Discovery of Novel Inhibitors Targeting trans-Golgi Network Acidification in Pancreatic Cancer

项目摘要 胰腺导管腺癌（PDAC）预计是癌症相关性肿瘤的第二大原因。 到2030年美国的死亡人数。PDAC的5年相对存活率仅为10%，死亡率最高 在最常见的癌症中，这一比例，强调迫切需要新的治疗策略， 临床结果。近年来，人们越来越认识到代谢重编程 在赋予PDAC细胞生存优势方面发挥作用，并且代谢过程的靶向被认为是一种有效的方法。 这是开发新疗法的一个有前途的领域。与正常细胞相反，癌细胞有利于糖酵解 这种现象被称为瓦尔堡效应。作为糖酵解的副产品，转化细胞 产生过量的H+离子。这种H+的胞质积累将对细胞适应性有害; 因此，肿瘤细胞已经进化出实现pH稳态的机制。使用PDAC作为模型系统， 我们发现，癌细胞中细胞溶质的pH值是通过细胞器中H+离子的螯合作用来调节的， 这些结果表明，跨高尔基体网络（trans-Golgi network，TGN）可以作为细胞溶质H+的“汇”。 重要的是，正常细胞不采用这种稳态机制。使用NHE 7 Na+/H+反向转运蛋白， 主要定位于TGN，我们已经表明，靶向NHE 7会导致胞质溶胶酸化， PDAC细胞中随后的细胞死亡，但正常细胞中没有。我们在小鼠中进一步验证了这些发现 PDAC模型以患者为中心的数据分析表明，TGN定位转运蛋白经常 在PDAC中过表达，并且它们的表达升高与更差的总体存活相关。稳健地 为了定量TGN内腔中的pH值，我们开发了一种创新的TGN靶向pH生物传感器测定法， 已被优化用于小分子化合物文库的高通量（HT）筛选，以鉴定新的 TGN-选择性酸化抑制剂。我们已经在一组临床试验中验证了我们的TGN pH生物传感器HT测定 和批准的药物，以及激酶抑制剂。为了补充我们的HT屏幕，我们设计了一个切割- 边缘的二级和三级检测管道，以进一步验证TGN选择性化合物。我们假设 新型高尔基体特异性小分子细胞器酸化抑制剂将选择性地防止癌细胞 实现适当的pH稳态，导致酸性细胞溶质pH和细胞死亡。在这一补助金中，我们将：1）执行 大规模HT筛选，以鉴定TGN酸化抑制剂并确认命中，2）验证确认的 使用二级测定和优先化命中支架的命中，和3）将化合物效应映射到TGN组分， 选择并表征最终探头。这项工作的成功完成将提供新的小分子 经验证可干扰TGN酸化并导致癌症选择性细胞死亡的化学探针， 起点是进一步开发成安全有效的PDAC治疗剂。

PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is projected to be the second leading cause of cancer-related deaths in the United States by 2030. With a 5-year relative survival rate of just 10%, PDAC has the highest death rate among the most common cancers, underscoring the urgent need for new therapeutic strategies that improve clinical outcomes. Recent years have witnessed a growing appreciation of the role that metabolic reprogramming plays in conferring survival advantages to PDAC cells, and the targeting of metabolic processes is considered a promising area for the development of novel therapies. In contrast to normal cells, cancer cells favor glycolysis for energy production, a phenomenon called the Warburg effect. As a glycolytic byproduct, transformed cells produce an excessive amount of H+ ions. This cytosolic accumulation of H+ would be detrimental to cell fitness; therefore, tumor cells have evolved mechanisms to achieve pH homeostasis. Using PDAC as a model system, we discovered that cytosolic pH in cancer cells is regulated by organellar sequestration of H+ ions via compartmental ion transport and that the trans-Golgi network (TGN) can act as a “sink” for cytosolic H+. Importantly, normal cells do not employ this homeostatic mechanism. Using the NHE7 Na+/H+ antiporter, which is primarily localized to the TGN, we have shown that targeting NHE7 causes acidification of the cytosol and subsequent cell death in PDAC cells but not normal cells. We have further validated these findings in mouse models of PDAC. Patient-centric data analyses show that TGN localized transporters are frequently overexpressed in PDAC and their elevated expression is correlated with worse overall survival. To robustly quantify pH in the lumen of the TGN, we have developed an innovative TGN-targeted pH biosensor assay, which has been optimized for high-throughput (HT) screening of small molecule compound libraries to identify novel TGN-selective acidification inhibitors. We have validated our TGN pH biosensor HT assay in a set of clinical trial and approved drugs, as well as kinase inhibitors. To complement our HT screen, we have designed a cutting- edge secondary and tertiary assay pipeline to further validate TGN-selective compounds. We hypothesize that novel Golgi–specific small molecule inhibitors of organelle acidification will selectively prevent cancer cells from achieving proper pH homeostasis, resulting in acidic cytosolic pH and cell death. In this grant we will: 1) perform a large-scale HT screen to identify inhibitors of TGN acidification and confirm the hits, 2) validate the confirmed hits using secondary assays and prioritize hit scaffolds, and 3) map compound effects to TGN components and select and characterize final probe(s). Successful completion of this work will provide novel small molecule chemical probes validated to interfere with TGN acidification and cause cancer-selective cell death, serving as starting points to be further developed into safe and effective therapeutic agents for PDAC.