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-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 (Eu(3+)). The labeling protocol did not significantly alter the gp120 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 gp120 in 96-well plates prior to the addition of Eu(3+)-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 identification of several aqueous natural product extracts that inhibited CV-N-gp120 binding and also had anti-HIV activity.
McMahon
J B
JB
Laboratory of Drug Discovery Research and Development, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702-1201, USA.
Beutler
J A
JA
O'Keefe
B R
BR
Goodrum
C B
CB
Myers
M A
MA
Boyd
M R
MR
D019380:Anti-HIV Agents / Q000378:metabolism*; D001426:Bacterial Proteins; D001667:Binding, Competitive; D001685:Biological Factors / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*
D019380:Anti-HIV Agents; D001426:Bacterial Proteins; D001685:Biological Factors; D002352:Carrier Proteins; D015699:HIV Envelope Protein gp120; D011994:Recombinant Proteins; C107733:cyanovirin N