Developing HTS assays for the functions of the chaperone GRP94

描述（由申请人提供）：GRP94是HSP90家族的ER伴侣，因为它控制分泌蛋白和膜蛋白的折叠而至关重要。这种伴侣蛋白的一个独特之处在于它的选择性：GRP94只控制少数底物蛋白的表达，这使它成为一个很好的药物靶标。尽管如此，这些底物在许多细胞-细胞相互作用中起着关键作用。其中一种底物是igf - 1，它是一种有丝分裂激素，其信号传导决定许多组织的生长和分化结果。它也在许多肿瘤细胞中以自分泌方式发出生长/死亡决定的信号。我们发现，在GRP94缺陷细胞中，IGF-I的加工和分泌受到抑制，表现出对GRP94活性的严格依赖。我们利用这一发现，开发了一种细胞活力取决于IGF-I分泌的实验，因此取决于活性GRP94的可用性。这是第一个基于哺乳动物细胞的GRP94功能测定，实际上是任何ER伴侣。这将有助于确定GRP94的不同功能域和活性位点，以及它的相互作用蛋白。由于其潜力，我们建议为该检测开发一种高通量格式，以使其适合筛选小分子文库。目标是找到选择性抑制GRP94的探针，而不影响同源蛋白HSP90，而不是目前可用的泛HSP90探针。为了补充这种基于细胞的分析，我们将转换为高通量的三种基于蛋白质的分析，测量GRP94的不同活性-由n端结构域介导的肽结合，由第二个酸性结构域介导的钙结合，以及从活性构象到非活性构象的转换，这报告了n端和中间结构域的动态。这将使我们能够将任何探针在基于细胞的测定中的活性与体外活性联系起来，因此我们不仅可以验证探针的特异性，还可以获得其作用机制的知识。与任何伴侣一样，GRP94的作用周期涉及由多个结构域介导的动态分子内和分子间相互作用。使用功能分析的高通量分子文库筛选是获得定义这些多重相互作用的探针的最佳方法，这是理解GRP94为何是一种重要的哺乳动物蛋白的重要一步。GRP94是一种重要的分子伴侣，控制特定蛋白质的分泌，包括对正常发育和许多肿瘤进展很重要的生长因子。本提案的总体目标是开发可用于筛选小分子文库的检测方法，以发现GRP94特异性药物。如果成功，这些研究将对开发基于调节GRP94功能的新治疗方法有价值。

DESCRIPTION (provided by applicant): GRP94, an ER chaperone of the HSP90 family, is essential because it controls the folding of secreted and membrane proteins. One unique feature of this chaperone, which makes it a good drug target, is its selectivity: GRP94 controls the expression of only few substrate proteins. Nonetheless, these substrates play critical roles in a number of cell-cell interactions. One such substrate is IGF-I, a mitogenic hormone whose signaling determines growth and differentiation outcomes in many tissues. It also signals in autocrine fashion growth/death decisions in many tumor cells. We discovered that processing and secretion of IGF-I are inhibited in GRP94-deficient cells, showing strict dependence on the activity of GRP94. We took advantage of this finding and developed an assay in which cell viability is conditional on IGF-I secretion and therefore on the availability of active GRP94. This is the first mammalian cell-based assay for the function of GRP94, and indeed any ER chaperone. It will help define distinct functional domains and active sites of GRP94, as well as its interacting proteins. Because of its potential, we propose to develop a high throughput format for this assay, in order to make it suitable for screening small molecule libraries. The goal will be to find probes that inhibit GRP94 selectively, without affecting the homologous protein HSP90, rather than the pan-HSP90 probes that are currently available. To complement this cell-based assay, we will convert to a high-throughput format three protein-based assays that measure distinct activities of GRP94 - peptide binding that is mediated by the N-terminal domain, calcium binding that is mediated by the second, acidic domain, and conversion from an active to inactive conformation, which reports on the dynamics of both the N-terminal and middle domains. This will allow us to relate the activity of any probe in the cell-based assay to the in vitro activities, and thereby we will not only verify the specificity of the probe, but also gain knowledge of its mechanism of action. Like any chaperone, the action cycle of GRP94 involves dynamic intra- and inter-molecular interactions mediated by multiple domains. A high throughput screen of molecular libraries using functional assays is the best approach to obtain probes defining these multiple interactions, an important step towards understanding why GRP94 is an essential mammalian protein. GRP94 is an essential molecular chaperone that controls the secretion of specific proteins, including growth factors that are important for normal development as well as for the progression of many tumors. The overall goal of this proposal is to develop assays can be used to screen small molecule libraries to discover GRP94 specific drugs. If successful, these studies will be valuable for the development of novel therapeutic approaches based on modulating GRP94 function.