Aspartate/asparagine-β-hydroxylase: a high-throughput mass spectrometric assay for discovery of small molecule inhibitors

The human 2-oxoglutarate dependent oxygenase aspartate/asparagine-β-hydroxylase (AspH) catalyses the hydroxylation of Asp/Asn-residues in epidermal growth factor-like domains (EGFDs). AspH is upregulated on the surface of malign cancer cells; increased AspH levels correlate with tumour invasiveness. Due to a lack of efficient assays to monitor the activity of isolated AspH, there are few reports of studies aimed at identifying small-molecule AspH inhibitors. Recently, it was reported that AspH substrates have a non-canonical EGFD disulfide pattern. Here we report that a stable synthetic thioether mimic of AspH substrates can be employed in solid phase extraction mass spectrometry based high-throughput AspH inhibition assays which are of excellent robustness, as indicated by high Z’-factors and good signal-to-noise/background ratios. The AspH inhibition assay was applied to screen approximately 1500 bioactive small-molecules, including natural products and active pharmaceutical ingredients of approved human therapeutics. Potent AspH inhibitors were identified from both compound classes. Our AspH inhibition assay should enable the development of potent and selective small-molecule AspH inhibitors and contribute towards the development of safer inhibitors for other 2OG oxygenases, e.g. screens of the hypoxia-inducible factor prolyl-hydroxylase inhibitors revealed that vadadustat inhibits AspH with moderate potency.

In the structures of AspH:2,4-PDCA (1; PDB ID: 5JTC) and KDM4B:2,4-PDCA (2; PDB ID: 4LXL) the binding of the 2,4-PDCA C-2 and C-4 carboxylates to protein residues differs significantly. In the case of AspH, the 2,4-PDCA C-2 carboxylate is positioned to interact with Arg688AspH and His690AspH (both 2.8 Å); the 2,4-PDCA C-4 carboxylate is positioned to form a salt bridge with Arg735AspH (2.4 and 3.1 Å) and is positioned to interact with Ser668AspH (2.7 Å). In the case of KDM4B, the 2,4-PDCA C-2 carboxylate is positioned to interact with Lys242KDM4B (3.0 Å), and faces towards the substrate binding pocket close to the substrate K9me3 group; the 2,4-PDCA C-4 carboxylate is positioned to interact with Tyr133KDM4B and Lys207KDM4B (2.6 and 2.7 Å). The spatial orientations of Tyr133KDM4B and Lys207KDM4B is apparently determined by interactions with Asn281KDM4B (3.1 and 2.9 Å). In addition to differences in their binding of 2,4-PDCA, the active sites of AspH and KDM4B differ with regard to both the number of residues interacting with the active site metal and the modes of substrate binding.

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Supporting Figure S3. Comparison of selected human 2OG oxygenase crystal structures in complex with pyridine-2,4-dicarboxylic acid (2,4-PDCA). Colour code: grey: AspH; green: factor inhibiting hypoxia-inducible transcription factor (FIH); salmon: nucleolar protein 66 (NO66); cyan: Jmjc lysinespecific demethylase 4B (KDM4B); slate blue: fat mass-and obesity-associated protein (FTO); magenta: AlkB homolog 5 (Alkbh5); yellow: carbon-backbone of 2,4-PDCA; violet: Mn; orange: Fe; brown: Ni; metallic: Zn; red: oxygen; blue: nitrogen.  4 crystal structures reveals that the precise binding modes of 2OG oxygenases to 2,4-PDCA differ significantly. Between 1 and 3 residues interact directly with the either one or both of the two 2,4-PDCA C-4 carboxylate oxygen atoms; 1 to 2 protein residues interact directly with either one or both of the two 2,4-PDCA C-2 carboxylate oxygen atoms; metal ion coordination sites not occupied by protein residues can be occupied by water molecules (protein ligands and water molecules are not shown for clarity). Knowledge of the different binding modes of 2OG oxygenases with 2,4-PDCA (and related compounds) might be exploited for the design of derivatives that selectively inhibit specific sets of 2OG oxygenases.
Supporting Table S1. Summary of the 48 small-molecules of the library of pharmacologically active compounds (LOPAC, Sigma-Aldrich) which manifest >95% inhibition of AspH activity at a fixed inhibitor concentration (20 µM).

AspH-Inhibitor
Inhibition [ Figure S4. Differential scanning fluorimetry (DSF) assay. DSF assays were performed in independent duplicates as follows: The assay buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 50 μM NiCl2) was gently mixed with SYPRO orange (1‰v/v) and freshly thawed His6-AspH315-758 was added to a final AspH-concentration of 2 μM. The resulting orange solution was carefully mixed and pipetted into a 96-well Thermoscientific PCR-plate (19 μL per well). Finally, either an inhibitor solution (0.4 mM in DMSO) or a control sample (pure DMSO) was added to each well (1 μL per well, final inhibitor concentration of 20 μM) and the resulting solutions were gently mixed using a pipette. The plate was sealed with an optical tape (Bio-Rag, iCycler iQ), centrifuged (5 sec, 1000 rpm), then placed into a Stratagene Mx3005P PCR machine (Agilent Technologies). The PCR-machine was heated with a rate of 1° C per cycle (starting at an initial temperature of 25 °C; 70 cycles total). Data were analyzed using Microsoft Excel and GraphPad Prism following a literature protocol 5 .
Shifts (ΔTm) of the AspH melting temperature (Tm) are given with respect to DMSO controls. The validated AspH inhibitors 2,4-PDCA and NOG (entries 1 and 2), of which crystal structures in complex with AspH have been reported 1 , show a notable increase in the AspH Tm, presumably by stabilizing the AspH active site. The presence of candesartan cilexetil (entry 3) in the assay buffer has no significant effect on the AspH Tm. PBIT (entry 4) seems to strongly destabilize AspH, potentially by binding to one or more of its nucleophilic residues. The positive effect of IOX1 (entry 5) on the AspH Tm is similar to that of NOG, suggesting that crystallization of AspH in the presence of IOX1 could afford co-crystals. Vadadustat seems to slightly destabilize AspH (entry 6). Vadadustat -0.9 ± 0.2 a) Mean average of two independent runs (n = 2; mean ± standard deviation, SD).

Entry AspH-Inhibitor
Supporting Figure S5. Minimum significant ratio (MSR) analysis for the AspH inhibitors shown in Table 3. The mean ratio (MR), the MR confidence limits (RLs), the minimum significant ratio (MSR), and the limits of agreement (LsA) were calculated using Microsoft Excel 6,7 . General reproducibility acceptance criteria for assay validation have been reported to be: MSR ≤ 3 and 0.33 < LsA < 3 6 . The MR should ideally be 1 for a reproducible assay, the RLs should include 1 6 .
Data of the small-molecule AspH inhibitors displayed in Table 3  a) Values in brackets indicate high resolution data shell.