Project 3: Structural Basis for grp94 Drug Development and Chaperone Function

项目摘要 Grp 94是一种ER HSP 90分子伴侣，其广泛的致癌作用最近才被确定。 然而，对grp 94结构和机制的理解却落后于其他HSP 90。GRP94 显示出与其它细胞HSP 90旁系同源物的高度序列和结构同源性。然而，尝试 为了证明胞质和ER旁系同源物之间的互补在两种情况下都一直失败， 方向，这表明，维持高水平的专门ER的巨大代谢负担- 特异性HSP 90具有独特的机制目的。我们建议，这种独特的机制反映在其 结构该项目的目标是了解是什么使grp 94在HSP 90家族中独一无二，并利用 这些特征在设计作为癌症治疗剂的新抑制剂中的应用。我们将在四个方面回答这个问题。 明确的目标。第一个是表征和开发下一代grp 94选择性抑制剂。连同 博士Gabriela Chiosis，项目2的负责人，我们已经表征了一组基于嘌呤（PU）的化合物， 选择性地结合GRP 94并显示抗癌活性。更高的亲和力和更高的选择性化合物， 需要提高它们的治疗潜力并用作GRP 94功能的化学探针。二是 以确定PU配体与grp 94选择性结合的结构基础。我们将研究如何 促进选择性抑制剂结合的GRP 94中的构象变化设法发生在一个部分中， 另一个问题是，什么样的特定序列和结构要素使grp 94反应与众不同？第三，我们的目标是 确定单个结构域如何有助于独特的生物化学和功能特性， grp94.与项目1的负责人李子海博士合作，我们将研究单个grp 94域如何 有助于这种蛋白质的整体专业化和功能，因为它使其客户蛋白质成熟， GARP。第四，我们将探讨翻译后修饰（PTM）对grp 94客户端结合的作用， 活动这些实验应该为开发新的抗癌疗法铺平道路 并阐明了关于GRP 94作用的中心机制问题。

PROJECT SUMMARY Grp94 is an ER HSP90 chaperone whose broad oncogenic roles have only recently been established. However, understanding of grp94 structure and mechanism has lagged behind that of other HSP90s. grp94 exhibits high sequence and structural homology to the other cellular HSP90 paralogs. Nevertheless, attempts to demonstrate complementation between the cytosolic and ER paralogs have consistently failed in both directions, suggesting that the enormous metabolic burden of maintaining high levels of a specialized ER- specific HSP90 has a distinct mechanistic purpose. We propose that this distinct mechanism is reflected in its structure. The goal of this project is to understand what makes grp94 unique in the HSP90 family and to exploit these features in the design of new inhibitors as cancer therapeutics. We will address this question in four specific aims. The first is to characterize and develop next-generation grp94-selective inhibitors. Together with Dr. Gabriela Chiosis, leader of Project 2, we have characterized a set of purine-based (PU) compounds that bind selectively to grp94 and exhibit anti-cancer activity. Higher affinity and more selective compounds are needed to improve their therapeutic potential and to serve as chemical probes of grp94 function. The second is to determine the structural basis for selective binding of PU ligands to grp94. We will investigate how the conformational changes in grp94 that foster selective inhibitor binding manage to occur in one paralog and not another – what specific sequence and structural elements make the grp94 response distinct? Third, we aim to determine how individual structural domains contribute to the unique biochemical and functional properties of grp94. In collaboration with Dr. Zihai Li, leader of Project 1, we will examine how individual grp94 domains contribute to the overall specialization and function of this paralog as it matures its client proteins such as GARP. Fourth, we will probe the role of post-translational modifications (PTMs) on grp94 client binding and activity. Together these experiments should pave the way for the development of new anti-cancer therapeutics and illuminate central mechanistic questions about grp94 action.