Identification of Inhibitors that Stabilize ER Degradation Substrates

描述（由申请人提供）：新合成的内质网（ER）蛋白接受内质网质量控制机器的检查，以确保只有正确折叠的分子才能运输到最终目的地。内质网蛋白由于遗传缺陷而无法达到其天然状态，通过一个称为位错的多步骤过程从内质网输出到细胞质中，随后是蛋白酶体降解，通常称为内质网相关降解（ERAD）。遗传缺陷阻止该蛋白达到其天然构象的人类疾病包括肺部疾病（如肺气肿和囊性纤维化）、血液疾病（如蛋白C缺乏症）和神经系统疾病（如神经性ceroid lipofuscinses和Fabri病）。此外，霍乱和蓖麻毒素等毒素利用蛋白质从内质网转运到细胞质，从而进入细胞质并改变细胞代谢。引人注目的是，病毒已经进化到利用ERAD途径逃避免疫检测并在宿主体内持续存在。人巨细胞病毒（HCMV）独特的短（US）2和US11基因产物利用ERAD介导免疫蛋白主要组织相容性复合体（MHC） I类重链的破坏，以限制细胞毒性T细胞对病毒的清除。事实上，如何从哺乳动物细胞内质网中提取蛋白质最初是用表达HCMV US2-和us11的细胞来表征的，这种细胞系统继续识别关键的ERAD成分。US2和us11介导的I类降解的一个重要特征是I类降解的快速动力学（t1/2 ~5 min）。利用由增强的绿色荧光蛋白和I类重链组成的I类重链嵌合体，利用US2和US11细胞中I类重链较短的半衰期进行高通量筛选。两种被称为eeyarestatin I和II的化合物被鉴定为稳定I类重链以及其他错误折叠的蛋白质，T细胞受体1链和11-抗胰蛋白酶变体。筛选更大和更多样化的化学文库将允许发现稳定内质网降解底物的其他化合物。这些抑制剂将是对抗由错误折叠的内质网蛋白和利用ERAD途径的病原体引起的遗传疾病的有效药物。

DESCRIPTION (provided by applicant): Newly synthesized endoplasmic reticulum (ER) proteins are subjected to inspection by the ER quality control machinery to ensure that only properly folded molecules can traffic to their final destination. ER proteins that cannot reach their native state due to genetic defects are exported out of the ER and into the cytosol by a multi- step process referred to as dislocation, followed by proteasome degradation generally referred to as ER-associated degradation (ERAD). Human diseases whose genetic defects prevent the protein from reaching its native conformation include pulmonary diseases (e.g. emphysema and cystic fibrosis), blood disorders (e.g. protein C deficiency), and neurological disorders (e.g. neuronal ceroid lipofuscinoses and Fabri disease). In addition, toxins such as cholera and ricin toxin have exploited the transport of proteins from the ER to cytosol to gain access to the cytosol and alter cellular metabolism. Strikingly, viruses have evolved to utilize the ERAD pathway to evade immune detection and persist within the host. Human cytomegalovirus (HCMV) unique short (US)2 and US11 gene products have co-opted ERAD to mediate the destruction of the immunological protein major histocompatibility complex (MHC) class I heavy chain to limit viral clearance by cytotoxic T cells. In fact, how proteins are extracted from the ER in mammalian cells was initially characterized using HCMV US2- and US11-expressing cells and this cell system continues to identify key ERAD components. An important feature of US2 and US11-mediated class I destruction is the fast kinetics (t1/2 ~5 min) of class I degradation. The short half-life of class I heavy chains in US2 and US11 cells was adapted into a high-throughput screen using a class I heavy chain chimera consisting of enhanced green fluorescence protein and class I heavy chain. Two compounds referred to as eeyarestatin I and II were identified to stabilize class I heavy chains as well as other misfolded proteins, T cell receptor 1 chain and an 11-antitrypsin variant. The screening of larger and more diverse chemical libraries would allow for the discovery of additional compounds that stabilize ER degradation substrates. These inhibitors would be effective agents against genetic disorders caused by misfolded ER proteins and pathogens that utilize the ERAD pathway.