Abstract
Human aspartate/asparagine-β-hydroxylase (AspH) is a 2-oxoglutarate (2OG) dependent oxygenase that catalyses the hydroxylation of Asp/Asn-residues of epidermal growth factor-like domains (EGFDs). AspH is reported to be upregulated on the cell surface of invasive cancer cells in a manner distinguishing healthy from cancer cells. We report studies on the effect of small-molecule active pharmaceutical ingredients (APIs) of human cancer therapeutics on the catalytic activity of AspH using a high-throughput mass spectrometry (MS)-based inhibition assay. Human B-cell lymphoma-2 (Bcl-2)-protein inhibitors, including the (R)-enantiomer of the natural product gossypol, were observed to efficiently inhibit AspH, as does the antitumor antibiotic bleomycin A2. The results may help in the design of AspH inhibitors with the potential of increased selectivity compared to the previously identified Fe(II)-chelating or 2OG-competitive inhibitors. With regard to the clinical use of bleomycin A2 and of the Bcl-2 inhibitor venetoclax, the results suggest that possible side-effects mediated through the inhibition of AspH and other 2OG oxygenases should be considered.
Highlights
Human aspartate/asparagine-β-hydroxylase (AspH, BAH, HAAH) belongs to the family of 2-oxoglutarate (2OG) dependent oxygenases[1] and catalyses the post-translational hydroxylation of specific aspartyland asparaginyl-residues in human epidermal growth factor-like do mains (EGFDs) using 2OG and O2 as co-substrates and Fe(II) as a co factor (Fig. 1).[2,3] Hypoxia is reported to regulate the expression levels of human AspH4,5 and upregulated levels of AspH have been detected on the cell surface of invasive human cancers such as hepatocellular carcinoma,[6,7] breast cancer,[8] and pancreatic cancer.[9]
A compound library composed of 316 small-molecules, which are either active pharmaceutical ingredients (APIs) of approved human cancer therapeutics or of human cancer therapeutics under current or previous clinical investigation, was investigated for AspH inhibition under the previously established AspH inhibition assay conditions.[36]
Complete suppression of AspH activity was observed in case of (R)-gossypol, while obatoclax was the least efficient AspH inhibitor out of these four compounds (~20% AspH activity remaining at 20 μM; Table 1, entry 11)
Summary
Human aspartate/asparagine-β-hydroxylase (AspH, BAH, HAAH) belongs to the family of 2-oxoglutarate (2OG) dependent oxygenases[1] and catalyses the post-translational hydroxylation of specific aspartyland asparaginyl-residues in human epidermal growth factor-like do mains (EGFDs) using 2OG and O2 as co-substrates and Fe(II) as a co factor (Fig. 1).[2,3] Hypoxia is reported to regulate the expression levels of human AspH4,5 and upregulated levels of AspH have been detected on the cell surface of invasive human cancers such as hepatocellular carcinoma,[6,7] breast cancer,[8] and pancreatic cancer.[9]. Despite the combined evidence suggesting that AspH is a promising medicinal chemistry target for the development of small-moleculebased cancer therapeutics, comparatively few AspH inhibition studies using small-molecules are reported, with most of these relying either on the use of likely non-selective[19,20] or partially selective[21] small-mole cules or on the use of L-ascorbic acid (LAA)-derived small-mole cules.[18,22,23,24] This likely reflects the historic lack of simple and reliable assays to monitor recombinant human AspH activity in vitro. Pioneering assays employed (native) partially purified bovine or murine AspH and monitored AspH activity by analysing either 2OG turnover or the amino acid content of EGFD substrate peptides after acidic hydrolysis using mass spectrometry (MS).[20,25,26,27]
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