Maeda Kenji, Das Debananda, Yin Philip D, Tsuchiya Kiyoto, Ogata-Aoki Hiromi, Nakata Hirotomo, Norman Rachael B, Hackney Lauren A, Takaoka Yoshikazu, Mitsuya Hiroaki
Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
J Mol Biol. 2008 Sep 12;381(4):956-74. doi: 10.1016/j.jmb.2008.06.041. Epub 2008 Jun 20.
C-C chemokine receptor 5 (CCR5), a member of G-protein-coupled receptors, serves as a coreceptor for human immunodeficiency virus type 1 (HIV-1). In the present study, we examined the interactions between CCR5 and novel CCR5 inhibitors containing the spirodiketopiperazine scaffolds AK530 and AK317, both of which were lodged in the hydrophobic cavity located between the upper transmembrane domain and the second extracellular loop (ECL2) of CCR5. Although substantial differences existed between the two inhibitors--AK530 had 10-fold-greater CCR5-binding affinity (K(d)=1.4 nM) than AK317 (16.7 nM)-their antiviral potencies were virtually identical (IC(50)=2.1 nM and 1.5 nM, respectively). Molecular dynamics simulations for unbound CCR5 showed hydrogen bond interactions among transmembrane residues Y108, E283, and Y251, which were crucial for HIV-1-gp120/sCD4 complex binding and HIV-1 fusion. Indeed, AK530 and AK317, when bound to CCR5, disrupted these interhelix hydrogen bond interactions, a salient molecular mechanism enabling allosteric inhibition. Mutagenesis and structural analysis showed that ECL2 consists of a part of the hydrophobic cavity for both inhibitors, although AK317 is more tightly engaged with ECL2 than AK530, explaining their similar anti-HIV-1 potencies despite the difference in K(d) values. We also found that amino acid residues in the beta-hairpin structural motif of ECL2 are critical for HIV-1-elicited fusion and binding of the spirodiketopiperazine-based inhibitors to CCR5. The direct ECL2-engaging property of the inhibitors likely produces an ECL2 conformation, which HIV-1 gp120 cannot bind to, but also prohibits HIV-1 from utilizing the "inhibitor-bound" CCR5 for cellular entry--a mechanism of HIV-1's resistance to CCR5 inhibitors. The data should not only help delineate the dynamics of CCR5 following inhibitor binding but also aid in designing CCR5 inhibitors that are more potent against HIV-1 and prevent or delay the emergence of resistant HIV-1 variants.
C-C趋化因子受体5(CCR5)是G蛋白偶联受体家族的成员之一,作为人类免疫缺陷病毒1型(HIV-1)的共受体发挥作用。在本研究中,我们检测了CCR5与含有螺二酮哌嗪支架AK530和AK317的新型CCR5抑制剂之间的相互作用,这两种抑制剂均位于CCR5上跨膜结构域与第二细胞外环(ECL2)之间的疏水腔内。尽管这两种抑制剂之间存在显著差异——AK530对CCR5的结合亲和力(K(d)=1.4 nM)比AK317(16.7 nM)高10倍——但其抗病毒效力几乎相同(IC(50)分别为2.1 nM和1.5 nM)。对未结合CCR5的分子动力学模拟显示,跨膜残基Y108、E283和Y251之间存在氢键相互作用,这些相互作用对于HIV-1-gp120/sCD4复合物结合和HIV-1融合至关重要。实际上,AK530和AK317与CCR5结合时,会破坏这些螺旋间氢键相互作用,这是一种实现变构抑制的显著分子机制。诱变和结构分析表明,ECL2构成了两种抑制剂疏水腔的一部分,尽管AK317与ECL2的结合比AK530更紧密,这解释了尽管K(d)值不同,但它们具有相似的抗HIV-1效力。我们还发现,ECL2的β-发夹结构基序中的氨基酸残基对于HIV-1引发的融合以及螺二酮哌嗪类抑制剂与CCR5的结合至关重要。抑制剂直接与ECL2结合的特性可能会产生一种HIV-1 gp120无法结合的ECL构象,但同时也会阻止HIV-1利用“与抑制剂结合的”CCR5进入细胞——这是HIV-1对CCR5抑制剂产生抗性的一种机制。这些数据不仅有助于描绘抑制剂结合后CCR5的动态变化,还将有助于设计对HIV-1更有效的CCR5抑制剂,并预防或延缓耐药HIV-1变体的出现。