Exceptional potency and structural basis of a T1249-derived lipopeptide fusion inhibitor against HIV-1, HIV-2, and simian immunodeficiency virus

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.

Keywords: HIV-1; HIV-2; T1249; T20; antiviral agent; crystal structure; fusion inhibitor; human immunodeficiency virus (HIV); inhibitor; lipopeptide; membrane fusion; peptides; structure-function.

Figure 1.

Schematic view of HIV-1 gp41 and its NHR- and CHR-derived peptides. A, the functional domains of gp41 protein. The gp41 numbering of HIV-1_HXB2 was used. FP, fusion peptide; TM, transmembrane domain. The positions and amino acid sequences corresponding to the T20-resistant site and pocket-forming site in the NHR are marked in blue. The positions and amino acid sequences corresponding to the M-T hook structure, PBD, and tryptophan-rich motif (TRM) in the CHR are marked in green, red, and purple, respectively. B, primary structure of gp41 CHR-derived viral fusion inhibitors. The dashed lines in T1249 indicate the missed residues corresponding to the functional domains of the CHR; the C16 in parentheses represents a fatty acid group; PEG8 represents a linker of 8-unit polyethylene glycol.

Figure 2.

Inhibitory activities of T1249-based inhibitors against HIV-1 infection. A, inhibitory activities of inhibitors on HIV-1 NL4–3 (subtype B, X4). B, inhibitory activities of inhibitors on HIV-1 JRCSF (subtype B, R5). C, inhibitory activities of inhibitors on HIV-1 R3A (subtype B, R5X4). The experiments were performed in triplicate and repeated three times. The percentage inhibition of the inhibitors and IC₅₀ values were calculated. Data are expressed as means ± S.D. (error bars). The solid lines represent sigmoidal non-linear regression analysis. The mean numbers are presented in parentheses for convenience.

Figure 3.

Inhibitory activities of LP-46 and control inhibitors against HIV-1–mediated cell entry and fusion. A, inhibitory activities of LP-46 and control inhibitors on NL4-3 pseudovirus. B, inhibitory activities of LP-46 and control inhibitors on HXB2 Env-mediated cell-cell fusion. The experiments were performed in triplicate and repeated three times. The percentage inhibition of the inhibitors and IC₅₀ values were calculated. Data are expressed as means ± S.D. (error bars). The solid lines represent sigmoidal non-linear regression analysis. The mean numbers are presented in parentheses for convenience.

Figure 4.

Secondary structure and stability of LP-46 determined by CD spectroscopy. The α-helicity (A) and thermostability (B) of the isolated T1249 and LP-46 were measured in PBS, with a final concentration of inhibitor at 20 μm. The α-helicity (C) and thermostability (D) of T1249, LP-46, and LP-40 in complexes with the NHR-derived peptide N44 were measured in deionized water, with the final concentration of each inhibitor at 10 μm. The experiments were performed two times, and representative data are shown.

Figure 5.

Crystal structure of 6-HB formed by N44 and LP-46. A, the crystal structure of N44/LP-46 is presented in ribbon models vertically by PyMOL. N44 peptides are colored in gray with the PFS marked in orange, and LP-46 inhibitors are colored in slate. B, comparison of the binding footprints of LP-46 and LP-40 with N44 by superimposing. The LP-46 inhibitors are colored slate, whereas the LP-40 inhibitors are colored green. Both the N and C termini of N44 and inhibitors are labeled.

Figure 6.

Interactions between N44 and LP-46. A, binding of the introduced pocket-binding sequence in LP-46 with the NHR pocket site. The N44 trimer is shown as a surface model in gray with the pocket region marked in light orange. LP-46 inhibitors are shown as ribbon models and colored in slate. Residues related to hydrophobic interactions in LP-46 are shown as stick models in yellow. B, hydrophobic contacts of LP-46 with the N44 helices in ribbon models. The N44 peptides are colored in gray with the residues related to hydrophobic interactions marked in orange, whereas the LP-46 inhibitors are in slate. C, intrahelical bonds in the LP-46 helix. The N44 helices are colored in gray, and the LP-46 inhibitors are colored in slate. The residues involving hydrogen bond formation are shown as stick models with labels. Hydrogen bonds are indicated in dashed black lines. D, interhelical bonds between N44 and LP-46. The N44 helices are colored in gray, and the LP-46 inhibitors are colored in slate. The residues involved in interhelical hydrogen bond formation are shown as stick models with labels. Hydrogen bonds are indicated in dashed black lines.