Mutation of Asp¹⁷¹and Asp²⁶²of the Chemokine Receptor CXCR4 Impairs Its Coreceptor Function for Human Immunodeficiency Virus-1 Entry and Abrogates the Antagonistic Activity of AMD3100

Abstract

The bicyclam AMD3100 is a highly potent and selective CXCR4 antagonist with strong antiviral activity against human immunodeficiency virus (HIV)-1 and HIV-2, which use CXCR4 as coreceptor for host cell entry. Here, we investigated the interaction of AMD3100 with CXCR4 at the molecular level by mutational analysis. We established a set of stably transfected U87.CD4 cell lines expressing different mutant forms of CXCR4 (i.e., CXCR4[WT], CXCR4[D171N], CXCR4[D262N], CXCR4[D171N,D262N], and CXCR4[H281A]), to compare the activity of the compound against mutated versus wild-type CXCR4. We found that the antagonistic action of AMD3100 against CXCR4--as assessed by the inhibitory effects of the compound on stromal cell-derived factor (SDF-1) binding to its receptor and on SDF-1-induced intracellular calcium signaling, and by displacement of the CXCR4-specific antibody, clone 12G5--was greatly reduced by substitution of Asp(171) and/or Asp(262) by neutral asparagine residue(s). Both aspartates, but most particularly Asp(262), also proved essential for the anti-HIV-1 activity of AMD3100 against the viruses NL4.3, IIIB, and HE. In contrast, substitution of His(281) by a neutral alanine potentiated the antagonistic and antiviral effects of the compound in the different assay systems. Importantly, compared with the wild-type receptor, CXCR4[D262N] was much less effective, whereas CXCR4[D171N,D262N] completely failed as a coreceptor for infection by HIV-1 NL4.3. Thus, the negatively charged aspartate residues at positions 171 and 262, located in transmembrane domains 4 and 6 of the 7-transmembrane receptor, respectively, may represent crucial sites for electrostatic interaction of the positive charges of the bicyclams, as well as for the highly basic V3 loop of the gp120 envelope protein of certain HIV-1 strains.

Replication-Competent Variants of Human Immunodeficiency Virus Type 2 Lacking the V3 Loop Exhibit Resistance to Chemokine Receptor Antagonists

“…Although V3 shows extensive amino acid diversity across HIV and SIV phylogeny, unlike V1/V2 and V4, which can tolerate insertions and deletions (4,10,22,46), the V3 length is highly conserved and is typically 34 or 35 amino acids (8,16,18,22). This conservation is consistent with the view that a critical V3 length is required to contact the chemokine receptor (22).Small-molecule inhibitors of CCR5 and CXCR4 have been described, which bind to a pocket defined by transmembrane helices (53) or membrane-proximal regions of the ECLs (17,19,58), respectively, and inhibit viral entry. Although their mechanism of action is not fully understood, rather than block-* Corresponding author.…”

Identification of Allosteric Peptide Agonists of CXCR4

“…Mutagenesis experiments suggest that the second extracellular loop, transmembrane IV, and transmembrane VI of CXCR4 are critical for its interaction with AMD3100 (47,48). In principle, this allows novel agonists to bind to the N terminus or to other regions of the receptor that are not necessary for antagonist(s) interactions.…”

Evaluation of SDF‐1/CXCR4‐induced Ca2+ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry

“…It shows activity against a wide variety of X4 and even R5/X4 HIV strains (20,(23)(24)(25)(26). Recently, our group demonstrated that two aspartate residues at positions 171 and 262 of the chemokine receptor CXCR4 are crucial for the high-affinity binding of AMD3100 to CXCR4 (27).…”