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: 8014942
• 负责人: ELIAS LOLIS
• 金额: $ 39.91万
• 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/8014942
• 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; Health; 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; 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(申请人提供)：CXCR4是一种G蛋白偶联受体，由唯一的趋化因子激动剂CXCL12激活，其功能是在胚胎发育期间导致细胞迁移到适当的解剖位置，并对成年患者的压力做出反应。CXCR4还参与了许多癌症的生长和/或转移，是HIV-1的共同受体，突变的CXCR4会导致一种称为突发综合征的免疫抑制情况。疱疹病毒8型释放的趋化因子v-MIP-II是CXCR4的拮抗剂。CXCL12和vMIP-II是同源分子，因为它们的序列和结构定义了趋化因子超家族的成员，并有望与趋化因子受体相互作用。最近，一种非同源蛋白--巨噬细胞移动抑制蛋白(MIF)被报道具有激活CXCR4的功能。我们验证了MIF和CXCR4相互作用。有趣的是，CXCL12和MIF的一些生物学功能重叠，这可能是由于CXCR4的激活所致。除了CXCL12、vMIP-II和MIF与CXCR4的结合外，还从一个16万个突变的文库中鉴定出两种变构多肽激动剂，该文库是从一株经过转基因表达CXCR4的酵母菌株中挑选出来的。本申请的目的是(1)利用X射线结晶学或核磁共振技术确定CXCR4与(A)CXCL12(B)MIF和(C)vMIP-II络合的N端区域的三维结构，(2)利用酿酒酵母研究CXCR4和CXCR4配体之间的相互作用部位，以及(3)鉴定和表征CXCR4激动剂和拮抗剂，以及对已经通过高通量筛选(HTS)MIF催化部位鉴定的小分子抑制剂进行动力学、结晶学和生物学表征。我们的合作者Joshua Rubin博士(华盛顿大学医学院)发现，在这种疾病的小鼠模型中，CXCR4拮抗剂AMD3100及其衍生物可以防止这些人类脑瘤的生长，但这种分子及其类似物在人类长期使用(用于抗艾滋病毒治疗)时是有毒的。研究还发现，在胶质母细胞瘤小鼠模型中，CXCR4的拮抗作用需要增加cAMP，而cAMP磷酸二酯酶抑制剂罗利普兰与由于cAMP增加而产生的CXCR4拮抗作用具有相似的治疗效果。我们已经确定了五种磷酸二酯酶抑制剂(包括罗利普兰)，它们可以抑制MIF。罗利普兰可能具有抑制胶质母细胞瘤的双重作用：抑制CXCR4激动剂MIF和抑制cAMP磷酸二酯酶活性。我们将鉴定HTS鉴定的另外18种MIF抑制剂的KI和对一组磷酸二酯酶的特异性，并选择不抑制cAMP磷酸二酯酶的分子来研究MIF在激活CXCR4或响应CXCR4激活的cAMP磷酸二酯酶活性中的作用。这些研究不仅将启发我们对CXCR4在胶质母细胞瘤中的理解，而且将为进一步研究或未来的药物开发提供更深入的了解，并为进一步的研究或未来的药物开发提供试剂。公共卫生相关性：G蛋白偶联受体CXCR4参与了几种癌症的转移和生长，是HIV-1的两个主要辅助受体之一，当C末端突变时，是被称为突发综合征的免疫抑制疾病的部分原因。这项应用旨在了解蛋白质作为CXCR4激动剂或拮抗剂的作用机制，以及这些蛋白质的小分子抑制剂对这些功能和CXCR4的影响。