Structure-Activity Relationships and Transcriptomic Analysis of Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitors

Abstract

To evaluate the differences in action of commercially available 2-oxoglutarate mimetics and "branched-tail" oxyquinoline inhibitors of hypoxia-inducible factor prolyl hydroxylase (HIF PHD), the inhibitors' IC₅₀ values in the activation of HIF1 ODD-luciferase reporter were selected for comparative transcriptomics. Structure-activity relationship and computer modeling for the oxyquinoline series of inhibitors led to the identification of novel inhibitors, which were an order of magnitude more active in the reporter assay than roxadustat and vadadustat. Unexpectedly, 2-methyl-substitution in the oxyquinoline core of the best HIF PHD inhibitor was found to be active in the reporter assay and almost equally effective in the pretreatment paradigm of the oxygen-glucose deprivation in vitro model. Comparative transcriptomic analysis of the signaling pathways induced by HIF PHD inhibitors showed high potency of the two novel oxyquinoline inhibitors (#4896-3249 and #5704-0720) at 2 μM concentrations matching the effect of 30 μM roxadustat and 500 μM dimethyl oxalyl glycine in inducing HIF1 and HIF2-linked pathways. The two oxyquinoline inhibitors exerted the same activation of HIF-triggered glycolytic pathways but opposite effects on signaling pathways linked to alternative substrates of HIF PHD 1 and 3, such as p53, NF-κB, and ATF4. This finding can be interpreted as the specificity of the 2-methyl-substitute variant for HIF PHD2.

Keywords: 2-oxoglutarate dioxygenase; adaptaquin; hypoxia; iron chelation; neuradapt; transcription factor.

Figure 1

Chemical structures of HIF prolyl hydroxylase inhibitors mimicking αKG (enarodustat, vadadustat, roxadustat, N-oxalyl glycine) and HIF peptide (adaptaquin and neuradapt) binding modes. The oxyquinoline ring provides two iron ligands and the “branched-tails” interfere with HIF peptide binding. Red ovals show iron ligands. Blue ovals show the αKG mimicking part. The arrangement of N-oxalyl glycine (replacing αKG) and HIF peptide with respect to the active site iron in HIF PHD2 (PDB: 3HQR).

Figure 2

Potency of HIF prolyl hydroxylase inhibitors in HIF1 ODD-luc reporter assay (a,b), the effect of exogenous iron on reporter activation by roxadustat (c), and neuradapt (d). Assay conditions under Materials and Methods.

Figure 3

Structure–activity relationships derived from the titration experiments. All compounds are commercially available from ChemDiv (Skolkovo, Russia).

Figure 4

Docking of neuradapt enantiomers into HIF PHD2 crystal structure (PDB: 2G19). Asp254 interacts with the pyridine nitrogen, and Tyr 310 restricts rotation of pyridine and phenyl rings in the “branched-tail”(see Table S1 in Supplement for docking energy values).

Figure 5

Effect of substitutions in the second and fifth position of the oxyquinoline core on HIF1 ODD-luc reporter activation. 5-Cl-Neuradapt was custom synthesized (See Figure S1). Other compounds are commercially available from ChemDiv (Skolkovo, Russia).

Figure 6

HIF PHD inhibitors protect SH-SY5Y cells subject to OGD: timeline (a); cell viability measured after 24 h of incubation using the MTT assay (b); 24 h pretreatment (c). Data show mean and SEM normalized to control (n = 6): * p < 0.05, ** p < 0.01 versus insult by a one-way ANOVA test; HIF PHD inhibitors structures (d); The absence of toxicity of the studied compounds at 48 h incubation (e).

Figure 6

HIF PHD inhibitors protect SH-SY5Y cells subject to OGD: timeline (a); cell viability measured after 24 h of incubation using the MTT assay (b); 24 h pretreatment (c). Data show mean and SEM normalized to control (n = 6): * p < 0.05, ** p < 0.01 versus insult by a one-way ANOVA test; HIF PHD inhibitors structures (d); The absence of toxicity of the studied compounds at 48 h incubation (e).

Figure 7

Overlapping genes upon 5 h treatment of human neuroblastoma SH-SY5Y cell line with 0.5 mM DMOG, 30 μM roxadustat (shown as FG), 2 μM compounds #4896-3249 and #5704-0720, and 2 h hypoxic treatment followed by 5 h incubation. (a) Activation, (b) Repression. 1.5-fold change cut-off. Experimental details under Materials and Methods.

Figure 8

Opposite pattern on heatmaps for Biocarta p53 (a) and EPO/NF-κB (b) pathways in response to the treatment with compounds #4896-3249 and #5704-0720. All triplicates for each compound are shown. Experimental details under Materials and Methods.