Development of a Cyanovirin-N-HIV-1 gp120 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-hum an immunodeficiency virus (HIV) protein, to the HIV envelope glycoprotein g p120, 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 (Eu3+). The labeling protocol did not significa ntly alter the gp120 binding properties or the antiviral activity of CV-N. This report describes the assay development, validation, and results of scr eening a large library of aqueous and organic natural product extracts. The extracts were incubated with immobilized recombinant gp120 in 96-well plat es 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 Eu 3+. 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 ident ification of several aqueous natural product extracts that inhibited CV-N-g p120 binding and also had anti-HIV activity.