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

人类癌症的治疗迫切需要有效的治疗方法；重要的是确定能够区分健康细胞和恶性细胞的特征特征，以开发选择性的抗癌药物：据报道，2-氧戊二酸依赖的加氧酶天冬氨酸/天冬酰胺-β-羟基酶(ASPH)在各种癌细胞的表面过表达，并与其细胞运动性增强有关；然而，对ASPH在正常细胞和癌细胞中的作用知之甚少-到目前为止还没有选择性的小分子ASPH抑制剂被描述。拟进行的研究实验旨在：(A)对ASPH及其一些(共)底物进行动力学表征，(B)确定底物EGFD中所需的二硫键连接模式，以及(C)提供新型ASPH抑制剂。ASPH相对于其底物/共底物(包括O2)的酶动力学将通过稳态(LCMS)和停流/流动猝灭(UV-Vis，LCMS)实验进行研究。特别是，EGF结构域的优先二硫键模式对ASPH催化和动力学的影响将被确定：稳定的ASPH底物的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的合成和烯烃的歧化反应，借助动态组合化学和非变性质谱学，将识别有机小分子ASPH-配体。有希望的配体的稳定衍生物将通过有机合成来获得。(稳定的)ASPH配体的抑制浓度将被确定，随后将评估它们作为抗癌治疗药物的潜力。这些抑制剂将被用来证实ASPH如何促进细胞运动的拟议机制：在用抑制剂处理细胞之前和之后，将分析Notch及其配体和下游靶点的表达水平(qRT-PCR，Western blotting)。

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.