Design and Characterization of Cholesterylated Peptide HIV-1/2 Fusion Inhibitors with Extremely Potent and Long-Lasting Antiviral Activity

Abstract

HIV infection requires lifelong treatment with multiple antiretroviral drugs in a combination, which ultimately causes cumulative toxicities and drug resistance, thus necessitating the development of novel antiviral agents. We recently found that enfuvirtide (T-20)-based lipopeptides conjugated with fatty acids have dramatically increased in vitro and in vivo anti-HIV activities. Herein, a group of cholesterol-modified fusion inhibitors were characterized with significant findings. First, novel cholesterylated inhibitors, such as LP-83 and LP-86, showed the most potent activity in inhibiting divergent human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). Second, the cholesterylated inhibitors were highly active to inhibit T-20-resistant mutants that still conferred high resistance to the fatty acid derivatives. Third, the cholesterylated inhibitors had extremely potent activity to block HIV envelope (Env)-mediated cell-cell fusion, especially a truncated minimum lipopeptide (LP-95), showing a greatly increased potency relative to its inhibition on virus infection. Fourth, the cholesterylated inhibitors efficiently bound to both the cellular and viral membranes to exert their antiviral activities. Fifth, the cholesterylated inhibitors displayed low cytotoxicity and binding capacity with human serum albumin. Sixth, we further demonstrated that LP-83 exhibited extremely potent and long-lasting anti-HIV activity in rhesus monkeys. Taken together, the present results help our understanding on the mechanism of action of lipopeptide-based viral fusion inhibitors and facilitate the development of novel anti-HIV drugs.IMPORTANCE The peptide drug enfuvirtide (T-20) remains the only membrane fusion inhibitor available for treatment of viral infection, which is used in combination therapy of HIV-1 infection; however, it exhibits relatively low antiviral activity and a genetic barrier to inducing resistance, calling for the continuous development for novel anti-HIV agents. In this study, we report cholesterylated fusion inhibitors showing the most potent and broad anti-HIV activities to date. The new inhibitors have been comprehensively characterized for their modes of action and druggability, including small size, low cytotoxicity, binding ability to human serum albumin (HSA), and, especially, extremely potent and long-lasting antiviral activity in rhesus monkeys. Therefore, the present studies have provided new drug candidates for clinical development, which can also be used as tools to probe the mechanisms of viral entry and inhibition.

Keywords: HIV-1; HIV-2; T-20; fusion inhibitor; lipopeptide.

FIG 1

Schematic diagram of HIV-1 gp41 and its peptide derivatives. The gp41 numbering of HIV-1_HXB2 is used. FP, fusion peptide; NHR, N-terminal heptad repeat; CHR, C-terminal heptad repeat; TRM, tryptophan-rich motif; TM, transmembrane domain; CT, cytoplasmic tail. The positions and sequences corresponding to the T-20-resistance mutation site and the pocket-forming site in the NHR are marked in blue. The positions and sequences of the M-T hook structure, pocket-binding domain (PBD), and tryptophan-rich motif (TRM) in the CHR are marked in green, red, and purple, respectively. Chol, C₁₆, and C₁₈ in parentheses represent cholesterol, palmitic acid, and stearic acid, respectively; PEG8 represents a flexible linker of 8-unit polyethylene glycol. Engineered residues in newly designed T-20 sequence-based lipopeptides are marked in pink.

FIG 2

Secondary structure and stability of cholesterylated peptide fusion inhibitors. (A) The α-helicity (left) and thermostability (right) of inhibitors in isolation and (B) the α-helicity (left) and thermostability (right) of inhibitors in complexes with the target mimic peptide N39 were determined by CD spectroscopy. The final concentration of the isolated inhibitors was 20 μM and of the complexed inhibitors was 10 μM in PBS. The experiments were repeated 2 times, and representative data are shown.

FIG 3

Binding ability of fusion inhibitors with the target cell membrane. The lipopeptide inhibitors LP-52, LP-80, LP-83, and LP-86 and the control inhibitors T-20, C34, and P-52 were preincubated with TZM-b1 cells, followed by thorough washes, and their sustained activities in inhibiting the infectious molecular clones HIV-1_NL4-3 (A) and HIV-1_JRCSF (B) were measured. LP-52, LP-80, LP-83, and LP-86 were used at 0.5 nM, while T-20, C34, and P-52 were used at 750, 25, and 200 nM, respectively. (C to F) The IC₅₀ values of LP-83 and LP-86, administered with or without the washing steps, on HIV-1_NL4-3 and HIV-1_JRCSF were determined. The experiments were performed 3 times, and data are expressed as means ± standard deviations (SDs).

FIG 4

Visualization of lipopeptide inhibitors bound to the target cell membrane. Different concentrations of FITC-labeled P-52 (A), LP-52 (B), LP-80 (C), and LP-83 (D) were preincubated with TZM-b1 cells for 30 min, followed by washes, and the fluorescence intensities of membrane-attached inhibitors were observed under a confocal microscope.

FIG 5

Binding ability of fusion inhibitors with the viral membrane. All the inhibitors were preincubated with the infectious virus HIV-1_NL4-3 (A) or HIV-1_JRCSF (B). After thorough washes, the antiviral activities of the virus-bound inhibitors were measured. LP-52, LP-80, LP-83, and LP-86 were used at 0.5 nM, while T-20, C34, and P-52 were used at 250, 25, and 25 nM, respectively. (C to F) The IC₅₀ values of LP-83 and LP-86, administered with or without the washing steps, on HIV-1_NL4-3 and HIV-1_JRCSF were determined. The experiments were performed 3 times, and data are expressed as means ± SDs.

FIG 6

Binding ability of lipopeptide inhibitors with human serum albumin. (A) Reactivity of diverse lipopeptide inhibitors with the mouse anti-P52 monoclonal clonal antibody 12H1 in ELISA. A lipopeptide was precoated on the ELISA plate well, and 12H1 was tested at 10 μg/ml. (B) Different concentrations of P-52, LP-52, LP-80, LP-83, or LP-94 were incubated with precoated HSA, followed by washes, and bound inhibitors were detected by 12H1 antibody and HRP-conjugated anti-mouse IgG. (C) The inhibitory activities of lipopeptides on HIV-1_NL4-3 in the presence and absence of HSA. (D) The inhibitory activities of lipopeptides on HIV-1_JRCSF in the presence and absence of HSA.

FIG 7

Ex vivo anti-HIV activities of lipopeptide fusion inhibitors. Each inhibitor (LP-52, LP-80, and LP-83) was subcutaneously injected into six rhesus monkeys at 3 mg/kg, and monkey sera were harvested at different time points before and after injection. The inhibitory activities of sera from monkeys administered LP-52 (A), LP-80 (B), and LP-83 (C) on HIV-1_NL4-3 were measured by a single-cycle infection assay, and serum dilutions required for 50% inhibition of virus infection were calculated. (D) Comparison of HIV-inhibitory activities in the monkey sera containing different inhibitors. Since the same group of monkeys was used, the data for T-20, LP-19, LP-40, LP-50, and LP-51 were obtained from previous studies (19, 25).