Fatty acid synthase inhibitors and prostate cancer

描述（由申请人提供）：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表达。我们已经发现，奥利司他，FDA批准的药物，是一种新的抑制剂的硫酯酶（TE）结构域的FAS。奥利司他对FAS的抑制导致体外和体内肿瘤细胞的选择性杀伤。本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，内质网（ER）应激反应激活后，FAS抑制剂治疗的肿瘤细胞。ER应激反应的激活似乎是凋亡的上游，可能参与细胞死亡程序。此外，FAS抑制剂与毒胡萝卜素（已知激活ER应激的另一种药剂）的组合产生肿瘤细胞存活的协同降低。我们还确定了与奥利司他复合的TE结构域（FAS-TE）的晶体结构。奥利司他以与先前提出的底物结合模型相反的方式与活性位点结合。提出了三个具体的目标，以进一步探索这些观察，并提供一个重要的基础，设计和优化FAS抑制剂的癌症治疗。在具体目标1中，我们将使用肿瘤细胞系和具有通路特异性突变的转化小鼠胚胎成纤维细胞（MEF）来确定当FAS被抑制时PERK、IRE 1和ATF 6信号通路对ER应激反应的贡献。在具体目标2中，将确定毒胡萝卜素和缺氧增强FAS抑制剂的细胞毒性作用的能力。体内成像系统也将用于监测用FAS抑制剂处理的小鼠的肿瘤异种移植物中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射线晶体学来确定FAS-TE与奥利司他和奥利司他类似物Ebelactone B之间相互作用的结构基础。我们还将通过确定底物和产物的晶体结构并使用新的连续测定法分析结合沟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以及这些化合物如何有助于提高当前癌症药物的疗效提供宝贵的见解，特别是那些发生耐药性的药物。所提出的晶体结构将有助于奥利司他类似物的合理设计，其具有改善的靶向特异性、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.