Beutler, J.A., McMahon, J.B., Johnson, T.R., O'Keefe, B.R., Buzzell, R.A., Robbins, D., Gardella, R., Wilson, J., Boyd, M.R.: J.Biomolecular Screening 7(2): 105-110, 2002.
Abstract:
The HIV-1 envelope glycoprotein gp41 is an important mediator of viral entry into host cells. Previous studies showed that the virucidal protein cyanovirin-N bound to both gp120 and gp41, and that this binding was associated with its antiviral activity. We constructed an HTS assay based on the interaction of europium-labeled cyanovirin-N with recombinant glycosylated gp41 ectodomain to support identification of small molecule mimetics of cyanovirin-N which might be developed as antiviral drug leads. Primary screening of over 107,000 natural product extracts in the assay yielded 347 confirmed hits. Secondary assays eliminated extracts which bound directly to labeled cyanovirin- N or for which the simple sugars mannose and glcNAc blocked the interaction with gp41 (lectin activity). Extracts were further prioritized based on anti-HIV activity and other biological, biochemical and chemical criteria. The distribution of source organism taxonomy of active extracts was analyzed, as was the cross-correlation of activity between the CV-N/ gp41 binding competition assay and the previously reported CV-N/gp120 binding competition assay.
A limited set of extracts was selected for bioassay-guided fractionation. Additional studies showed that the binding of CV-N to both gp120 and gp41 was mediated through polar and electrostatic interactions with specific high-mannose oligosaccharides. These studies also showed that the binding interaction between gp41 and CV-N was 2 logs weaker than that between gp120 and CV-N, and that the gp41/CV-N interaction was 1:1 as opposed to the 5:1 stoichiometry for gp120/CV-N interaction. These recent results made it clear that gp41 was a better target to develop an HTS assay for finding CV-N mimetics.
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