High Throughput Chemical Genomic screening to identify novel ER stress-inducing

描述（由申请人提供）：尽管口腔鳞状细胞癌（OSCC）患者的手术和放疗技术取得了进步，但在过去的二十年里，生存率仍然没有提高，这表明我们治疗患者的能力已经达到了平台期。我们已经报道了蛋白酶体抑制在多种HNSCC细胞系中诱导内质网应激和C/ ebp同源蛋白（CHOP）介导的细胞凋亡，包括几种对顺铂不敏感的细胞系。内质网应激导致一种被称为未折叠蛋白反应（UPR）的生理反应，它由不同的平行遗传程序组成，这些程序试图恢复稳态蛋白质折叠或导致细胞凋亡，如果应激持续或强烈。我们假设我们可以使用高通量筛选（HTS）和大量不同的化学文库来鉴定新的小分子激活剂CHOP来诱导OSCC细胞凋亡。我们使用稳定转染的CHO-K1细胞开发了一种互补的基于细胞的检测方法，这些细胞分别报告了UPR的PERK/ elf20 /CHOP（凋亡）和IRE1/XBP1（适应性）途径。鉴定特异性只激活PERK/elF2a/CHOP而不激活IRE1/XBP1的化合物使我们能够排除许多通常有毒的化合物以及那些改变蛋白质合成和/或折叠的一般性质的化合物，例如Ca2+或氧化还原状态。已经确定了65种“击中”的化合物，这些化合物强有力地激活了CHOP，但不激活XBP1报告基因，具有可用于合成类似物和药物开发的化学特征。我们建议在一组OSCC细胞中测试这65种化合物以及市售类似物系列诱导CHOP表达和凋亡的能力。最有希望的候选人将在一个具有良好特征的啮齿动物异种移植模型中进行体内评估。拟议研究的长期目标是阐明这些新型小分子在OSCC细胞中诱导CHOP、抑制生长或诱导凋亡的能力，并通过我们的互补细胞筛选进一步筛选非常大的各种化学探针文库。通过这些研究确定的小分子UPR激动剂将为患有这种疾病的患者提供一种方便、经济的治疗手段，这种疾病的治疗方法仅限于最低限度有效的化疗、破坏性辐射或痛苦的手术，这些手术通常会干扰患者的说话、咀嚼甚至呼吸能力。公共卫生相关性：20多年来，头颈部肿瘤患者的生存率没有提高。我们筛选了一个包含66000个小分子的化学文库，以识别新的类药物化合物。拟议的研究将确定来自该屏幕的“撞击”干扰或杀死头颈部肿瘤细胞的能力。

DESCRIPTION (provided by applicant): Despite technological advances in surgery and radiotherapy for patients who suffer from oral squamous cell carcinoma (OSCC), the survival rate has remained un-improved during the last two decades indicating our ability to treat patients has reached a plateau. We have reported that proteasome inhibition induced ER stress and C/EBP-homologous protein- (CHOP) mediated apoptosis in a variety of HNSCC cell lines, including several that are not sensitive to cisplatin. ER stress leads to a physiological response known as the unfolded protein response (UPR) which consists of distinct parallel genetic programs that attempt to restore homeostatic protein folding or lead to apoptosis if the stress is prolonged or robust. We hypothesized that we could use high throughput screening (HTS) with large diverse chemical libraries to identify novel small molecule activators CHOP to induce apoptosis in OSCC cells. We have developed a complementary cellbased assay using stably transfected CHO-K1 cells that individually report on the PERK/elF2o/CHOP (apoptotic) and the IRE1/XBP1 (adaptive) pathways of the UPR. Identifying compounds that specifically activate only PERK/elF2a/CHOP and not IRE1/XBP1 has allowed us to rule out many compounds that are generally toxic as well as those that alter general properties of protein synthesis and/or folding, such as Ca2+ or redox status. 65 "hit" compounds have been identified that robustly activated the CHOP but not the XBP1 reporter with chemical signatures amenable for synthetic analoging and pharmaceutical development. We propose to test the ability of these 65 compounds along with commercially available analog series to induce CHOP expression and apoptosis in a panel of OSCC cells. The most promising candidates will be evaluated in vivo in a well-characterized rodent xenograft model. The long-term objectives of the proposed studies are to elucidate the ability of these novel small molecules to induce CHOP and inhibit growth or induce apoptosis in OSCC cells and to further screen very large diverse libraries of chemical probes with our complementary cell-based screen. Small molecule UPR agonisits identified through these studies will provide a convenient cost-effective means of treating patients suffering from a disease for which therapies are limited to minimally effective chemotherapies, damaging radiation or painful surgeries that often interfere with their ability to speak, chew or even breathe. PUBLIC HEALTH RELEVANCE: The survival rate for patients with tumors in the head and neck region has not improved in over 20 years. We screened a chemical library of 66,000 small molecules to identify novel drug-like compounds. The proposed studies will determine the ability of "hits" from this screen to interfere with the growth of or kill head and neck tumor cells.