It has been 25 years since the development of the first efficient HIV-1/AIDS treatment. Scientists now know more about the HIV-1 infection life cycle, and more than 30 antiretroviral drugs have been developed, including HIV-1 fusion inhibitors. Fundamental work was begun in the early 1990s and led to the development of a novel class of anti-HIV-1 drugs, culminating in a peptide known as T20, which is currently the only HIV-1 fusion inhibitor approved by the US Food and Drug Administration. However, more work needs to be done to perfect the development of peptide and small-molecule HIV fusion inhibitors, particularly those that target gp41. Herein we present a brief overview of the development of this class of anti-HIV-1 drug by focusing on the achievements, challenges, and lessons learned. We cite hallmark studies of the past and comment on future drug development.

A metallopeptide-based fluorescence assay has been designed for the detection of small-molecule inhibitors of human immunodeficiency virus type 1 gp41, the viral protein involved in membrane fusion. The assay involves two peptides representing the inner N-terminal-heptad-repeat (HR1) coiled coil and the outer Cterminal-heptad-repeat (HR2) helical domains of the gp41 six-helix bundle which forms prior to fusion. The two peptides span a hydrophobic pocket previously defined in the literature. The HR1 peptide is modified with a metal-ligated dye complex, which maintains structural integrity and permits association with a fluorophorelabeled HR2 peptide to be followed by fluorescence quenching. Compounds able to disrupt six-helix bundle formation can act as fusion inhibitors, and we show that they can be detected in the assay from an increase in the fluorescence that is correlated with the potency of the compound. Assay optimization and validation have resulted in a simple quantitative competitive inhibition assay for fusion inhibitors that bind in the hydrophobic pocket. The assay has an assay quality factor (Z) of 0.88 and can rank order inhibitors at 10 M concentration with K i s in the range of 0.2 M to 30 M, an ideal range for drug discovery. Screening of a small peptidomimetic library has yielded three new low-molecular-weight gp41 inhibitors. In vitro syncytium inhibition assays confirmed that the compounds inhibited cell-cell fusion in the low micromolar range. These lead compounds provide a new molecular scaffold for the development of fusion inhibitors.

Development of effective vaccines to prevent influenza, particularly highly pathogenic avian influenza (HPAI) caused by influenza A virus (IAV) subtype H5N1, is a challenging goal. In this study, we designed and constructed two recombinant influenza vaccine candidates by fusing hemagglutinin 1 (HA1) fragment of A/Anhui/1/2005(H5N1) to either Fc of human IgG (HA1-Fc) or foldon plus Fc (HA1-Fdc), and evaluated their immune responses and cross-protection against divergent strains of H5N1 virus. Results showed that these two recombinant vaccines induced strong immune responses in the vaccinated mice, which specifically reacted with HA1 proteins and an inactivated heterologous H5N1 virus. Both proteins were able to cross-neutralize infections by one homologous strain (clade 2.3) and four heterologous strains belonging to clades 0, 1, and 2.2 of H5N1 pseudoviruses as well as three heterologous strains (clades 0, 1, and 2.3.4) of H5N1 live virus. Importantly, immunization with these two vaccine candidates, especially HA1-Fdc, provided complete cross-clade protection against high-dose lethal challenge of different strains of H5N1 virus covering clade 0, 1, and 2.3.4 in the tested mouse model. This study suggests that the recombinant fusion proteins, particularly HA1-Fdc, could be developed into an efficacious universal H5N1 influenza vaccine, providing cross-protection against infections by divergent strains of highly pathogenic H5N1 virus.

Human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein surface subunit gp120 and transmembrane subunit gp41 play important roles in HIV-1 entry, thus serving as key targets for the development of HIV-1 entry inhibitors. T20 peptide (enfuvirtide) is the first U.S. FDA-approved HIV entry inhibitor; however, its clinical application is limited by the lack of oral availability. Here, we have described the structure and function of the HIV-1 gp120 and gp41 subunits and reviewed advancements in the development of small-molecule HIV entry inhibitors specifically targeting these two Env glycoproteins. We then compared the advantages and disadvantages of different categories of HIV entry inhibitor candidates and further predicted the future trend of HIV entry inhibitor development.

Albuvirtide (ABT) is a 3-maleimimidopropionic acid (MPA)-modified peptide HIV fusion inhibitor that can irreversibly conjugate to serum albumin. Previous studies demonstrated its in vivo long half-life and potent anti-HIV activity. Here, we focused to characterize its biophysical properties and evaluate its antiviral spectrum. In contrast to T20 (Enfuvirtide, Fuzeon), ABT was able to form a stable α-helical conformation with the target sequence and block the fusion-active six-helix bundle (6-HB) formation in a dominant-negative manner. It efficiently inhibited HIV-1 Env-mediated cell membrane fusion and virus entry. A large panel of 42 HIV-1 pseudoviruses with different genotypes were constructed and used for the antiviral evaluation. The results showed that ABT had potent inhibitory activity against the subtypes A, B and C that predominate the worldwide AIDS epidemics, and subtype B′, CRF07_BC and CRF01_AE recombinants that are currently circulating in China. Furthermore, ABT was also highly effective against HIV-1 variants resistant to T20. Taken together, our data indicate that the chemically modified peptide ABT can serve as an ideal HIV-1 fusion inhibitor.