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

Andrey A Poloznikov,Olga V Shurupova,Nancy Krucher,Vladimir I Tishkov,Irina G Gazaryan,Andrey F Asachenko,Eric P Chang,Anthony Narciso,Andrey I Osipyants,Sergey V Kazakov,Svyatoslav S Savin,Gregory R J Thatcher,Sue H Lee,Anna Yu Khristichenko,Sergey V Nikulin,Bobby Thomas,Dmitry M Hushpulian,Irina N Gaisina

doi:10.3390/antiox11020220

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.

Highlights

The cellular response to hypoxia is executed by the stabilization and activation of hypoxia-inducible factor (HIF), a transcription factor that launches the expression of dozens of genes necessary for survival under low oxygen, with erythropoietin (EPO) being the best-known target
The HIF1 ODD-luc reporter assay employs a cell line stably expressing a reporter vector providing the continuous production of an oxygen-dependent domain of HIF1α fused via its C-terminus to N-terminus of firefly luciferase
As we showed before [8], the enzyme step is rate-limiting in the fusion protein degradation, and the steady-state level of HIF1 ODD-luc fusion is determined by the activity of the intracellular pool of all three hypoxia-inducible factor prolyl hydroxylase (HIF PHD) enzymes

Summary

Introduction

The cellular response to hypoxia is executed by the stabilization and activation of hypoxia-inducible factor (HIF), a transcription factor that launches the expression of dozens of genes necessary for survival under low oxygen, with erythropoietin (EPO) being the best-known target. The HIF-triggered program includes apoptotic/pro-death factors, and depending on the availability of energy resources, the cell may either survive or die. HIF is a heterodimer, with the dimer stability controlled by an α-subunit (HIFα), which degrades in normoxia. At least three different alpha subunits are known, termed HIF1α, 2α, and 3α. They dimerize with the HIFβ subunit forming the corresponding transcription factors, e.g., HIF1, HIF2, HIF3. HIF1, and HIF2, posessing overlapping and distinct gene targets; HIF2 is considered the key factor regulating

Methods

Results

Conclusion