Kinetic Characterization of Aspartate/Asparagine-beta-Hydroxylase and Synthesis of Small Molecule Aspartate/Asparagine-beta-Hydroxylase-Inhibitors as Potential Anti-Cancer Agents
Organic Molecular Chemistry - Synthesis and Characterisation
Final Report Abstract
The human enzyme Aspartate/Asparagine-β-Hydroxylase (AspH) is a 2-oxoglutarate (2OG)-dependent dioxygenase which catalyzes the β-hydroxylation of specific Asp/Asn-residues in epidermal growth factor (EGF)-like domains (EGFD). It is overexpressed on the cell surface membrane of invasive human tumors (e. g. hepatocellular carcinoma) which correlates to a worse clinical prognosis. During the funding period of this DGF-fellowship, a reliable and robust in vitro assay to monitor AspH-activity was developed using a truncated AspH-construct and a simplified EGFD-based cyclic peptide as AspH-substrate. The assay was performed on a RapidFire-mass spectrometer suitable for high-throughput experimentation. A kinetic characterization of AspH with respect to its major (co-)substrates was performed using this novel AspH-assay; KM-concentrations of 2OG and Fe2+ were determined. These concentrations were rather low when compared to those of other 2OG-dependent dioxygenases indicating a relatively high affinity of AspH for its co-substrates. Furthermore, the assay was used to validate the utility of cyclic peptides to act as AspH-substrate mimetics: The hydroxylation-degrees of cyclic peptides based on the amino acid sequences of different substrate-EGFDs were in good agreement with the literature-reported EGFD-hydroxylation-degrees of these AspH-substrates. Therefore, the cyclic peptides were used as chemical probes to facilitate the identification of previously unknown AspH-substrates. The in vitro AspH-assay was modified to enable the screening of bioactive small-molecules as potential AspH-inhibitors: More than 2000 molecules were screened taking advantage of the highthroughput RapidFire-technology. Inter alia, derivatives of 2,4-pyridinedicarboxylic acid were identified as potent and selective AspH-inhibitors. To improve the synthetic accessibility of this compound class, a novel four-step synthesis for these molecules was designed permitting the performance of a proper structure-activity relationship study. Surprisingly, AspH was catalytically active in the absence of 2OG when suitable derivatives of this cofactor were added to the reaction mixture instead. A novel three step protocol was developed to enable the facile synthesis of a library of 2OG-analogues: These molecules were profiled against AspH, out of more than 35 molecules synthesized, approximately one third were acting either as alternative AspH-cofactors or AspH-inhibitors. The KM-concentrations of the alternative AspH- cofactors were 2 to 100 times higher than that of 2OG. The most potent AspH-inhibitor identified out of this class of compounds showed a potency against AspH (Inhibitory concentrations, IC50 of 0.4 μM at 2OG KM-concentration) even at physiologic concentrations of 2OG (IC50 of 3.8 μM).
