A high-throughput fluorescence polarization assay specific to the CD4 binding site of HIV-1 glycoproteins based on a fluorescein-labelled CD4 mimic
Résumé
The three-dimensional structure of CD4M33, a mimic of the host-cell receptor-antigen CD4 and a powerful inhibitor of CD4–gp120 (viral envelope glycoprotein 120) interaction and HIV-1 entry into cells [Martin, Stricher, Misse, Sironi, Pugniere, Barthe, Prado-Gotor, Freulon, Magne, Roumestand et al. (2003) Nat. Biotechnol. 21, 71–76], was solved by 1H-NMR and its structure was modelled in its complex with gp120. In this complex, CD4M33 binds in a CD4-like mode and inserts its unnatural and prominent Bip23 (biphenylalanine-23) side-chain into the gp120 interior ‘Phe43 cavity’, thus filling its volume. CD4M33 was specifically labelled with fluorescein and shown by fluorescence anisotropy to bind to different gp120 glycoproteins with dissociation constants in the nanomolar range. Fluorescent CD4M33 was also used in a miniaturized 384-well-plate assay to study direct binding to a large panel of gp120 glycoproteins and in a competition assay to study binding of CD4 or other ligands targeting the CD4 binding site of gp120. Furthermore, by using the fluorescently labelled CD4M33 and the [Phe23]M33 mutant, which possesses a natural Phe23 residue and thus cannot penetrate the gp120 Phe43 cavity, we show that a recently discovered small-molecule-entry inhibitor, BMS-378806, does not target the CD4 binding site nor the Phe43 cavity of gp120. The fluorescently labelled CD4M33 mimic, its mutants and their derivatives represent useful tools with which to discover new molecules which target the CD4 binding site and/or the Phe43 cavity of gp120 glycoproteins in a high-throughput fluorescence-polarization assay and to characterize their mechanism of action.