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

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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.