Project 2: Development of grp94-selective Inhibitors for Cancer

项目总结 背景和假设：虽然grp94在癌症中的表达与侵袭性疾病和 对于治疗的耐药性，人们对这些影响的机制知之甚少。我们无法研究grp94 遗传上易受控制的生物，如酵母和小分子grp94抑制剂的缺乏是 在很大程度上要对这种情况负责。到目前为止，对grp94的研究主要采用突变细胞系。 以及基因缺陷小鼠的研究。尽管功能强大，但这种方法是有限的，因为它解决了表型 在完全缺乏基因的情况下发生的变化，而且利用了工程细胞环境。 解决grp94在内源性细胞环境中的作用和生物学的替代策略，其中 Grp94是有限的，但不是没有的，因此是必要的。除了将grp94介导的机制链接到 相关的疾病状态，即肿瘤类型，这些试剂可能提供目前难以 可用的手段。 方法：到目前为止，发现grp94选择性配体和化学工具一直是具有挑战性的 因为HSP90类似物之间高度相似。我们在此提供初步证据 这表明发现具有对数选择性的grp94抑制剂比其他平行对数是可能的。使用 筛选内部生成的小分子文库和计算分析的组合 在项目3中进行的结构研究，我们已经确定了重要的结构决定因素 Grp94的配基选择性和高亲和力。我们建议利用这些信息作为跳板， 以grp94为导向的化学工具的发展，如grp94选择性配体和固定化固体载体， 生物素化和荧光标记的衍生物(目标1a)，并作为开发 类药物grp94抑制剂作为新的抗癌疗法转化到临床(目标1b)。我们还提议 这里使用grp94指导的工具包来研究grp94的特定疾病作用(目标2)。 意义：为了支持这一化学生物学研究方法，我们提供了初步数据。 这证实了它的力量。我们发现grp94在调节HER2酪氨酸激酶中扮演了一个新的角色。 质膜，特别是在这种蛋白过度表达的肿瘤中。我们还牵连到 Grp94在质膜上调节致癌信号转导中的作用，表明grp94是肿瘤信号转导的靶点。 HER2-过度表达乳腺癌。这些发现为grp94的发展提供了蓝图。 抑制剂作为一种新的靶向治疗依赖于信号增强的乳腺癌 通过质膜受体。重要的是，尽管在过去的二十年里，HER2是 最广泛研究的HSP90癌基因客户蛋白，机制和治疗的理解揭示 到目前为止，我们的化学生物学方法被遗漏了，这进一步证明了拥有 除了遗传和生化手段外，还有强大的化学工具包，用于理解生物学。

PROJECT SUMMARY Background and hypothesis: While grp94 expression in cancer is linked to aggressive disease and resistance to therapy, little is known on mechanisms underlying these effects. Our inability to study grp94 in genetically tractable organisms such as yeast and the unavailability of small molecule grp94 inhibitors are largely responsible for this state of affairs. Until now investigation of grp94 has mainly used mutant cell lines and gene deficient mouse studies. Although powerful, this approach is limited because it addresses phenotypic changes in the complete absence of a gene, and moreover, makes use of an engineered cellular environment. Alternative strategies that address the role and biology of grp94 in an endogenous cellular environment, where grp94 is limiting but not absent, are therefore needed. In addition to linking grp94-mediated mechanisms to the relevant disease state, i.e. tumor type, these reagents may provide evidence refractory to the currently available means. Approach: Discovery of grp94-selective ligands and chemical tools has been challenging thus far because of a high degree of analogy between the HSP90 paralogs. We here provide preliminary evidence showing that the discovery of grp94 inhibitors with log selectivity over the other paralogs is possible. Using a combination of screening an in-house generated small-molecule library and computational analyses backed by structural studies conducted with Project 3, we have identified important structural determinants that impart ligand selectivity and high affinity for grp94. We propose to use this information as a springboard for the development of grp94-directed chemical tools such as grp94 selective ligands and solid support immobilized, biotinylated and fluorescently labeled derivatives (Aim 1a) and as a starting point towards the development of drug-like grp94 inhibitors for translation to clinic as novel anti-cancer therapeutics (Aim 1b). We also propose here to use the grp94-directed toolset for the investigation of disease-specific roles of grp94 (Aim 2). Significance: In support of this investigative chemical biology approach, we provide preliminary data that confirm its power. We show that it identifies a novel role for grp94 in regulating HER2 tyrosine kinase at the plasma membrane, specifically in the case of tumors with overexpression of this protein. Also we implicate grp94 in regulating oncogenic signal transduction at the plasma membrane, indicating grp94 as a target in HER2-overexpressing breast cancer. These findings provide the blueprint for the development of grp94 inhibitors as a novel targeted therapy for the treatment of breast cancers dependent on increased signaling through plasma membrane receptors. Importantly, although HER2 was for the last two decades one of the most widely studied HSP90 onco-client protein, the mechanistic and the therapeutic understanding unveiled by our chemical biology approach was missed so far, proving further evidence to the importance of having a strong chemical toolset in addition to genetic and biochemical means for the understanding of biology.