Abstract
Enfuvirtide (T20) is the only viral fusion inhibitor approved for clinical use, but it has relatively weak anti-HIV activity and easily induces drug resistance. In succession to T20, T1249 has been designed as a 39-mer peptide composed of amino acid sequences derived from HIV-1, HIV-2, and simian immunodeficiency virus (SIV); however, its development has been suspended due to formulation difficulties. We recently developed a T20-based lipopeptide (LP-40) showing greatly improved pharmaceutical properties. Here, we generated a T1249-based lipopeptide, termed LP-46, by replacing its C-terminal tryptophan-rich sequence with fatty acid. As compared with T20, T1249, and LP-40, the truncated LP-46 (31-mer) had dramatically increased activities in inhibiting a large panel of HIV-1 subtypes, with IC50 values approaching low picomolar concentrations. Also, LP-46 was an exceptionally potent inhibitor against HIV-2, SIV, and T20-resistant variants, and it displayed obvious synergistic effects with LP-40. Furthermore, we showed that LP-46 had increased helical stability and binding affinity with the target site. The crystal structure of LP-46 in complex with a target surrogate revealed its critical binding motifs underlying the mechanism of action. Interestingly, it was found that the introduced pocket-binding domain in LP-46 did not interact with the gp41 pocket as expected; instead, it adopted a mode similar to that of LP-40. Therefore, our studies have provided an exceptionally potent and broad fusion inhibitor for developing new anti-HIV drugs, which can also serve as a tool to exploit the mechanisms of viral fusion and inhibition.
Highlights
Enfuvirtide (T20) is the only viral fusion inhibitor approved for clinical use, but it has relatively weak anti-HIV activity and induces drug resistance
To validate the function of the tryptophan-rich motif (TRM) in T1249, we synthesized the truncated peptide T1249-TRM by deleting the TRM from T1249 (Fig. 1), and their antiviral activities were compared with three replicative HIV-1 strains
A flexible linker (8-unit polyethylene glycol, PEG8) was introduced between the peptide sequence and the lipid moiety of LP-46; the resulting lipopeptide LP-47 showed an obviously reduced inhibitory activity, suggesting that a linker is not required for the antiviral activity of LP-46, similar to the T20-based lipopeptide LP-40
Summary
Envelope; CHR, C-terminal heptad repeat(s); NHR, N-terminal heptad repeat; 6-HB, six-helix bundle; PBD, pocket-binding domain; TRM, tryptophan-rich motif; DSP, dual split protein; CI, combination index; SIV, simian immunodeficiency virus. Preclinical and clinical studies demonstrated that T1249 exhibited significantly increased antiviral activity, including its inhibition on T20-resistant HIV-1 mutants and HIV-2 isolates (28 –31). By conjugating different lipids (fatty acid, cholesterol, sphingolipids) to the C terminus of short peptides that mainly target the NHR pocket site, we previously developed the lipopeptides LP-11 and LP-19 (Fig. 1), which did show markedly increased anti-HIV potency and in vivo half-lives [36, 37]. Our studies have generated the most potent and broad HIV-1/2 and SIV fusion inhibitor known to date, which provides an ideal candidate for drug development, and serves as a critical tool to investigate the mechanisms of viral fusion and inhibition
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