. Circular dichroism was performed to measure and compare the α-helical content and thermal stability of SAH-gp41 compounds and the corresponding unmodified peptides. Proteolysis studies were likewise performed to quantitate the resistance to degradation conferred by peptide stapling. Functional activity of SAH-gp41 compounds was assessed using luciferase-based HIV single-round infectivity assays.

Results:  Whereas native gp41 C-terminal heptad peptides are predominantly random coils in solution (i.e. <20% α-helicity), SAH-gp41 compounds demonstrate more than 3-fold enhancement of α-helical content. This stabilization of α-helical structure correlates with a dramatic enhancement in both thermal stability and protease resistance of SAH-gp41 compounds compared to their unmodified counterparts. For example, in chymotrypsin resistance assays, select SAH-gp41 compounds display a more than 50-fold prolongation of half-life compared to the unmodified peptides. Additionally, SAH-gp41 compounds exhibit up to 10 times greater potency in in vitro HIV infectivity assays.

Conclusions:  Insertion of chemical staples into HIV fusion inhibitor peptides restores their native α-helical structure, resulting in markedly improved thermal stability, protease resistance, and fusion inhibitory activity. Thus, peptide optimization by hydrocarbon stapling may yield more pharmacologically robust and clinically effective HIV fusion inhibitors.