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A novel assay to identify entry inhibitors that block binding of HIV-1 gp120 to CCR5
Authors:Zhao Qian  He Yuxian  Alespeiti Gabriel  Debnath Asim Kumar
Affiliation:a Laboratory of Molecular Modeling and Drug Design, Lindsley F. Kimball Research Institute of The New York Blood Center, New York, NY 10021, USA
b Laboratory of Viral Immunology, Lindsley F. Kimball Research Institute of The New York Blood Center, New York, NY 10021, USA
c Laboratory of Flow Cytometry, Lindsley F. Kimball Research Institute of The New York Blood Center, New York, NY 10021, USA
Abstract:HIV-1 infection is initiated by the interaction of the envelope glycoprotein gp120 with the cellular receptor CD4 that triggers conformational changes in gp120 necessary for subsequent interaction with a coreceptor CCR5 (or CXCR4). The CD4-induced (CD4i) conformation of gp120 can be mimicked by a full-length single chain (FLSC) protein consisting of gp120 linked with the D1D2 domains of CD4 by a 20-amino-acid linker. We have used this protein to establish a flow cytometry-based assay and an ELISA-based assay to identify inhibitors that block the binding of gp120 to CCR5. Both assays are specific for detecting the known CCR5 antagonist TAK-779, but the ELISA-based assay was more sensitive, simple, inexpensive, and rapid; thus, it can be adapted to high throughput screening (HTS). The ELISA-based method was validated with a diverse set of known antagonists, for example, TAK-779, AOP-RANTES, PSC-RANTES, and several mAbs.
Keywords:HIV   Envelope glycoprotein   Coreceptor   CCR5   Fusion   Entry inhibitor   Flow cytometry   Cell-based assay   ELISA
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