Hedgehog Palmitoylation as a Novel Target for Inhibiting Pancreatic Cancer

描述（由申请人提供）：胰腺癌是美国癌症相关死亡的第四大原因。R21项目中提出的研究目标是鉴定和开发阻断Sonic Hegehog（Shh）功能的药物，Shh是胰腺肿瘤发生的关键因素。Shh的异常表达与人胰腺癌的生物发生有关。为了正确地发出信号，Shh必须通过将16碳脂肪酸棕榈酸酯连接到其N-末端来修饰。我们的目标是利用Shh棕榈酰化作为一个潜在的阿喀琉斯之踵，通过靶向Hhat（刺猬酰基转移酶），催化棕榈酸酯附着到Shh的酶。由于棕榈酰化是Shh功能所必需的，因此阻断Shh棕榈酰化的Hhat抑制剂可以开发成在胰腺癌治疗中有效的新型化学治疗剂。我的实验室已经开发了一种依赖于Hhat活性的体外Shh棕榈酰化测定。已针对高通量筛选（HTS）优化了该测定法，以鉴定Hhat的小分子抑制剂。我们即将完成HTS（共80，000种化合物），并估计选择200-300种潜在的“命中”进行二次分析。我们现在寻求资金来确定抑制剂命中，这是最有效地阻止Shh驱动的胰腺癌细胞生长。目的1）对HTS“命中物”进行二次筛选，以鉴定新的和有效的Hhat介导的Shh棕榈酰化的小分子抑制剂。在HTS测定中产生>70%抑制和B评分>12的化合物将在二次HTS筛选中分析。然后将评估这些Hhat抑制剂抑制正常和胰腺癌细胞中Shh棕榈酰化的能力。目的2）从HTS中筛选出抑制胰腺癌细胞增殖的Hhat抑制剂。我们已经表明，shRNA介导的Hhat消耗抑制人胰腺癌细胞的锚定依赖性和非依赖性生长，并且还阻断旁分泌Shh信号传导。这些发现验证了Hhat作为确定的分子和治疗靶点的选择。目的2中的实验将评估来自HTS的Hhat抑制剂抑制人胰腺癌细胞增殖、存活和迁移的能力。将建立基于基质胶的三维培养系统，并确定Hhat抑制剂对自分泌和旁分泌Shh信号传导的影响。最有效的抑制剂将被开发成先导化合物，用于进一步的治疗开发。 公共卫生相关性：Hedgehog信号已被证明会驱动人类胰腺癌的生长。拟议的研究将帮助我们开发小分子抑制剂，阻断Hedgehog蛋白在癌细胞中的功能。这些化合物可能在临床上用作治疗人胰腺癌的抗肿瘤剂。

DESCRIPTION (provided by applicant): Pancreatic cancer is the fourth leading cause of cancer-related deaths in the US. The goal of the research proposed in this R21 project is to identify and develop drugs that block the function of Sonic Hegehog (Shh), a key contributor to pancreatic tumorigenesis. Aberrant expression of Shh is implicated in the biogenesis of human pancreatic cancer. In order to signal correctly, Shh must be modified by attachment of the 16-carbon fatty acid palmitate to its N-terminus. We aim to exploit Shh palmitoylation as a potential Achilles heel by targeting Hhat (Hedgehog acyltransferase), the enzyme that catalyzes attachment of palmitate to Shh. Since palmitoylation is required for Shh function, Hhat inhibitors that block Shh palmitoylation could be developed into novel chemotherapeutics that will be efficacious in the treatment of pancreatic cancer. My laboratory has developed an in vitro Shh palmitoylation assay that is dependent on Hhat activity. The assay has been optimized for High Throughput Screening (HTS) to identify small molecule inhibitors of Hhat. We are nearing completion of the HTS (a total of 80,000 compounds) and estimate selection of 200-300 potential "hits" for secondary analysis. We now seek funding to identify the inhibitor hits that are the most effective in blocking Shh-driven pancreatic cancer cell growth. Aim 1) To perform secondary screening of the HTS "hits" to identify novel and effective small molecule inhibitors of Hhat-mediated Shh palmitoylation. Compounds that yielded >70% inhibition and a B-score >12 in the HTS assay will be analyzed in secondary HTS screens. The ability of these Hhat inhibitors to inhibit Shh palmitoylation in normal and pancreatic cancer cells will then be assessed. Aim 2) To identify Hhat inhibitors from the HTS that inhibit proliferation of pancreatic cancer cells. We have shown that shRNA mediated depletion of Hhat inhibits anchorage- dependent and independent growth of human pancreatic cancer cells and also blocks paracrine Shh signaling. These findings validate the choice of Hhat as a defined molecular and therapeutic target. Experiments in Aim 2 will assess the ability of the Hhat inhibitors from the HTS to inhibit proliferation, survival and migration of human pancreatic cancer cells. Three-dimensional Matrigel-based culture systems will be established and the effects of Hhat inhibitors on autocrine and paracrine Shh signaling will be determined. The most effective inhibitors will be developed into lead compounds for further therapeutic development. PUBLIC HEALTH RELEVANCE: Hedgehog signaling has been shown to drive the growth of human pancreatic cancers. The proposed studies will help us develop small molecule inhibitors that block the functioning of Hedgehog proteins in cancer cells. These compounds could potentially be clinically useful as anti-tumor agents to treat human pancreatic cancer.