Viral Fusion Inhibitors

Until recently, only two proteins essential for the viral life cycle were targets of therapeutic intervention by three mechanistic classes of antiretroviral drugs. Reverse transcriptase is the target of nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs), while HIV protease is targeted by protease inhibitors (PIs). In March 2003, however, Enfuvirtide was approved as the first HIV fusion inhibitor. Enfuvirtide is a peptide consisting of residues 643-678 of the HIV-1 gp160 protein. These residues are part of the HR2 segment of gp41 that folds back onto the hydrophobic groove formed by the HR1 stalk. The mechanism of action is thought to involve competitive binding of Enfuvirtide to HR1 while gp41 is still in the pre-fusion state, thus inhibiting membrane fusion and viral entry. Interestingly, the first peptides inhibiting viral fusion were designed in the absence of high-resolution structural data on the fusion protein, and structural implications of the fusion process were merely based on secondary-structure prediction from the primary sequence of gp160 (Wild et al. 1992). Though Enfuvirtide lacks the three hydrophobic residues of HR2 that are in contact with HR1 (see Sect. 3.3), it has reasonably high in vitro antiviral activity with an IC50 value for HIV-1 entry of 50-60 nM (Wild et al. 1994). Peptides derived from the; HR1 region also inhibit viral entry, indicating that the believed mechanism of action is essentially correct (Wild et al. 1992; Jiang et al. 1994).

The wealth of structural details and structure-activity relationships available for coiled-coil interactions make Enfuvirtide and other peptides obvious candidates for rational drug development. Indeed, several peptide sequences modified accordingly have been shown to be more active in inhibiting viral entry, with IC50 values in the upper femtomolar to lower nanomolar range (Otaka et al. 2002; Eron et al. 2004; Dwyer et al. 2007). A peptide termed T-1249 appeared to be the most promising candidate for development of second-generation fusion inhibitors because it possesses greater in vitro potency than Enfuvirtide and is active on most Enfuvirtide-resistant strains, too (Eron et al. 2004). However, further development was put on hold because its poor solubility and stability more than outweigh its favorable safety, efficacy, and tolerability properties determined in phase I trials (Martínez-Carbonero 2004).

In an effort to design more potent inhibitors of viral fusion, Dwyer et al. (2007) applied modifications to an HR2 peptide known to increase helical content and oligomer stability of coiled-coil peptides. Having identified functionally critical residues by alanine scanning, they introduced glutamic acid and arginine residues at the core-flanking positions e and g such as to promote interhelical salt bridges stabilizing the coiled-coil assembly (see Sect. 1.2). Additionally, eight non-critical residues were replaced by alanine, which has one of the highest propensities to induce a-helix formation. In a last step, two alanine residues were included at a and d positions to increase the hydrophobic contact area of the core. The resulting peptide, referred to as T-2635, assembles into a homotrimeric coiled-coil structure at low micromolar concentrations and forms an extremely stable six-helix bundle with HR1, having a melting temperature of 86°C in 8 M urea. In vitro, T-2635 reveals greatly enhanced activity in comparison with Enfuvirtide against several, though not all, tested HIV-1 strains. Moreover, viral resistance evolves considerably slower for a close analogue of T-2635 as compared with Enfuvirtide (Dwyer et al. 2007).

Several small molecules inhibiting HIV-1 fusion with IC50 in the lower micromolar range have been discovered by high-throughput screening of molecular libraries. These compounds are believed to inhibit viral entry by interacting with the hydrophobic cavity of gp41-HR1, thereby destabilizing the central three-helix stalk and inhibiting formation of the post-fusion six-helix bundle (Jiang et al. 2004; Jin et al. 2005). Developing such small-molecule lead scaffolds into more potent HIV-1 fusion inhibitors is attracting enormous attention from both academic and industrial researchers as this could mark a major breakthrough in antiretro viral therapy.

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