Fatty acid synthase inhibitors and prostate cancer

描述(申请人提供)：脂肪酸合成酶(Fas)，合成脂肪酸棕榈酸酯的酶，在前列腺癌和许多其他上皮来源的癌症中过度表达。我们发现FDA批准的药物奥利司他是一种新型的Fas硫酯酶(TE)结构域抑制剂。奥利司他对Fas的抑制导致了体内和体外对肿瘤细胞的选择性杀伤。该项目的目标是确定奥利司他和其他Fas抑制剂抗肿瘤作用的基本细胞和生化机制。为此，我们已经证明了在Fas抑制剂治疗下，肿瘤细胞的内质网应激反应被激活。内质网应激反应的激活似乎是在细胞凋亡的上游，可能参与了细胞死亡程序。此外，Fas抑制剂与另一种已知的激活内质网应激的药物thapsigargin相结合，可协同降低肿瘤细胞的存活率。我们还测定了奥利司他络合物中TE结构域(Fas-TE)的晶体结构。奥利司他与活性部位结合的方式与先前提出的底物结合模型相反。提出了三个具体的目标，以进一步探索这些观察结果，并为设计和优化用于癌症治疗的Fas抑制剂提供关键基础。在特定的目标1中，我们将使用肿瘤细胞和转化的具有通路特异性突变的小鼠胚胎成纤维细胞(MEF)来确定当Fas被抑制时，PERK、IRE1和ATF6信号通路在ER应激反应中的作用。在特定的目标2中，将确定thapsigargin和低氧增强Fas抑制剂的细胞毒作用的能力。体内成像系统还将用于监测Fas抑制剂治疗的小鼠肿瘤移植瘤中ER应激驱动的荧光素酶活性。在具体目标3中，我们将使用X射线结晶学来确定Fas-TE与奥利司他之间相互作用的结构基础，以及奥利司他的类似物Ebelacone B。我们还将通过确定底物和产物的晶体结构，并使用一种新的、连续的方法分析结合槽内保守残基的定点突变体的活性，来测试底物是否以类似的方式与奥利司他结合。这些研究将为奥利司他和其他Fas抑制剂如何启动细胞凋亡提供宝贵的见解，以及这些化合物如何有助于提高当前癌症药物的疗效，特别是那些已经发生耐药的药物。所提出的晶体结构将有助于合理设计奥利司他类似物，提高靶向性、生物利用度和药代动力学，用于治疗各种癌症。

DESCRIPTION (provided by applicant): Fatty acid synthase (FAS), the enzyme that synthesizes the fatty acid palmitate, is overexpressed in prostate cancer and many other cancers of epithelial origin. We have discovered that orlistat, an FDA approved drug, is a novel inhibitor of the thioesterase (TE) domain of FAS. Inhibition of FAS by orlistat results in the selective killing of tumor cells in vitro and in vivo. The goal of this project is to determine the basic cellular and biochemical mechanisms of the anti-tumor effects of orlistat and other FAS inhibitors. To this end we have demonstrated that the endoplasmic reticulum (ER) stress response is activated in tumor cells upon FAS inhibitor treatment. Activation of the ER stress response appears to be upstream of apoptosis, perhaps engaging the cell death program. Moreover, the combination of FAS inhibitors with thapsigargin, another agent known to activate ER stress, yields a synergistic decrease in tumor cell survival. We have also determined the crystal structure of the TE domain (FAS-TE) in complex with orlistat. Orlistat binds to the active site in a manner contrary to a previously proposed model of substrate binding. Three specific aims are proposed to further explore these observations and to provide a critical foundation for the design and optimization of FAS inhibitors for cancer therapy. In Specific Aim 1 we will use tumor cell lines and transformed mouse embryonic fibroblasts (MEFs) with pathway specific mutations to determine the contribution of the PERK, IRE1 and ATF6 signaling pathways to the ER stress response when FAS is inhibited. In Specific Aim 2 the ability of thapsigargin and hypoxia to enhance the cytotoxic effects of FAS inhibitors will be determined. An in vivo imaging system will also be used to monitor ER stress-driven luciferase activity in tumor xenografts of mice treated with FAS inhibitors. In Specific Aim 3 we will use X-ray crystallography to determine the structural basis for the interactions between FAS-TE and orlistat and the orlistat analog Ebelactone B. We will also test whether substrate binds in a similar manner to orlistat by determining the crystal structures of substrate and product and analyzing the activity of site-directed mutants of conserved residues within the binding groove using a new, continuous assay. These studies will provide invaluable insights into how orlistat and other FAS inhibitors initiate apoptosis and how these compounds may be useful to increase the efficacy of current cancer drugs, particularly those where resistance has occurred. The proposed crystal structures will be instrumental to the rational design of analogs of orlistat with improved target specificity, bioavailability, and pharmacokinetics for the treatment of a variety of cancers.