A high-throughput fluorescence polarization assay specific to the CD4 binding site of HIV-1 glycoproteins based on a fluorescein-labelled CD4 mimic

TY - JOUR

T1 - A high-throughput fluorescence polarization assay specific to the CD4 binding site of HIV-1 glycoproteins based on a fluorescein-labelled CD4 mimic

AU - Stricher, François

AU - Martin, Loïc

AU - Barthe, Philippe

AU - Pogenberg, Vivian

AU - Mechulam, Alain

AU - Menez, André

AU - Roumestand, Christian

AU - Veas, Francisco

AU - Royer, Catherine

AU - Vita, Claudio

PY - 2005/8/15

Y1 - 2005/8/15

N2 - 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.

AB - 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.

KW - CD4 Antigens/chemistry

KW - Drug Delivery Systems

KW - Fluorescence Polarization Immunoassay/methods

KW - HIV Envelope Protein gp120/chemistry

KW - HIV-1/chemistry

KW - Molecular Mimicry

KW - Mutation

KW - Protein Binding

KW - Protein Conformation

U2 - 10.1042/BJ20041953

DO - 10.1042/BJ20041953

M3 - SCORING: Journal article

C2 - 15836438

VL - 390

SP - 29

EP - 39

JO - BIOCHEM J

JF - BIOCHEM J

SN - 0264-6021

IS - Pt 1

ER -