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Development of a Cyanovirin-N-HIV-1 gpl20 Binding Assay for High Throughput Screening of Natural Product Extracts by Time-Resolved Fluorescence

The unique, high-affinity binding of cyanovirin-N (CV-N), a potent anti-human immunodeficiency virus (HIV) protein, to the HIV envelope glycoprotein gp120, was exploited to develop an HTS assay in an attempt to discover small-molecule mimetics of CV-N. A competition binding assay was developed using CV-N labeled with europium (Eu31). The labeling protocol did not significantly alter the gpl20 binding properties or the antiviral activity of CV-N. This report describes the assay development, validation, and results of screening a large library of aqueous and organic natural product extracts. The extracts were incubated with immobilized recombinant gpl20 in 96-well plates prior to the addition of Eu3+-labeled CV-N. Following a wash step, bound CV-N was measured by dissociation-enhanced time-resolved fluorometry of Eu3+. The assay proved to be robust, rapid, and reproducible, and was used to screen over 50,000 natural product extracts, and has resulted in the identification of several aqueous natural product extracts that inhibited CV-N-gp120 binding and also had anti-HIV activity.