Regulation of CXCR4 by cognate and non-cognate ligands

同源和非同源配体对 CXCR4 的调节

• 批准号: 7635937
• 负责人: ELIAS LOLIS
• 金额: $ 40.72万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-15 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7635937
• 关键词: AMD3100; Adult; Adverse effects; Agonist; Alleles; Anti-HIV Therapy; Binding; Binding Sites; Biological Process; Brain; Brain Neoplasms; CXCL12 gene; CXCR4 gene; Catalytic Domain; Cell Line; Cellular Assay; Chemicals; Collaborations; Complex; Crystallization; Cyclic AMP; Defect; Disease; Disease model; Embryonic Development; Future; G Protein-Coupled Receptor Genes; G-Protein-Coupled Receptors; Genomics; Glioblastoma; Goals; Growth; HIV-1; Heart; Herpesviridae; Human; Immunosuppressive Agents; In Vitro; Infection; Length; Letters; Libraries; Ligands; Location; Lymphoblastic Leukemia; Malignant Neoplasms; Mediating; Metastatic Neoplasm to the Breast; Mus; Mutate; Mutation; N-terminal; Neoplasm Metastasis; Neoplasm Transplantation; Orphan; Peptides; Phosphodiesterase Inhibitors; Physiological; Play; Positioning Attribute; Property; Proteins; Reagent; Recording of previous events; Regulation; Reporting; Role; Rolipram; Saccharomyces cerevisiae; Signal Transduction; Site; Source; Specificity; Stress; Structure; Syndrome; System; Therapeutic Effect; Universities; Washington; Work; X-Ray Crystallography; Yeasts; analog; cell motility; chemokine; chemokine receptor; design; drug development; effective therapy; extracellular; gastrointestinal; high throughput screening; in vivo; inhibitor/antagonist; insight; interest; macrophage; macrophage migration inhibitory factor receptor; medical schools; member; migration; mouse model; mutant; novel; phosphoric diester hydrolase; prevent; protein function; public health relevance; receptor; receptor binding; research study; response; small molecule; success; three dimensional structure; tumor; vMIP-II

DESCRIPTION (provided by applicant): CXCR4 is a G-protein coupled receptor activated by a sole chemokine agonist, CXCL12, and functions to cause the migration of cells to the appropriate anatomical location during embryonic development and in response to stress in adults. CXCR4 is also involved in growth and/or metastasis of a number of cancers, is a co-receptor for HIV-1, and mutant CXCR4 causes an immunosuppressive condition known as WHIM syndrome. A chemokine released by herpesvirus-8, v-MIP-II, is an antagonist of CXCR4. CXCL12 and vMIP-II are cognate molecules because there are members of the chemokine superfamily as defined by their sequence and structure and would be expected to interact with chemokine receptors. More recently, a non-cognate protein, macrophage migration inhibitory protein (MIF), was reported to functionally activate CXCR4. We verified that MIF and CXCR4 interact with each other. It is interesting to note that some of the biological functions of CXCL12 and MIF overlap raising the possibility that this may be due to activation of CXCR4. In addition to CXCR4 binding by CXCL12, vMIP-II, and MIF, two allosteric peptide agonists were identified from a library of 160,000 mutants selected from a strain of yeast that was genetically modified to express a functional CXCR4. This application aims (1) to determine the three-dimensional structures of the N-terminal region of CXCR4 complexed to (a) CXCL12 (b) MIF, and (c) vMIP-II using either X-ray crystallography or NMR, (2) to use S. cerevisiae to study the sites of interactions between the ligands of CXCR4 and CXCR4, and (3) to identify and characterize CXCR4 agonists and antagonists, as well as to kinetically, crystallographically, and biologically characterize small molecules inhibitors already identified by high throughput screening (HTS) of the catalytic site of MIF. Our collaborator, Dr. Joshua Rubin (Washington University School of Medicine) found that the CXCR4 antagonist AMD3100 and its derivatives prevent growth of these human brain tumors in a murine model of the disease, but this molecule and its analogues are toxic when administered in humans for long-term use (for anti-HIV therapy). It was also found that CXCR4 antagonism in the glioblastoma mouse model required an increase on cAMP and that the cAMP phosphodiesterase inhibitor rolipram had similar therapeutic effects as CXCR4 antagonism due to an increase in cAMP. We have identified five phosphodiesterase inhibitors, (including rolipram) that inhibit MIF. It is possible that rolipram has dual effects in inhibiting glioblastoma: inhibiting the CXCR4 agonist MIF and inhibiting the cAMP phosphodiesterase activity. We will characterize another 18 MIF inhibitors identified by HTS with respect to Ki and specificity against a panel of phosphodiesterases and choose molecules with no inhibition of cAMP phosphodiesterase for studying the role of MIF in activating CXCR4 or in cAMP phosphodiesterase activity in response to CXCR4 activation in this tumor. These studies will not only enlighten our understanding of CXCR4 in glioblastoma, but provide a greater insight into the potential mechanisms by which this GPCR is regulated by different ligands, and provide reagents for further studies or for future drug development. PUBLIC HEALTH RELEVANCE: The G-protein coupled receptor CXCR4 is involved in the metastasis and growth of several cancers, serves as one of two major co-receptors for HIV-1, and, when mutated at the C-terminus, is partly responsible for the immunosuppressive disorder known as WHIM syndrome. This application seeks to understand the mechanism by which proteins function as agonists or antagonists of CXCR4 and the effects that small molecules inhibitors of these proteins have on these functions and on CXCR4.

描述（由申请人提供）：CXCR 4是一种由唯一趋化因子激动剂CXCL 12激活的G蛋白偶联受体，其功能是在胚胎发育期间和成年人对应激的反应中引起细胞迁移到适当的解剖位置。CXCR 4还参与许多癌症的生长和/或转移，是HIV-1的共受体，并且突变的CXCR 4引起称为WHIM综合征的免疫抑制病症。由疱疹病毒-8释放的趋化因子v-MIP-II是CXCR 4的拮抗剂。CXCL 12和vMIP-II是同源分子，因为存在由其序列和结构定义的趋化因子超家族的成员，并且预期与趋化因子受体相互作用。最近，据报道，一种非同源蛋白，巨噬细胞迁移抑制蛋白（MIF），在功能上激活CXCR 4。我们验证了MIF和CXCR 4相互作用。值得注意的是，CXCL 12和MIF的一些生物学功能重叠，这可能是由于CXCR 4的激活。除了通过CXCL 12、vMIP-II和MIF结合CXCR 4之外，还从160，000个突变体的文库中鉴定了两种变构肽激动剂，所述突变体选自经遗传修饰以表达功能性CXCR 4的酵母菌株。本申请旨在（1）使用X射线晶体学或NMR确定与（a）CXCL 12（B）MIF和（c）vMIP-II复合的CXCR 4的N-末端区域的三维结构，（2）使用S.酿酒酵母中研究CXCR 4和CXCR 4的配体之间的相互作用位点，和（3）鉴定和表征CXCR 4激动剂和拮抗剂，以及从动力学、晶体学和生物学上表征已经通过MIF催化位点的高通量筛选（HTS）鉴定的小分子抑制剂。我们的合作者，约书亚鲁宾博士（华盛顿大学医学院）发现，CXCR 4拮抗剂AMD 3100及其衍生物可以防止这些人类脑肿瘤在小鼠模型中的生长，但这种分子及其类似物在人类长期使用时是有毒的（用于抗HIV治疗）。还发现成胶质细胞瘤小鼠模型中的CXCR 4拮抗作用需要cAMP的增加，并且cAMP磷酸二酯酶抑制剂咯利普兰由于cAMP的增加而具有与CXCR 4拮抗作用相似的治疗效果。我们已经确定了五个磷酸二酯酶抑制剂，（包括咯利普兰），抑制MIF。咯利普兰可能在抑制胶质母细胞瘤中具有双重作用：抑制CXCR 4激动剂MIF和抑制cAMP磷酸二酯酶活性。我们将通过HTS鉴定另外18种MIF抑制剂的Ki和对磷酸二酯酶的特异性，并选择不抑制cAMP磷酸二酯酶的分子，用于研究MIF在激活CXCR 4中的作用或在该肿瘤中响应CXCR 4激活的cAMP磷酸二酯酶活性。这些研究不仅将启发我们对胶质母细胞瘤中CXCR 4的理解，而且还将为不同配体调控这种GPCR的潜在机制提供更深入的了解，并为进一步研究或未来药物开发提供试剂。公共卫生相关性：G蛋白偶联受体CXCR 4参与多种癌症的转移和生长，作为HIV-1的两种主要共受体之一，并且当在C末端突变时，部分负责称为WHIM综合征的免疫抑制性疾病。本申请试图理解蛋白质作为CXCR 4的激动剂或拮抗剂发挥作用的机制，以及这些蛋白质的小分子抑制剂对这些功能和CXCR 4的影响。