Mutation of Asp (171) and Asp (262) of the chemokine receptor CXCR4 impairs its coreceptor function for human immunodeficiency virus-1 entry and abrogates the antagonistic activity of AMD3100

The bicyclam AMD3100 is a highly potent and selective CXCR4 antagonist with strong antiviral activity against human immunode-ficiency 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 anal-ysis. 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 inhibi-tory 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-specifi

Mutations at the CXCR4 interaction sites for AMD3100 influence anti-CXCR4 antibody binding and HIV-1 entry

AbstractThe interaction of the CXCR4 antagonist AMD3100 with its target is greatly influenced by specific aspartate residues in the receptor protein, including Asp171 and Asp262. We have now found that aspartate-to-asparagine substitutions at these positions differentially affect the binding of four different anti-CXCR4 monoclonal antibodies as well as the infectivity of diverse human immunodeficiency virus type 1 (HIV-1) strains and clinical isolates. Mutation of Asp262 strongly decreased the coreceptor efficiency of CXCR4 for wild-type but not for AMD3100-resistant HIV-1 NL4.3. Thus, resistance of HIV-1 NL4.3 to AMD3100 is associated with a decreased dependence of the viral gp120 on Asp262 of CXCR4, pointing to a different mode of interaction of wild-type versus AMD3100-resistant virus with CXCR4

Metal ion enhanced binding of AMD3100 to Asp262 in the CXCR4 receptor

The affinity of AMD3100, a symmetrical nonpeptide antagonist composed of two 1,4,8,11-tetraazacyclotetradecane (cyclam) rings connected through a 1,4-dimethylene(phenylene) linker to the CXCR4 chemokine receptor was increased 7, 36, and 50-fold, respectively, by incorporation of the following: Cu2+, Zn2+, or Ni2+ into the cyclam rings of the compound. The rank order of the transition metal ions correlated with the calculated binding energy between free acetate and the metal ions coordinated in a cyclam ring. Construction of AMD3100 substituted with only a single Cu2+ or Ni2+ ion demonstrated that the increase in binding affinity of the metal ion substituted bicyclam is achieved through an enhanced interaction of just one of the ring systems. Mutational analysis of potential metal ion binding residues in the main ligand binding crevice of the CXCR4 receptor showed that although binding of the bicyclam is dependent on both Asp171 and Asp262, the enhancing effect of the metal ion was selectively eliminated by substitution of Asp262 located at the extracellular end of TM-VI. It is concluded that the increased binding affinity of the metal ion substituted AMD3100 is obtained through enhanced interaction of one of the cyclam ring systems with the carboxylate group of Asp262. It is suggested that this occurs through a strong concomitant interaction of one of the oxygen's directly with the metal ion and the other oxygen to one of the nitrogens of the cyclam ring through a hydrogen bond