Regulation of CXCR4 by cognate and non-cognate ligands

同源和非同源配体对 CXCR4 的调节

基本信息

批准号：
8416439
负责人：
ELIAS LOLIS
金额：
$ 38.12万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
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/8416439
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

项目摘要

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.

描述（由申请人提供）：CXCR4是由唯一的趋化因子激动剂，CXCL12激活的G蛋白偶联受体，并且功能使细胞在胚胎发育过程中迁移到适当的解剖位置，并响应成人的压力。 CXCR4还参与了许多癌症的生长和/或转移，是HIV-1的共受体，突变体CXCR4会导致免疫抑制疾病，称为WHIM综合征。疱疹病毒8（V-MIP-II）释放的趋化因子是CXCR4的拮抗剂。 CXCL12和VMIP-II是同源分子，因为趋化因子超家族的成员由其序列和结构定义，并且有望与趋化因子受体相互作用。最近，据报道，非认知蛋白巨噬细胞抑制蛋白（MIF）在功能上激活CXCR4。我们验证了MIF和CXCR4相互作用。有趣的是，CXCL12和MIF的某些生物学功能与CXCR4的激活可能引起的可能性有关。除了CXCL12，VMIP-II和MIF的CXCR4结合外，还从从160,000个突变体的文库中鉴定出了两种变构肽激动剂，从酵母菌株中选择，这些酵母是经过遗传修饰以表达功能性CXCR4的。该应用旨在（1）确定CXCR4的N末端区域的三维结构，该结构使用X射线晶体学或NMR或NMR（2）使用S. cerevisiae来研究CREVisiae的相互作用（cxcl12（b）Mif和（c）VMIP-II，以研究CCR4和CXCR4和Cx4的相互作用（2） CXCR4激动剂和拮抗剂，以及在动力学，结晶和生物学上表征已经通过MIF催化位点的高吞吐量筛选（HTS）鉴定的小分子抑制剂。我们的合作者约书亚·鲁宾（Joshua Rubin）（华盛顿大学医学院）发现，CXCR4拮抗剂AMD3100及其衍生物在该疾病的鼠模型中阻止了这些人脑肿瘤的生长，但该分子及其类似物在人类长期使用时（用于抗HIV治疗）时具有毒性。还发现，胶质母细胞瘤小鼠模型中的CXCR4拮抗作用需要增加cAMP，并且营地磷酸二酯酶抑制剂rolipram具有与CXCR4拮抗作用相似的治疗作用，这是由于CXCR4拮抗作用而引起的。我们已经确定了五种抑制MIF的磷酸二酯酶抑制剂（包括Rolipram）。 rolipram可能在抑制胶质母细胞瘤中具有双重影响：抑制CXCR4激动剂MIF并抑制CAMP磷酸二酯酶活性。我们将表征HTS在Ki方面鉴定出的另外18个MIF抑制剂，以及针对磷酸二酯酶的特异性，并选择分子，而无需抑制CAMP磷酸二酯酶在研究CXCR4激活CXCR4或CAMP磷酸二二酯酶在此thumor中激活CXCR4或CAMP磷酸二酯酶活性中的作用。这些研究不仅会启发我们对胶质母细胞瘤中CXCR4的理解，而且还可以更深入地了解该GPCR受不同配体调节的潜在机制，并为进一步的研究或未来的药物开发提供试剂。