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CC-Chemokine-CCR5相互作用的影响，我们在活细胞中进行了研究，我们新建立了一个测定系统，以研究细胞CCR5的动力学，并量化CC-Chemokine诱导的内在化水平。在CCR5抑制剂存在下YFPCCR5的迁移率在光漂白（FRAP）成像后通过荧光恢复检查。在这两个末端，我们产生了一个含有CCR5编码基因的质粒，该基因在CCR5（YFPCCR5）的C末端连接，转染了粘附的人星形胶质瘤衍生的U373-MAGI（UM）细胞，并使用质粒的稳定的CCR5级别的平台，并获得了稳定的CCR5级别，并获得了稳定的ccr5级别的平衡，并获得了稳定的ccr5级别，并获得了稳定的ccr5级别，并获得了稳定的ccr5级别。细胞膜上的CCR5。通过限制稀释技术，我们获得了快速增长的表达CCR5克隆YFPCCR5-UM16。我们拳头询问YFPCCR5是否在生理上与野生型CCR5（WTCCR5）相似，就CC-emokine诱导的CCR5内在化而言。为此，将YFPCCR5-UM16细胞暴露于CCL5，这是最有效的CC-femokine，在共聚焦显微镜下孵育40分钟，并记录了YFPCCR5-UM16细胞的图像。数据表明，与先前发表的数据相比，CC化学因子诱导的CCR5内在化发生了相对缓慢但稳定地发生的，即CC化学因子诱导的CCR5的内在化发生在20分钟内发生，如使用CCR5特异性的单克抗体所检查的，在人类CCR5表达的中汉斯特汉斯特汉斯特汉斯特·汉斯特杂型细胞中。差异可以通过人类CCR5在人类细胞和仓鼠细胞膜上的动力学可能有所不同来解释。由于YFPCCR5的较大尺寸和可能的功能变化，因此YFP对CCR5的附着可能会减慢YFPCCR5-UM16细胞中CCR5的内在化。我们还使用当前的测定系统与YFPCCR5-UM16细胞一起研究了三种CCR5抑制剂对CC-emokine-CCR5相互作用的影响。为了量化CCR5抑制剂对CC变异成分诱导的抗HIV-1活性的内在化的影响，我们确定了趋化因子诱导的内在化的ED50值比HIV-1抑制的IC50值比三个抑制剂APL，APL，Take779和MVC的HIV-1抑制作用的比率。 APL，TAK779和MVC（CCL5）的比率为16.4、1.1和0.9，表明APL更允许CCL5引起CCR5内在化，而MIP-1Alpha-和Beta诱导的内在化的比率为0.8至2.5。这些数据表明APL发挥其抗HIV-1活性，以保留CCL5-CCR5相互作用。值得注意的是，据报道，我们在这项研究中检查的所有三种CCR5抑制剂都将被置于CCR5内CCR5的相同疏水口袋中，尽管在结构和生物Cr的结构差异上，但由于疏水性腔体的形状和大小差异，但在细胞外结构域和跨膜型领域之间的界面近端有所不同。因此，可以预期这三种抑制剂的不同特征，而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如何产生这些差异，这应该有助于设计更有效且有效的CCR5抑制剂。我们还继续进行了长期的努力，以设计，合成和确定对R5-HIV-1发挥有效抗病毒活性的新型CCR5抑制剂。最近，我们获得了5种以上新型的CCR5抑制剂，这些抑制剂对临床R5-HIV-1菌株有效活跃。一些此类CCR5抑制剂的IC50值低至0.36 nm。现在，我们要优先考虑一些具有代表性的新型CCR5抑制剂，并在病毒学上，免疫学，生化和结构上表征这种抑制剂。