Study of Structures of CCR5 and Its Interactions with CCR5 Inhibitors

CCR5的结构及其与CCR5抑制剂相互作用的研究

• 批准号: 8349332
• 负责人: Hiroaki Mitsuya
• 金额: $ 9.92万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349332
• 关键词: Acquired Immunodeficiency Syndrome; Adverse effects; Anti-Retroviral Agents; Antiviral Agents; Astrocytoma; Biochemical; Biological Assay; CCR5 gene; CD4 Positive T Lymphocytes; Cell membrane; Cells; Chemokine (C-C Motif) Receptor 5; Chinese Hamster; Clinical; Confocal Microscopy; Coupled; Data; Dilution Techniques; Disease; Extracellular Domain; Fluorescence Recovery After Photobleaching; GTP-Binding Proteins; Genes; Goals; HIV-1; Hamsters; Human; Image; Immune; Incubated; Individual; Infection; Inhibitory Concentration 50; Intervention; Lead; Life; Magnetism; Membrane; Molecular; Monoclonal Antibodies; Ovarian; Plasmids; Population; Property; Publishing; RANTES; Regimen; Reporting; Risk; Shapes; Small Inducible Cytokine A3; Sorting - Cell Movement; Structure; System; Testing; Transmembrane Domain; Tube; United States Food and Drug Administration; West Nile virus; adverse outcome; beta-Chemokines; chemokine; chemokine receptor; design; extracellular; gamma-Chemokines; inhibitor/antagonist; liver function; member; novel; receptor; scaffold

In the present project, in an attempt to determine the effects of CCR5 inhibitors on CC-chemokine-CCR5 interactions was also examined in living cells, we newly established an assay system to investigate the dynamics of cellular CCR5 and quantify the levels of CC-chemokine-induced internalization. The mobility of YFPCCR5 in the presence of CCR5 inhibitors was examined with fluorescence recovery after photobleaching (FRAP) imaging. To these two ends, we generated a plasmid carrying the CCR5-encoding gene connected to YFP at the C-terminus of CCR5 (YFPCCR5), transfected adherent human astroglioma-derived U373-MAGI (UM) cells with the plasmid, sorted out stable CCR5-expressing UM cells using magnetic sorting, and obtained clonal populations that expressed consistent levels of CCR5 on the cell membrane. With the limiting dilution technique, we obtained a rapid-growing, CCR5-expressing clone, YFPCCR5-UM16. We fist asked whether YFPCCR5 functioned as physiologically similar to wild-type CCR5 (WTCCR5) with regard to CC-chemokine-induced CCR5 internalization. To this end, YFPCCR5-UM16 cells were exposed to CCL5, the most potent CC-chemokine, incubated for 40 min under confocal microscopy, and the images of YFPCCR5-UM16 cells were digitally recorded. The data demonstrated that CC-chemokine-induced CCR5 internalization occurs relatively slowly but steadily over 40 min in contrast to previously published data that CC-chemokine-induced internalization of CCR5 occurred within 20 min as examined using CCR5-specific monoclonal antibodies in human CCR5-expressing Chinese hamster ovarian cells. The difference could be explained by that the dynamics of human CCR5 on the membrane of human cells and hamster cells could differ. It was also possible that the attachment of YFP to CCR5 could slow down the internalization of CCR5 in YFPCCR5-UM16 cells due to the bulkier size of YFPCCR5 and possible functional alterations. We also examined the effects of three CCR5 inhibitors on CC-chemokine-CCR5 interactions using the current assay system with YFPCCR5-UM16 cells. To quantify the effects of CCR5 inhibitors against CC-chemokine-induced internalization in regard to their anti-HIV-1 activity, we determined the ratios of the ED50 values for chemokine-induced internalization over the IC50 values for HIV-1 inhibition for the three inhibitors, APL, TAK779, and MVC. The ratios for APL, TAK779, and MVC with CCL5 were 16.4, 1.1, and 0.9, indicating that APL is more permissive to allow CCL5 to elicit CCR5 internalization, while the ratios for MIP-1alpha- and beta-induced internalization with all three chemokines ranged from 0.8 to 2.5. These data suggest that APL exerts its anti-HIV-1 activity preserving the CCL5-CCR5 interactions. It is noteworthy that all three CCR5 inhibitors we examined in this study have been reported to get lodged in the same hydrophobic pocket of CCR5 located within CCR5 in the proximity of the interface between the extracellular domain and the trans-membrane domain, although the shape and size of the hydrophobic cavity substantially differ due to the structural and biochemical difference of CCR5 inhibitors. Thus, the different features of inhibition by the three inhibitors are to be expected while further detailed structural molecular analyses of the CCR5 inhibitors-CCR5 interactions will elucidate how these differences are produced, which should help design more potent and effective CCR5 inhibitors. We also continued our long-standing efforts to design, synthesize, and identify novel CCR5 inhibitors that exert potent antiviral activity against R5-HIV-1. We have recently obtained more than 5 novel CCR5 inhibitors that are potently active against clinical R5-HIV-1 strains. Some of such CCR5 inhibitors have IC50 values of as low as 0.36 nM. We are now to prioritize a few representative novel CCR5 inhibitors and virologically, immunologically, biochemically, and structurally characterize such inhibitors.

在本项目中，为了确定 CCR5 抑制剂对活细胞中 CC-趋化因子-CCR5 相互作用的影响，我们新建立了一个测定系统来研究细胞 CCR5 的动态并量化 CC-趋化因子的水平-诱导内化。通过光漂白后荧光恢复 (FRAP) 成像检查 YFPCCR5 在 CCR5 抑制剂存在下的迁移率。为此，我们生成了携带CCR5编码基因并在CCR5的C端连接到YFP的质粒（YFPCCR5），用该质粒转染贴壁的人星形胶质瘤来源的U373-MAGI（UM）细胞，筛选出稳定的CCR5 -使用磁力分选表达UM细胞，并获得在细胞膜上表达一致水平的CCR5的克隆群体。通过有限稀释技术，我们获得了一个快速生长的、表达CCR5的克隆，YFPCCR5-UM16。我们首先询问在 CC 趋化因子诱导的 CCR5 内化方面，YFPCCR5 的功能是否与野生型 CCR5 (WTCCR5) 生理上相似。为此，将YFPCCR5-UM16细胞暴露于CCL5（最有效的CC趋化因子），在共聚焦显微镜下孵育40分钟，并以数字方式记录YFPCCR5-UM16细胞的图像。数据表明，CC 趋化因子诱导的 CCR5 内化在 40 分钟内相对缓慢但稳定地发生，而之前发表的数据则表明 CC 趋化因子诱导的 CCR5 内化在 20 分钟内发生，正如在人 CCR5 中使用 CCR5 特异性单克隆抗体所检查的那样。表达中国仓鼠卵巢细胞。这种差异可以解释为人类 CCR5 在人类细胞和仓鼠细胞膜上的动态可能不同。由于 YFPCCR5 体积较大且可能存在功能改变，YFP 与 CCR5 的附着也可能会减慢 YFPCCR5-UM16 细胞中 CCR5 的内化。我们还使用 YFPCCR5-UM16 细胞的当前检测系统检查了三种 CCR5 抑制剂对 CC-趋化因子-CCR5 相互作用的影响。为了量化 CCR5 抑制剂对 CC 趋化因子诱导内化的抗 HIV-1 活性的影响，我们确定了三种抑制剂趋化因子诱导内化的 ED50 值与 HIV-1 抑制的 IC50 值的比率。抑制剂、APL、TAK779 和 MVC。 APL、TAK779 和 MVC 与 CCL5 的比率分别为 16.4、1.1 和 0.9，表明 APL 更容易允许 CCL5 引发 CCR5 内化，而 MIP-1α 和 β 诱导的内化与所有三种趋化因子的比率范围为 0.8 至 2.5。这些数据表明 APL 发挥其抗 HIV-1 活性，保留了 CCL5-CCR5 相互作用。值得注意的是，据报道，我们在本研究中检查的所有三种 CCR5 抑制剂都被滞留在位于 CCR5 内胞外结构域和跨膜结构域之间的界面附近的 CCR5 的同一个疏水口袋中，尽管形状和结构不同。由于CCR5抑制剂的结构和生化差异，疏水腔的大小显着不同。因此，三种抑制剂的不同抑制特征是可以预期的，同时对 CCR5 抑制剂-CCR5 相互作用的进一步详细的结构分子分析将阐明这些差异是如何产生的，这将有助于设计更有效的 CCR5 抑制剂。我们还继续长期努力设计、合成和鉴定新型 CCR5 抑制剂，这些抑制剂对 R5-HIV-1 发挥有效的抗病毒活性。我们最近获得了超过 5 种新型 CCR5 抑制剂，它们对临床 R5-HIV-1 毒株具有有效活性。一些此类 CCR5 抑制剂的 IC50 值低至 0.36 nM。我们现在优先考虑一些有代表性的新型 CCR5 抑制剂，并从病毒学、免疫学、生化和结构上表征这些抑制剂。