HTP chemical genomic screens to identify positive regulators of the Unfolded Prot

描述（由申请人提供）：我们对病理机制的理解取得了巨大进展，最近揭示了分泌途径中有缺陷的蛋白质加工是许多遗传和环境疾病的一个组成部分。从糖尿病、阿尔茨海默病、帕金森病到血友病和溶酶体贮积病，各种疾病状态都以折叠缺陷或内质网（ER）运输受损为特征。非常重要的是，最近发现蛋白质合成的失调可能是多种人类癌症致病性转移的关键因素。当错误折叠的蛋白质在内质网内积聚时，细胞激活未折叠蛋白反应（UPR）来清除错误折叠的蛋白质并恢复稳态蛋白质加工。当压力延长或强烈时，UPR采用一种导致细胞死亡的遗传途径。这些拟议的研究的重点是确定和表征普遍定期审议这两个不同环节的药物样小分子调节剂。我们将研究以下假设：1)激活UPR的IRE1-XBP1（适应性）臂将促进在细胞因错误折叠或分泌不良蛋白质积累而负担的病理中延长恢复机会；2) PERK-eIF21-CHOP（凋亡）臂的特异性和稳健激活会加剧UPR并导致分泌机制过度活跃的恶性细胞死亡。在我们的初步研究中，我们成功筛选了一个6.6万个化合物文库，其中包含稳定表达荧光素酶构建体的中国仓鼠卵巢（CHO）细胞系，这些荧光素酶构建体分别报告了UPR的IRE1-XBP1或PERK-eIF21-CHOP臂。在与一个由药物、有机/合成和计算化学家组成的专家团队合作后，我们确定了一些有效调节普遍定期审议的热门话题。我们现在建议通过重复筛选具有更大化学多样性和复杂性的文库来扩展我们的探针数据库，目标是确定更特异性和更有效的upr调节探针。

DESCRIPTION (provided by applicant): Tremendous advances in our understanding of pathologic mechanisms have recently revealed that defective protein processing within the secretory pathway is an integral component of many genetic and environmental diseases. Diverse disease states ranging from diabetes, Alzheimer's disease, and Parkinson's disease, to hemophilia and lysosomal storage diseases have all been characterized by folding defects or impaired transport from the endoplasmic reticulum (ER). Very importantly, it has recently come to light that deregulation of protein synthesis is likely a key component in the pathogenic metastasis of a variety human cancers. When misfolded protein accumulates in the ER lumen, the cell activates the Unfolded Protein Response (UPR) to clear the malfolded proteins and restore homeostatic protein processing. When a stress is prolonged or robust, the UPR employs a genetic pathway that results in cell death. These proposed studies are focused to identify and characterize drug-like small molecule regulators of these two distinct arms of the UPR. We will investigate the hypotheses that: 1) Activation of the IRE1-XBP1 (adaptive) arm of the UPR will facilitate a prolonged recovery opportunity in pathologies where cells are burdened with an accumulation of mis-folded or poorly secreted proteins; and 2) Specific and robust activation of the PERK-eIF21-CHOP (apoptotic) arm will exacerbate the UPR and lead to cell death in malignant cells with hyper-active secretory machinery. In our preliminary studies, we have successfully screened a 66,000 compound library with Chinese Hamster Ovary (CHO) cell lines stably expressing luciferase constructs that report individually on the IRE1-XBP1 or PERK-eIF21-CHOP arms of the UPR. Having worked with a team of expert Medicinal, Organic/Synthetic and Computational Chemists we have identified some hits that productively modulate the UPR. We now propose to expand our probe database by repeating the screen with libraries of greater chemical diversity and complexity with the goal of identifying more specific and potent UPR-regulating probes. PUBLIC HEALTH RELEVANCE: The long-term objective of the proposed studies is to identify novel small molecules that specifically activate adaptive or cell death arms of the unfolded protein response (UPR). We will characterize: 1) the potential of the adaptive activators as therapeutic agents for treating diseases caused by aberrant protein folding and/or trafficking; and 2) the ability of the apoptosis-inducing compounds to kill head and neck cancer cells. Small molecule UPR agonists/antagonists identified through the proposed studies will provide a convenient cost-effective means of treating patients suffering from diseases for which therapies are limited to minimally effective gene therapy or extensive costly enzyme replacement therapies.