Kinetic Characterization of Aspartate/Asparagine-beta-Hydroxylase and Synthesis of Small Molecule Aspartate/Asparagine-beta-Hydroxylase-Inhibitors as Potential Anti-Cancer Agents

迫切需要用于治疗人类癌症的有效疗法;重要的是鉴定允许区分健康和恶性细胞的特征以开发选择性抗癌药物：已经报道，2-酮戊二酸依赖性加氧酶天冬氨酸/天冬酰胺基-β-羟化酶（AspH）在各种癌细胞的表面上过表达，并且与它们的细胞运动性的增强相关;然而，人们对AspH在正常细胞和癌细胞中的功能知之甚少，迄今为止还没有描述过选择性小分子AspH抑制剂。拟议的研究实验旨在：（a）进行AspH及其一些（共）底物的动力学表征，（B）确定底物EGFD中所需的二硫键连接模式和（c）提供新的AspH抑制剂。将通过稳态（LCMS）和停止流动/流动淬灭（UV-Vis，LCMS）实验来研究氧（包括O2）。特别是，EGF结构域的优选二硫键模式对AspH催化和动力学的影响将被确定：AspH底物的稳定合成类似物将作为探针来研究所需的二硫键模式。这些稳定的类似物的合成将涉及固相肽合成和烯烃复分解反应，借助动态组合化学和非变性质谱，将鉴定小的有机分子AspH-配体。有前途的配体的稳定衍生物将通过有机合成获得。将测定（稳定的）AspH配体的抑制浓度，随后将评价其作为抗癌治疗剂的潜力。抑制剂将用于确认AspH如何促进细胞运动性的拟定机制：在用抑制剂处理细胞之前和之后，将分析Notch、其配体和下游靶标的表达水平（qRT-PCR、蛋白质印迹法）。

Effective therapies for the treatment of human cancer are urgently needed; it is important to identify characteristic features that allow to differentiate between healthy and malign cells to develop selective anti-cancer drugs: It has been reported, that the 2-oxoglutarate-dependent oxygenase Aspartate/Asparaginyl-beta-Hydroxylase (AspH) is overexpressed on the surface of various cancer cells and associated with the enhancement of their cell motility; however, little is known about the function of AspH in normal and cancer cells - no selective small molecule AspH-inhibitors have been described so far. The proposed research experiments are aimed at: (a) performing a kinetic characterization of AspH and some of its (co-)substrates, (b) determining the required disulfide connectivity pattern in the substrate EGFDs and (c) providing novel AspH-inhibitors.The enzyme kinetics of AspH with respect to its substrates/co-substrates (including O2) will be investigated by steady state (LCMS) and stopped flow/flow quench (UV-Vis, LCMS) experiments. Particularly, the influence of preferred disulfide patterns of EGF-domains on AspH-catalysis and -kinetics will be determined: Stable synthetic analogues of AspH-substrates will serve as probes to investigate the required disulfide patterns. The synthesis of these stable analogues will involve solid phase peptide synthesis and olefin metathesis reactions.With the help of dynamic combinatorial chemistry and non-denaturing mass spectrometry, small organic molecule AspH-ligands will be identified. Stable derivatives of promising ligands will be accessed by organic synthesis. The inhibitory concentration of the (stabilized) AspH-ligands will be determined and their potential as anti-cancer therapeutics will subsequently be evaluated. The inhibitors will be used to confirm the proposed mechanism of how AspH promotes cell motility: the expression levels of Notch, its ligands and downstream targets will be analyzed (qRT-PCR, western blotting) before and after treatment of cells with the inhibitors.