Abstract
Human immunodeficiency virus (HIV) gp41 plays a key role in viral fusion; the N- and C-terminal heptad repeats (N-HR and C-HR) of gp41 form a stable 6-helical conformation for fusion. Therefore, HR-derived peptides, such as enfuvirtide (T-20), inhibit HIV-1 fusion by acting as decoys, and have been used for the treatment of HIV-1 infection. However, the efficacy of T-20 is attenuated by resistance mutations in gp41, including V38A and N43D. To suppress the resistant variants, we previously developed electrostatically constrained peptides, SC34 and SC34EK, and showed that both exhibited potent anti-HIV-1 activity against wild-type and T-20-resistant variants. In this study, to clarify the resistance mechanism to this next generation of fusion inhibitors, we selected variants with resistance to SC34 and SC34EK in vitro. The resistant variants had multiple mutations in gp41. All of these mutations individually caused less than 6-fold resistance to SC34 and SC34EK, indicating that there is a significant genetic barrier for high-level resistance. Cross-resistance to SC34 and SC34EK was reduced by a simple difference in the polarity of two intramolecular electrostatic pairs. Furthermore, the selected mutations enhanced the physicochemical interactions with N-HR variants and restored activities of the parental peptide, C34, even to resistant variants. These results demonstrate that our approach of designing gp41-binding inhibitors using electrostatic constraints and information derived from resistance studies produces inhibitors with enhanced activity, high genetic barrier, and distinct resistance profile from T-20 and other inhibitors. Hence, this is a promising approach for the design of future generation peptide fusion inhibitors.
Highlights
Based on the nature of the mechanism of Human immunodeficiency virus (HIV) fusion, peptides corresponding to N-terminal heptad repeat (N-HR) or C-terminal heptad repeat (C-HR) of HIV fusion acted as decoys and interfered with formation of the 6-helix bundle [6, 7]
Selection of SC34-resistant HIV-1 in Vitro—To determine the resistance profile of SC34, SC34-resistant variants were selected by a dose-escalating method and susceptibility of the obtained variants was determined by the multinuclear activation of a galactosidase indicator (MAGI) assay
Inhibitory Activity of Modified Fusion Inhibitors—We have previously demonstrated that introduction of resistance mutations to T-20 restores anti-HIV-1 activity against T-20-resistant variants [18], suggesting that this strategy can result in the design of peptides with improved potential for the treatment of resistant HIV-1
Summary
N-HR, N-terminal heptad repeat; C-HR, C-terminal heptad repeat; MAGI, multinuclear activation of a galactosidase indicator. In addition to engineering stabilizing electrostatic interactions we have established a second strategy to design improved peptide fusion inhibitors. This strategy is based on the introduction of resistance mutations into the sequence of the original peptide inhibitor. These new changes enhance the antiviral potency against resistant variants. The novel strategy to design inhibitor peptides utilizing resistance mutations has resulted in antivirals that can suppress variants resistant to the parental peptides. Our results demonstrate that peptides that are designed to have specific electrostatic constraints and include changes that are based on resistance information have significantly improved properties in terms of potency and cross-resistance
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
