Small molecular weight eNOS inhibitors for the treatment of pancreatic cancer

描述（由申请人提供）：胰腺癌是一种毁灭性的疾病，需要新的治疗方法。为此，胰腺癌的分子标志是小GTPase KRAS的激活突变。不幸的是，很难从药理学上抑制KRas，这促使我们确定KRas肿瘤发生所需的药物蛋白。为此，我们发现eNOS是一氧化氮合酶家族（由eNOS、nNOS和iNOS组成）中产生一氧化氮（NO）的三种蛋白之一，介导致癌的KRAS肿瘤发生。具体来说，eNOS被致癌Ras激活，而且，在人胰腺癌细胞系中敲低eNOS，或在小鼠中基因消融eNOS基因，大大延缓了致癌Ras驱动的肿瘤生长。最重要的是，NOS酶的小分子量抑制剂已经开发出来，并在感染性休克和心源性休克的人体临床试验中进行了测试。利用NOS抑制剂治疗其他疾病的发展，我们在最具侵袭性的胰腺癌小鼠模型中测试并发现一种这样的通用NOS抑制剂阻碍了人类胰腺癌细胞系的肿瘤生长并延长了生存期。因此，eNOS是胰腺癌的一个真正的可药物靶点。没有enos特异性的小分子量抑制剂。虽然一般的NOS抑制剂是有吸引力的探索分子，至少在最初，因为它们已经在人体中进行了测试，但它们也抑制iNOS和nNOS。两个论点表明开发enos特异性抑制剂的重要性。首先，eNOS是参与胰腺癌的NOS家族成员。其次，随着越来越多的NOS家族成员基因敲除导致更严重的表型，与专门针对eNOS的抑制剂相比，一般NOS抑制剂可能具有不良的脱靶效应。鉴于此，我们建议筛选eNOS抑制剂。我们开发了一种基于细胞的检测方法，适用于高通量分析，可区分细胞eNOS、iNOS和nNOS酶活性。我们现在建议利用这种基于细胞的实验来筛选小分子库，以确定它们比nNOS和iNOS更优先抑制细胞eNOS的能力。这些研究的完成将提供新的eNOS抑制剂，作为特异性靶向这种酶治疗胰腺癌的第一步。

DESCRIPTION (provided by applicant): Pancreatic cancer is a devastating disease in need of novel therapeutics. To this end, the molecular hallmark of pancreatic cancer is an activating mutation in the small GTPase KRAS. Unfortunately, it has been difficult to pharmacologically inhibit KRas, prompting us to identify druggable proteins required for KRas oncogenesis. To this end, we discovered eNOS, one of three proteins in the Nitric Oxide Synthase family (composed of eNOS, nNOS and iNOS) that generate nitric oxide (NO), mediates oncogenic KRAS tumorigenesis. Specifically, eNOS is activated by oncogenic Ras, and moreover, knockdown of eNOS in human pancreatic cancer cell lines, or genetic ablation of the eNOS gene in mice, greatly retarded oncogenic Ras-driven tumor growth. Most importantly, small molecular weight inhibitors of NOS enzymes have been developed and tested in human clinical trials of septic and cardiogenic shock. Capitalizing on the development of NOS inhibitors for the treatment of other diseases, we tested and found that one such general NOS inhibitor impeded tumor growth of human pancreatic cancer cell lines and extended survival in the most aggressive mouse model of pancreatic cancer. Thus, eNOS is a bona fide target for pancreatic cancer that is druggable. There are no eNOS-specific small molecular weight inhibitors. While general NOS inhibitors are attractive molecules to explore, at least initially since they have already been tested in humans, they nevertheless also inhibit iNOS and nNOS. Two arguments suggest the importance of developing eNOS-specific inhibitors. First, eNOS is the NOS family member involved in pancreatic cancer. Second, as knockout of progressively more NOS family members results in more severe phenotypes, general NOS inhibitors may have undesirable off-target effects compared to inhibitors that specifically target eNOS. Given this, we propose to screen for eNOS inhibitors. We have developed a cell-based assay suitable for high throughput analysis that differentiates between cellular eNOS, iNOS and nNOS enzyme activity. We now propose to utilize this cell-based assay to screen a library of small molecules for their ability to inhibit cellular eNOS preferentially over nNOS and iNOS. Completion of these studies will provide novel eNOS inhibitors as a first step to specifically target this enzyme in pancreatic cancers. PUBLIC HEALTH RELEVANCE: Pancreatic cancer is a devastating disease in need of novel therapeutics. In this regard, our efforts to identify small molecular weight inhibitors of eNOS, an enzyme that is required for pancreatic tumorigenesis, provides a new avenue to treat this fatal disease.