A novel assay to identify entry inhibitors that block binding of HIV-1 gp120 to CCR5.
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.
Laboratory of Molecular Modeling and Drug Design, Lindsley F. Kimball Research Institute of The New York Blood Center, New York, NY 10021, USA.
Qian
Q
Zhao
Yuxian
Y
He
Gabriel
G
Alespeiti
Asim Kumar
AK
Debnath
D000577:Amides / Q000494:pharmacology; D000818:Animals; D019380:Anti-HIV Agents / Q000494:pharmacology; D001596:Benzylamines; D065100:CCR5 Receptor Antagonists; D015704:CD4 Antigens / Q000378:metabolism; D002460:Cell Line; D000080027:Cyclams; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods; D015699:HIV Envelope Protein gp120 / Q000378:metabolism*; D015658:HIV Infections / Q000821:virology; D015497:HIV-1 / Q000187:drug effects* / Q000472:pathogenicity; D006571:Heterocyclic Compounds / Q000494:pharmacology; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D019713:Receptors, CCR5 / Q000378:metabolism*; D019718:Receptors, CXCR4 / Q000037:antagonists & inhibitors; D014774:Virulence; D014779:Virus Replication / Q000187:drug effects
D000577:Amides; D019380:Anti-HIV Agents; D001596:Benzylamines; D065100:CCR5 Receptor Antagonists; D015704:CD4 Antigens; D000080027:Cyclams; D015699:HIV Envelope Protein gp120; D006571:Heterocyclic Compounds; D000644:Quaternary Ammonium Compounds; D019713:Receptors, CCR5; D019718:Receptors, CXCR4; C119369:TAK 779; C088327:plerixafor