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 是一种由唯一趋化因子激动剂 CXCL12 激活的 G 蛋白偶联受体，其功能是在胚胎发育过程中和成人应激反应中导致细胞迁移到适当的解剖位置。 CXCR4 还参与多种癌症的生长和/或转移，是 HIV-1 的共同受体，突变的 CXCR4 会导致称为 WHIM 综合征的免疫抑制病症。疱疹病毒 8 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，因为根据其序列和结构定义，它们属于趋化因子超家族的成员，并且预计会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) 可以功能性激活 CXCR4。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 结合外，还从 160,000 个突变体文库中鉴定出两种变构肽激动剂，该突变体选自经过基因改造以表达功能性 CXCR4 的酵母菌株。本应用的目的是 (1) 使用 X 射线晶体学或 NMR 确定与 (a) CXCL12 (b) MIF 和 (c) vMIP-II 复合的 CXCR4 N 末端区域的三维结构，(2 ) 使用酿酒酵母研究 CXCR4 配体和 CXCR4 之间的相互作用位点，以及 (3) 鉴定和表征 CXCR4 激动剂和拮抗剂对已通过 MIF 催化位点高通量筛选 (HTS) 鉴定的小分子抑制剂进行动力学、晶体学和生物学表征。我们的合作者 Joshua Rubin 博士（华盛顿大学医学院）发现，CXCR4 拮抗剂 AMD3100 及其衍生物可以在小鼠模型中预防这些人类脑肿瘤的生长，但该分子及其类似物在人体给药时具有毒性。长期使用（用于抗艾滋病毒治疗）。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，并且由于cAMP增加，cAMP磷酸二酯酶抑制剂咯利普兰具有与CXCR4拮抗作用相似的治疗效果。我们已经鉴定出五种抑制 MIF 的磷酸二酯酶抑制剂（包括咯利普兰）。咯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。我们将表征 HTS 鉴定的另外 18 种 MIF 抑制剂的 Ki 和针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 中的作用或在响应 CXCR4 激活的 cAMP 磷酸二酯酶活性中的作用。这个肿瘤。这些研究不仅将启发我们对胶质母细胞瘤中CXCR4的理解，而且可以更深入地了解这种GPCR受不同配体调节的潜在机制，并为进一步研究或未来药物开发提供试剂。