Regulation of CXCR4 by cognate and non-cognate ligands

描述（由申请人提供）：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和成人应激反应中引起细胞向适当的解剖位置迁移。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会导致一种称为WHIM综合征的免疫抑制状况。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，因为它们的序列和结构定义了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，一种非同源蛋白巨噬细胞迁移抑制蛋白（MIF）被报道可以功能性激活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。除了CXCL12、vMIP-II和MIF结合的CXCR4外，还从从酵母菌株中选择的160,000个突变体中鉴定了两种变压肽激动剂，这些突变体经过基因改造以表达功能性CXCR4。本应用程序旨在(1)利用x射线晶体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S. cerevisiae研究CXCR4和CXCR4配体之间相互作用的位点，(3)识别和表征CXCR4激动剂和拮抗剂，以及从动力学、晶体学上，并对已经通过高通量筛选（HTS）鉴定的MIF催化位点的小分子抑制剂进行生物学表征。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及其衍生物在小鼠疾病模型中阻止这些人类脑肿瘤的生长，但该分子及其类似物在长期使用（用于抗hiv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拮抗剂需要增加cAMP， cAMP磷酸二酯酶抑制剂罗利普兰与CXCR4拮抗剂由于cAMP的增加而具有相似的治疗效果。我们已经确定了五种抑制MIF的磷酸二酯酶抑制剂（包括罗利普兰）。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。我们将根据Ki和针对一组磷酸二酯酶的特异性对HTS鉴定的另外18种MIF抑制剂进行表征，并选择不抑制cAMP磷酸二酯酶的分子来研究MIF在激活CXCR4或cAMP磷酸二酯酶活性方面的作用，以响应该肿瘤中CXCR4的激活。这些研究不仅将启发我们对胶质母细胞瘤中CXCR4的认识，还将进一步深入了解该GPCR受不同配体调控的潜在机制，并为进一步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CXCR4参与多种癌症的转移和生长，是HIV-1的两个主要共受体之一，并且，当c端发生突变时，部分负责被称为WHIM综合征的免疫抑制性疾病。本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。

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.