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Antimicrobial Agents and Chemotherapy, June 2009, p. 2444-2449, Vol. 53, No. 6
0066-4804/09/$08.00+0     doi:10.1128/AAC.00150-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Stable Extended Human Immunodeficiency Virus Type 1 gp41 Coiled Coil as an Effective Target in an Assay for High-Affinity Fusion Inhibitors{triangledown}

Lifeng Cai,1,{dagger} Edina Balogh,1 and Miriam Gochin1,2*

Department of Basic Sciences, Touro University—California, Vallejo, California 94592,1 Department of Pharmaceutical Chemistry, University of California—San Francisco, San Francisco, California 941432

Received 2 February 2009/ Returned for modification 24 March 2009/ Accepted 27 March 2009

The human immunodeficiency virus type 1 (HIV-1) gp41 coiled-coil domain is an important target for fusion inhibitors, including the peptide T20, which has been approved as a drug against HIV-1. Research into nonpeptide fusion inhibitors has focused primarily on a hydrophobic pocket located within the coiled coil and has so far yielded compounds with relatively weak fusion inhibitory activity. Here, we describe metal ion-assisted stabilization of an extended 39-residue construct of gp41, which includes residues of the hydrophobic pocket and also of an extended groove N terminal to the hydrophobic pocket. We show that the presence of a metal ion and the high-affinity interaction between the receptor construct and cognate C-peptides result in a simple and highly selective assay for fusion inhibitors that may be used to scan large compound libraries. The long construct presents multiple potential binding sites along the extended coiled-coil groove. We demonstrate the modular use of assay probes to detect whether compounds bind in the hydrophobic pocket or elsewhere along the groove. Rapid detection and quantitation of hits can lead to the discovery of compounds binding to different sites along the groove and provide structure-activity relationship data for optimization. Compounds binding to adjacent sites could be linked to form more potent fusion inhibitors.

* Corresponding author. Mailing address: Basic Sciences, Touro University—California, 1310 Johnson Lane, Mare Island, Vallejo, CA 94592. Phone: (707) 638-5482. Fax: (707) 638-5255. E-mail: mgochin{at}touro.edu

{triangledown} Published ahead of print on 13 April 2009.

{dagger} Present address: Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065.

Antimicrobial Agents and Chemotherapy, June 2009, p. 2444-2449, Vol. 53, No. 6
0066-4804/09/$08.00+0     doi:10.1128/AAC.00150-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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