Drug-like small molecule modulators of the Integrated Stress Response

描述(申请人提供)：早期分泌途径中的蛋白质错误折叠通过两种不同的机制对其产生病理影响：内质网(ER)质量控制机制保留和降解错误折叠的突变蛋白质，导致功能丧失表型(例如溶酶体储存病)。错误折叠蛋白的产生和积累会导致内质网应激，导致细胞功能障碍和死亡，从而威胁细胞器的完整性，这被认为是导致神经退行性疾病、糖尿病和其他衰老疾病的原因之一。内质网中的蛋白质折叠环境由少量的信号转导通路控制，这些信号转导通路以一种刻板的方式响应内质网应激，介导未折叠蛋白反应(UPR)。最近的证据表明，UPR中的信号水平是由全球参数和ER不同蛋白质客户的平均需求决定的。因此，UPR不太可能微调到任何一种导致突变的疾病的特定紧急情况。最近有证据表明，在内质网应激条件下，敲除在UPR中起作用的正常基因可以提高存活率。这些动态平衡失稳的例子表明，UPR可以在病理生理条件下进行治疗。翻译起始因子2a的受控磷酸化和去磷酸化(ElF2a)是UPR的一个被广泛理解和潜在可塑性的臂，被称为整合应激反应(ISR)。我们建议识别像小分子这样的药物，它们将增强ISR的活性，而其他药物将降低ISR的活性。对ISR的瞬时抑制可能会压倒内质网的质量控制机制，促进酶活性突变蛋白向其功能隔间运输，从而减轻相关功能表型的损失。ISR失活也可能被证明有利于基于细胞培养的生物疗法的生产，这些生物疗法用于治疗由酶替代疗法错误折叠引起的疾病。ISR的激活剂可能会保护细胞和器官免受内质网应激的致命后果，并可能被证明在治疗衰老疾病方面有用。为了实现这些目标，我们将开发高通量筛选(HTS)，用于抑制内质网应激诱导的elF2a激酶激活的化合物，并开发二次筛选，以评估这些化合物的效力、特异性、生物利用度和非靶标效应。在一个平行的链中，我们将开发HTS分析elF2a去磷酸化抑制剂和其他elF2a激酶激活剂，这些方法通过非规范机制发挥作用，在不造成应激的情况下激活ISR。因此，这项提议的长期目标是创建一个药理学平台，用于操纵细胞对错误折叠的蛋白质的反应。鉴于蛋白质错误折叠在人类疾病中的普遍作用，这些药物很可能成为未来医生治疗武器的一部分。

DESCRIPTION (provided by applicant): Protein misfolding in the early secretory pathway exerts it pathological affects by two distinct mechanisms: Retention and degradation of misfolded mutant proteins by the endoplasmic reticulum's (ER) quality control machinery leads to loss-of-function phenotypes (exemplified by lysosomal storage diseases). Production and accumulation of the misfolded protein threatens the integrity of the organelle by causing ER stress, leading to cell dysfunction and death, which is believed to contribute to neurodegenerative disorders, diabetes mellitus and other diseases of aging. The protein folding environment in the ER is controlled by a small number of signal transduction pathways and these respond to ER stress in a stereotyped fashion, mediating the unfolded protein response (UPR). Recent evidence suggests that the level of signaling in the UPR is defined by global parameters and by the averaged needs of the ER's diverse protein clientele. It is unlikely therefore that the UPR is finely tuned to the specific exigencies of any one mutation-causing disease. Evidence for this has recently presented itself as circumstances in which knockout of normal genes that function in the UPR improved survival under conditions of ER stress. Such examples of failure of homeostasis indicate that the UPR can be manipulated therapeutically in pathophysiological conditions. Regulated phosphorylation and dephosphorylation of translation initiation factor 2a (elF2a) is a well understood and potentially malleable arm of the UPR, referred to as the integrated stress response (ISR). We propose to identify drug like small molecules that will enhance and others that will reduce the ISR's activity. Transient inhibition of the ISR might overwhelm the quality control mechanism of the ER and promote trafficking of enzymatically active mutant proteins to their functional compartment, alleviating the associated loss of function phenotypes. ISR inactivation might also prove beneficial in the cell culture based production of biotherapeutics used to treat diseases caused by misfolding by enzyme replacement therapy. Activators of the ISR are likely to protect cells and organs against the lethal consequences of ER stress and may prove useful in treating diseases of aging. To accomplish these goals we will develop high throughput screens (HTS) for compounds that inhibit the ER stress inducible elF2a kinase PERK and secondary screens to evaluate the potency, specificity, bioavailability and off-target effects of the compounds. In a parallel strand we will develop HTS assays for inhibitors of elF2a dephosphorylation and others for activators of elF2a kinases, which function by non-canonical mechanisms and activate the ISR without causing stress. The long-term goal of this proposal is therefore to create a pharmacological platform for manipulating the cellular response to misfolded proteins. Given the pervasive role of protein misfolding in human diseases, such agents are likely to become part of the therapeutic armamentarium of future physicians.