Structural and inhibition studies of 2-oxoglutarate and iron dependent oxygenase enzymes using selected natural product compounds

Abstract

The 2-oxoglutarate and iron-dependent oxygenases (2OGXs) have been shown to catalyse a wide range of oxidative reactions. This study evaluated the inhibitory activity of selected natural product compounds, mainly from the genus Garcinia, against two human hydroxylase 2OGX enzymes involved in regulating the cellular response to oxygen levels: Factor Inhibiting Hypoxia-Inducible Factor (FIH) and Prolyl Hydroxylase Domain-Containing Protein 2 (PHD2). Rapidfire mass spectrometry-based assay measuring peptide substrates and products was used to screen six potential bioactive natural product inhibitors against FIH and PHD2, namely the prenylated xanthone α-mangostin, the iminosugars 1-deoxynojirimycin (DNJ) and 1-deoxymannojirimycin (DMJ), the potassium hydroxycitrate salts (2S,3S)-(-)-KHCA and (2S,3R)-(+)-KHCA. α-Mangostin showed inhibition towards PHD2, which was stronger than NOG, the positive control (Km and Vmax for the negative control, NOG, and α-mangostin were 162.4 nM and 4.3 nM s-1, 159.1 nM and 2.5 nM s-1, and 93.2 nM and 2.15 nM s-1, respectively). Michaelis-Menten profiles of α-mangostin also displayed an uncompetitive mode of inhibition towards PHD2. None of the selected natural products showed inhibition towards FIH comparable to NOG. The enzyme inhibition results from the time course and LC-MS endpoint assays were consistent. α-Mangostin exhibited higher inhibition against PHD2 (IC50: 5.1 μM) compared to NOG (IC50: 21.0 μM). However, α-mangostin showed much lower inhibition against FIH (IC50: 10.6 μM) than NOG (IC50: 370.2 nM). The IC50 values of α-mangostin at a higher iron concentration against PHD2 and FIH were 4.8 μM and 18.9 μM, respectively, suggesting that iron chelation by α-mangostin in solution does not affect enzyme inhibition. The FIH-α-mangostin co-crystal that was obtained displayed poor-quality diffraction (reduced resolution of 4 Å and high mosaicity of 1.5 degree). On the other hand, the PHD2-α-mangostin co-crystal synchrotron acquired X-ray diffraction data was processed to a resolution of 2.37 Å and overall completeness of 99.6%. Iterative cycles of model building in COOT and refinement in PHENIX were carried out until the Rwork/Rfree values of the PHD2-α-mangostin converged to 0.2087/0.2395. α-Mangostin was not visible in the PHD2 structure obtained from crystals grown in the presence of α-mangostin. This supports the Michaelis-Menten inhibition profiles indicating that α-mangostin mode of action is via uncompetitive inhibition. Furthermore, there was no reduction in the active site metal occupancy. In conclusion, the inhibition of PHD2 enzymatic activity by α-mangostin occurs via an unclear mechanism that needs to be elucidated in future studies.