The human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein gp4l is an important mediator of viral entry into host cells. Previous studies showed that the virucidal protein cyanovirin-N (CV-N) bound to both gpl20 and gp4l, and that this binding was associated with its antiviral activity. We constructed an HTS assay based on the interaction of europium-labeled CV-N with recombinant glycosylated gp4l ectodomain to support identification of small-molecule mimetics of CV-N that 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 that bound directly to labeled CV-N or for which the simple sugars mannose and N-acetylglucosamine blocked the interaction with gp4l (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-gp4l binding competition assay and the previously reported CV-N-gpl20 binding competition assay. A limited set of extracts was selected for bioassay-guided fractionation.
References
1.
Weng Y, Yang Z, Weiss CD: Structure-function studies of the self-assembly domain of the human immunodeficiency virus type 1 transmembrane protein gp41. J Virol2000;74:5368-5372.
2.
Eckert DM, Malashkevich VN, Hong LH, et al: Inhibiting HIV-1 entry: discovery of D-peptide inhibitors that target the gp4l coiled-coil pocket. Cell1999;99:103-115.
3.
D'Souza MP, Cairns JS, Plaeger SF: Current evidence and future directions for targeting HIV entry: therapeutic and prophylactic strategies. JAMA2000;284:215-222.
4.
Nagashima KA, Thompson DA, Rosenfield SI, et al: Human immunodeficiency virus type 1 entry inhibitors PRO 542 and T-20 are potently synergistic in blocking virus-cell and cell-cell fusion. J Infect Dis2001;183:1121-1125.
5.
De Clercq E: Novel compounds in preclinical/early clinical development for the treatment of HIV infections. Rev Med Virol2000;10:255-277.
6.
Blair WS, Lin PF, Meanwell NA, et al: HIV-l entry an expanding portal for drug discovery. Drug Discov Today2000;5:183-194.
7.
Zhao X, Singh M, Malashkevich VN, et al: Structural characterization of the human respiratory syncytial virus fusion protein core. Proc Natl Acad Sci U S A2000;97:14172-14177.
8.
Boyd MR, Gustafson KR, McMahon JB, et al: Discovery of cyanovirin-N, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gpl20: potential applications to microbicide development. Antimicrob Agents Chemother1997;41:1521-1530.
9.
Gustafson KR, Sowder RC II, Henderson LE, et al: Isolation, primary sequence determination, and disulfide bond structure of cyanovirin-N, an anti-HIV (human immunodeficiency virus) protein from the cyanobacterium, Nostoc ellipsosporum. Biochem Biophys Res Commun1997;238:223-228.
10.
Dey B, Lerner DL, Lusso P, et al: Multiple antiviral activities of cyanovirin-N: blocking of gpl20 interaction with CD4 and coreceptor, and inhibition of diverse enveloped viruses. J Virol2000;74:4562-4569.
11.
Esser MT, Mori T, Mondor I, et al: Cyanovirin-N binds to gp120 to interfere with CD4-dependent human immunodeficiency virus type 1 virion binding, fusion, and infectivity but does not affect the CD4 binding site on gpl20 or soluble CD4-induced conformational changes in gpl2O. J Virol1999;73:4360-4371.
12.
O'Keefe BR, Shenoy S, Muschik JM, et al: Analysis of the interaction between the virucidal protein cyanovirin-N and the HIV-1 envelope glycoproteins gpl2O and gp41. Mol Pharmacol2000;58:982-992.
13.
Bolmstedt A, O'Keefe BR, Shenoy SR, et al: Cyanovirin-N defines a new class of antiviral agent targeting N-linked, high-mannose glycans in an oligosaccharide-dependent manner. Mol Pharmacol2001;49:949-954.
14.
Shenoy SR, OKeefe BR, Bolmstedt AJ, et al: Selective interactions of the HIV-inactivating protein, cyanovirin-N, with high-mannose oligosaccharides on gpl20 and other glycoproteins. J Pharmacol Exp Ther2001;297:704-710.
15.
McMahon JB, Beutler JA, O'Keefe BR, et al: Development of a cyanovirin-N/HIV-1 gpl20 binding assay for high throughput screening of natural products by time-resolved fluorescence. J Biomol Screen2000;5:169-176.
16.
Gulakowski RJ, McMahon JB, Staley PG, et al: A semiautomated multiparameter approach for anti-HIV drug screening. J Virol Methods1991;33:87-100.
17.
Charan RD, Munro MHG, O'Keefe BR, et al: Isolation and characterization of Myrianthus holstii lectin, a potent HIV-1 inhibitory protein from the plant Myrianthus holstii. J Nat Prod2000;63:1170-1174.
18.
Van Damme EJM, Peumans WJ, Pusztai A, et al: Handbook of Plant Lectins: Properties and Biomedical Applications. Chichester, UK: John Wiley & Sons, 1998.
