Liquid surface curvature variations in microplate wells were harnessed for quantitative surfactant measurement.

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 C-terminal-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 fluorophore-labeled 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 μM concentration with K _i s in the range of 0.2 μM to 30 μM, 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.

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ∼2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein - mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), Nterminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors.