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
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
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DOI：
10.1124/mol.60.1.164
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发表时间：
2001-07-01
影响因子：
3.6
通讯作者：
Schols, D
中科院分区：
文献类型：
--
作者：
Hatse, S;Princen, K;Schols, D
双环胺AMD 3100是一种高效和选择性的CXCR 4拮抗剂，对人类免疫缺陷病毒（HIV）-1和HIV-2具有很强的抗病毒活性，HIV-1和HIV-2使用CXCR 4作为辅助受体进入宿主细胞。在此，我们通过突变分析在分子水平上研究了AMD 3100与CXCR 4的相互作用。我们建立了一组稳定转染的U87.CD4细胞系，表达不同突变形式的CXCR 4（即，CXCR 4 [WT]、CXCR 4 [D171 N]、CXCR 4 [D262 N]、CXCR 4 [D171 N、D262 N]和CXCR 4 [H281 A]），以比较化合物针对突变型与野生型CXCR 4的活性。我们发现，通过化合物对基质细胞衍生因子（SDF-1）与其受体结合的抑制作用和对SDF-1诱导的细胞内钙信号传导的抑制作用以及通过置换CXCR 4特异性抗体（克隆12 G5）评估的AMD 3100对CXCR 4的拮抗作用，通过用中性天冬酰胺残基置换Asp（171）和/或Asp（262）大大降低。两种天冬氨酸盐，尤其是天冬氨酸（262），也被证明是AMD 3100抗HIV-1病毒NL4.3、IIIB和HE活性所必需的。相反，用中性丙氨酸取代His（281）增强了化合物在不同测定系统中的拮抗和抗病毒作用。重要的是，与野生型受体相比，CXCR 4 [D262 N]的有效性要低得多，而CXCR 4 [D171 N，D262 N]作为HIV-1 NL4.3感染的辅助受体完全失败。因此，分别位于7-跨膜受体的跨膜结构域4和6中的位置171和262处的带负电荷的天冬氨酸残基可能代表双环胺的正电荷的静电相互作用以及某些HIV-1毒株的gp 120包膜蛋白的高度碱性V3环的关键位点。
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.