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.

Design of potent membrane fusion inhibitors against SARS-CoV-2, an emerging coronavirus with high fusogenic activity

“…Emerging studies demonstrate that lipid conjugation is a viable strategy to design peptide-based viral fusion inhibitors with enhanced antiviral activity and in vivo stability. The resulting lipopeptides are considered to interact preferentially with the viral and cell membranes, thus raising the local concentration of the inhibitors at the site where viral fusion occurs (20-25). According to our previous experiences, here we modified the HR2 peptide IBP01 by adding a cholesterol group to its C-terminal, resulting in a lipopeptide termed IPB02, as illustrated in Fig.…”

“…Peptides were synthesized on rink amide 4-methylbenzhydrylamine (MBHA) resin using a standard solid-phase 9-flurorenylmethoxycarbonyl (FMOC) protocol as described previously (20). Lipopeptides were produced by conjugating cholesterol succinate monoester to the C-terminal lysine residue.…”

“…However, the previously reported fusion inhibitor peptides often display low antiviral activities, with a 50% inhibitory concentration (IC 50 ) at macromolar (μM) range. In the past decade, we have dedicated our efforts to develop viral fusion inhibitors with improved pharmaceutical profiles, generating a group of lipopeptides with extremely potent antiviral activity (20-25). To fighting the COVID-19 pandemic, here we have applied our expertise to develop fusion inhibitors against SARS-CoV-2 infection.…”

A Membrane-Anchored Short-Peptide Fusion Inhibitor Fully Protects Target Cells from Infections of Human Immunodeficiency Virus Type 1 (HIV-1), HIV-2, and Simian Immunodeficiency Virus

“…GPI-2P23 efficiently blocks HIV-1 Env-mediated cell-cell fusion. We previously adapted a dual-split-protein (DSP)-based cell-cell fusion assay to evaluate the inhibitory activities of various HIV fusion inhibitors (45), in which 293FT cells expressing CCR5/ CXCR4/DSP 8 -11 were used as a target (referred to as 293FT Target ). To characterize the inhibitory activities of the membrane-anchored inhibitors against HIV Env-mediated cell-cell fusion, here we transduced 293FT Target cells with lentiviral vectors encoding GPI-2P23, GPI-C34, or GPI-4B10.…”

Assessment of in vitro bioactivities of Pis v 1 (2S albumin) and Pis v 2.0101 (11S globulin) proteins derived from pistachio (Pistacia vera L.)

“…Our data showed that both allergens, markedly affected CV-B2-5 viability. Viral membrane fusion inhibitory activity of these proteins may be related to their lipopeptide-based structure [37]. It was indicated that tryptophan-rich, C8 (Ac-Trp-Glu-Asp-Trp-Val-Gly-Trp-Ile-NH2) plays a major role against the membrane-proximal external region of the feline immune deficiency virus (FIV) [8].…”