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Research ArticleArticle

Mutation of Asp171 and Asp262 of the Chemokine Receptor CXCR4 Impairs Its Coreceptor Function for Human Immunodeficiency Virus-1 Entry and Abrogates the Antagonistic Activity of AMD3100

Sigrid Hatse, Katrien Princen, Lars-Ole Gerlach, Gary Bridger, Geoffrey Henson, Erik De Clercq, Thue W. Schwartz and Dominique Schols
Molecular Pharmacology July 2001, 60 (1) 164-173; DOI: https://doi.org/10.1124/mol.60.1.164
Sigrid Hatse
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Katrien Princen
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Lars-Ole Gerlach
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Gary Bridger
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Geoffrey Henson
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Erik De Clercq
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Thue W. Schwartz
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Dominique Schols
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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 Asp171 and/or Asp262 by neutral asparagine residue(s). Both aspartates, but most particularly Asp262, also proved essential for the anti-HIV-1 activity of AMD3100 against the viruses NL4.3, IIIB, and HE. In contrast, substitution of His281 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.

  • Abbreviations

    HIV
    human immunodeficiency virus
    SDF-1
    stromal cell-derived factor-1
    PBS
    phosphate-buffered saline
    FBS
    fetal bovine serum
    PE
    phycoerythrin
    mAb
    monoclonal antibody
    RT-PCR
    reverse transcription polymerase chain reaction
    LTR
    long terminal repeat
    TM
    transmembrane domain
    Ag
    antigen
    GAPDH
    glyceraldehyde 3-phosphate dehydrogenase
    WT
    wild-type
    • Received December 19, 2000.
    • Accepted March 29, 2000.
    • The American Society for Pharmacology and Experimental Therapeutics
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    Molecular Pharmacology: 60 (1)
    Molecular Pharmacology
    Vol. 60, Issue 1
    1 Jul 2001
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    Research ArticleArticle

    Mutation of Asp171 and Asp262 of the Chemokine Receptor CXCR4 Impairs Its Coreceptor Function for Human Immunodeficiency Virus-1 Entry and Abrogates the Antagonistic Activity of AMD3100

    Sigrid Hatse, Katrien Princen, Lars-Ole Gerlach, Gary Bridger, Geoffrey Henson, Erik De Clercq, Thue W. Schwartz and Dominique Schols
    Molecular Pharmacology July 1, 2001, 60 (1) 164-173; DOI: https://doi.org/10.1124/mol.60.1.164

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    Research ArticleArticle

    Mutation of Asp171 and Asp262 of the Chemokine Receptor CXCR4 Impairs Its Coreceptor Function for Human Immunodeficiency Virus-1 Entry and Abrogates the Antagonistic Activity of AMD3100

    Sigrid Hatse, Katrien Princen, Lars-Ole Gerlach, Gary Bridger, Geoffrey Henson, Erik De Clercq, Thue W. Schwartz and Dominique Schols
    Molecular Pharmacology July 1, 2001, 60 (1) 164-173; DOI: https://doi.org/10.1124/mol.60.1.164
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