Abstract
The three hypoxia-inducible factor (HIF) prolyl-4-hydroxylase domain (PHD) 1–3 enzymes confer oxygen sensitivity to the HIF pathway and are novel therapeutic targets for treatment of renal anemia. Inhibition of the PHDs may further be beneficial in other hypoxia-associated diseases, including ischemia and chronic inflammation. Several pharmacologic PHD inhibitors (PHIs) are available, but our understanding of their selectivity and its chemical basis is limited. We here report that the PHI JNJ-42041935 (JNJ-1935) is structurally similar to the firefly luciferase substrate D-luciferin. Our results demonstrate that JNJ-1935 is a novel inhibitor of firefly luciferase enzymatic activity. In contrast, the PHIs FG-4592 (roxadustat) and FG-2216 (ICA, BIQ, IOX3, YM 311) did not affect firefly luciferase. The JNJ-1935 mode of inhibition is competitive with a Ki of 1.36 μM. D-luciferin did not inhibit the PHDs, despite its structural similarity to JNJ-1935. This study provides insights into a previously unknown JNJ-1935 off-target effect as well as into the chemical requirements for firefly luciferase and PHD inhibitors and may inform the development of novel compounds targeting these enzymes.
Highlights
The transcription factor hypoxia-inducible factor (HIF) is the master regulator of the cellular transcriptional response to oxygen deprivation [1]
We compared the effects of the PHD inhibitors (PHIs) JNJ-42041935 (JNJ-1935), FG-4592 and FG-2216 on HIF-1α and HIF-2α stabilization and HIF activity (Supplementary Fig. S1)
Renilla luciferase bioluminescence showed some significant but not biologically relevant (0.9–1.5-fold) differences following the various compound treatments in the same samples (Fig. 1B). These results indicated that the PHIs did not affect Renilla luciferase activity, because the expression of Renilla luciferase was constitutive and not controlled by HIF or, the PHIs (Supplementary Fig. S1)
Summary
The transcription factor hypoxia-inducible factor (HIF) is the master regulator of the cellular transcriptional response to oxygen deprivation (hypoxia) [1]. HIF-α subunits are regulated in an oxygen-dependent manner [1]. Three prolyl-4-hydroxylase domain (PHD1–3) enzymes hydroxylate two prolyl residues within the oxygen-dependent degradation (ODD) domain of HIF-α, leading to an increased affinity of hydroxylated HIF-α to the von Hippel-Lindau (VHL) protein [3]. Inhibition of the PHDs, for example by hypoxia or pharmacologic agents, stabilizes HIF-α, leading to enhanced HIF-dependent gene expression [3]. In chronic kidney disease (CKD), Epo production is no longer sufficient to maintain a normal hematocrit, leading to anemia [6]. Several different pharmacologic HIF PHD inhibitors (PHIs) are in advanced clinical trials for treatment of renal anemia [6,7] and the first compound (FG-4592/ roxadustat) has recently been approved for treatment of patients in China [8]. Pre-clinical analyses show that PHIs may be beneficial in other hypoxia-associated diseases, including chronic inflammation, fibrosis, ischemia and possibly even cancer [6,9,10,11,12]
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