Discovery of DS44470011: An oral hypoxia-inducible factor prolyl hydroxylase inhibitor for the treatment of renal anemia.

Inhibition of the hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) represents a promising strategy for discovering next-generation treatments for renal anemia. We identified a pyrimidine core with HIF-PHD inhibitory activity based on scaffold hopping of FG-2216 using crystal structures of HIF-PHD2 in complex with compound. By optimizing the substituents at the 2- and 6- positions of the pyrimidine core, we discovered DS44470011, which improves the effectiveness of erythropoietin (EPO) release in cells. Oral administration of DS44470011 to cynomolgus monkeys increased plasma EPO levels.

Inhibition of JMJD6 by 2-Oxoglutarate Mimics.

Studies on the inhibition of the human 2-oxoglutarate dependent oxygenase JMJD6, which is a cancer target, by 2-oxoglutarate mimics / competitors, including human drugs, drug candidates, and metabolites relevant to cancer are described. JMJD6 assays employed NMR to monitor inhibitor binding and use of mass spectrometry to monitor JMJD6-catalysed lysine hydroxylation. Notably, some clinically applied prolyl hydroxylase inhibitors also inhibit JMJD6. The results will help enable the development of inhibitors selective for human oxygenases, including JMJD6.

Discovery of a Potent and Orally Bioavailable Hypoxia-Inducible Factor 2α (HIF-2α) Agonist and Its Synergistic Therapy with Prolyl Hydroxylase Inhibitors for the Treatment of Renal Anemia.

Activation of hypoxia-inducible factor 2 (HIF-2) has emerged as a potent renal anemia treatment strategy. Here, the benzisothiazole derivative 26 was discovered as a novel HIF-2α agonist, which first demonstrated nanomolar activity (EC50 = 490 nM, Emax = 349.2%) in the luciferase reporter gene assay. Molecular dynamics simulations indicated that 26 could allosterically enhance HIF-2 dimerization. Furthermore, compound 26 had a good pharmacokinetic profile (the oral bioavailability in rats was 41.38%) and an in vivo safety profile (the LD50 in mice was greater than 708 mg·kg-1). In the in vivo efficacy assays, the combination of 26 and the prolyl hydroxylase inhibitor, AKB-6548, was confirmed for the first time to synergistically increase the plasma erythropoietin level in mice (from 260 to 2296 pg·mL-1) and alleviate zebrafish anemia induced by doxorubicin. These results provide new insights for HIF-2α agonists and the treatment of renal anemia.

Microbiota-derived butyrate is an endogenous HIF prolyl hydroxylase inhibitor.

The gut microbiota is essential for human health. Microbial supply of short-chain fatty acids (SCFAs), particularly butyrate, is a well-established contributor to gut homeostasis and disease resistance. Reaching millimolar luminal concentrations, butyrate is sequestered and utilized in the colon as the favored energy source for intestinal epithelia. Given the steep oxygen gradient across the anoxic lumen and the highly oxygenated lamina propria, the colon provides a particularly interesting environment to study oxygen sensing. Previous studies have shown that the transcription factor hypoxia-inducible factor (HIF) is stabilized in healthy colonic epithelia. Here we show that butyrate directly inhibits HIF prolyl hydroxylases (PHDs) to stabilize HIF. We find that butyrate stabilizes HIF in vitro despite eliminating ß-oxidation and resultant oxygen consumption. Using recombinant PHD protein in combination with nuclear magnetic resonance and enzymatic biochemical assays, we identify butyrate to bind and function as a unique, noncompetitive inhibitor of PHDs relative to other SCFAs. Butyrate inhibited PHD with a noncompetitive Ki of 5.3 ± 0.5 mM, a physiologically relevant concentration. We also confirm that microbiota-derived butyrate is necessary to stabilize HIF in mice colonic tissue through antibiotic-induced butyrate depletion and reconstitution experiments. Our results suggest that the co-evolution of mammals and mutualistic microbiota has selected for butyrate to impact a critical gene regulation pathway that can be extended beyond the mammalian gut. As PHDs are a major target for drug development in the stabilization of HIF, butyrate holds great potential as a well-tolerated endogenous inhibitor with far-reaching therapeutic impact.

Structural Basis of Prolyl Hydroxylase Domain Inhibition by Molidustat.

Human prolyl-hydroxylases (PHDs) are hypoxia-sensing 2-oxoglutarate (2OG) oxygenases, catalysis by which suppresses the transcription of hypoxia-inducible factor target genes. PHD inhibition enables the treatment of anaemia/ischaemia-related disease. The PHD inhibitor Molidustat is approved for the treatment of renal anaemia; it differs from other approved/late-stage PHD inhibitors in lacking a glycinamide side chain. The first reported crystal structures of Molidustat and IOX4 (a brain-penetrating derivative) complexed with PHD2 reveal how their contiguous triazole, pyrazolone and pyrimidine/pyridine rings bind at the active site. The inhibitors bind to the active-site metal in a bidentate manner through their pyrazolone and pyrimidine nitrogens, with the triazole π-π-stacking with Tyr303 in the 2OG binding pocket. Comparison of the new structures with other PHD inhibitor complexes reveals differences in the conformations of Tyr303, Tyr310, and a mobile loop linking ß2-ß3, which are involved in dynamic substrate binding/product release.

Enarodustat: First Approval.

The orally active hypoxia inducible factor-proly hydroxylase (HIF-PH) inhibitor enarodustat (ENAROY®, Japan Tobacco) is being developed as an alternative to injectable erythropoietin stimulating agents such as epoetin and darbepoetin for the treatment of anaemia associated with chronic kidney disease (CKD). The drug is approved in Japan and clinical development is ongoing in the USA and South Korea. This article summarizes the milestones in the development of enarodustat leading to this first approval for anaemia associated with CKD.

Daprodustat: First Approval.

Daprodustat (DUVROQ) is a small molecule inhibitor of hypoxia-inducible factor prolyl hydroxylase (PHD) developed by GlaxoSmithKline for the treatment of anaemia in patients with chronic kidney disease (CKD). Inhibition of PHD prevents degradation of hypoxia-inducible factor (HIF), leading to the production of erythropoietin and subsequent induction of erythropoiesis. In June, daprodustat received its first approval in Japan for the treatment of renal anaemia. Clinical studies of daprodustat are underway in multiple countries worldwide. This article summarizes the milestones in the development of daprodustat leading to this first approval for the treatment of renal anaemia.

Inhibition of firefly luciferase activity by a HIF prolyl hydroxylase inhibitor.

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.

Mannose Binding Lectin Is Hydroxylated by Collagen Prolyl-4-hydroxylase and Inhibited by Some PHD Inhibitors.

Background: Mannose-binding lectin (MBL) is an important component of innate immune defense. MBL undergoes oligomerization to generate high mol weight (HMW) forms which act as pattern recognition molecules to detect and opsonize various microorganisms. Several post-translational modifications including prolyl hydroxylation are known to affect the oligomerization of MBL. Yet, the enzyme(s) which hydroxylate proline in the collagen-like domain residues have not been identified and the significance of prolyl hydroxylation is incompletely understood. Methods: To investigate post-translational modifications of MBL, we stably expressed Myc-DDK tagged MBL in HEK293S cells. We used pharmacologic and genetic inhibition of 2-oxoglutarate-dependent dioxygenases (2OGDD) to identify the enzyme required for prolyl hydroxylation of MBL. We performed mass spectrometry to determine the effects of various inhibitors on MBL modifications. Results: Secretion of HMW MBL was impaired by inhibitors of the superfamily of 2OGDD, and was dependent on prolyl-4-hydroxylase subunit α1. Roxadustat and vadadustat, but not molidustat, led to significant suppression of hydroxylation and secretion of HMW forms of MBL. Conclusions: These data suggest that prolyl hydroxylation in the collagen-like domain of MBL is mediated by collagen prolyl-4-hydroxylase. Reduced MBL activity is likely to be an off-target effect of some, but not all, prolyl hydroxylase domain (PHD) inhibitors. There may be advantages in selective PHD inhibitors that would not interfere with MBL production.

Discovery of prolyl hydroxylase 2 inhibitors with new chemical scaffolds as in vivo active erythropoietin inducers through a combined virtual screening strategy.

Hypoxia-inducible factor (HIF) is identified to be a promising target to mediate the response to hypoxia. Its stability and activation are negatively controlled by prolyl hydroxylase 2 (PHD2). Thus, PHD2 inhibition has been perceived as a promising anti-anemia therapy. In this study, we carried out a structure-based virtual screening followed by in vitro and in vivo biological validation, with the goal to identify novel PHD2 inhibitors. As a result, a set of hits with new chemical scaffolds were revealed to be active in vitro for PHD2 inhibition. Compounds 2 and 3 were revealed to be capable of stabilizing HIF-α and stimulating erythropoietin (EPO) expression in cell-based assays. Notably, further in vivo assays revealed that 2 was capable of elevating the EPO plasma levels in C57BL/6 mice model. These findings provide new chemical scaffolds for further development of PHD2 inhibitors.

Structure-Activity Relationship and Crystallographic Studies on 4-Hydroxypyrimidine HIF Prolyl Hydroxylase Domain Inhibitors.

The 2-oxoglutarate-dependent hypoxia inducible factor prolyl hydroxylases (PHDs) are targets for treatment of a variety of diseases including anaemia. One PHD inhibitor is approved for use for the treatment of renal anaemia and others are in late stage clinical trials. The number of reported templates for PHD inhibition is limited. We report structure-activity relationship and crystallographic studies on a promising class of 4-hydroxypyrimidine-containing PHD inhibitors.

Photoactivatable Prolyl Hydroxylase 2 Inhibitors for Stabilizing the Hypoxia-Inducible Factor with Light.

HIF prolyl hydroxylase 2 (PHD2) inhibitors represent a novel approach for treating HIF-related diseases. This study reports the first application of photoremovable protecting group to the photoactivatable inhibitor (7) of PHD2. It allows the inhibitory activity for PHD2 to be controlled by light irradiation, subsequently stabilizing HIF and promoting expression of the target gene. Light activation to stabilize HIF offers promising potentials for the tissue-specific therapies for HIF-related disease by light irradiation onto target tissues.

Inhibition of a viral prolyl hydroxylase.

The hydroxylation of prolyl-residues in eukaryotes is important in collagen biosynthesis and in hypoxic signalling. The hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are drug targets for the treatment of anaemia, while the procollagen prolyl hydroxylases and other 2-oxoglutarate dependent oxygenases are potential therapeutic targets for treatment of cancer, fibrotic disease, and infection. We describe assay development and inhibition studies for a procollagen prolyl hydroxylase from Paramecium bursaria chlorella virus 1 (vCPH). The results reveal HIF PHD inhibitors in clinical trials also inhibit vCPH. Implications for the targeting of the human PHDs and microbial prolyl hydroxylases are discussed.

"Branched Tail" Oxyquinoline Inhibitors of HIF Prolyl Hydroxylase: Early Evaluation of Toxicity and Metabolism Using Liver-on-a-chip.

BACKGROUND: "Branched tail" oxyquinolines, and adaptaquin in particular, are potent HIF prolyl hydroxylase inhibitors showing promising results in in vivo hemorrhagic stroke models. The further improvement of the potency resulted in identification of a number of adaptaquin analogs. Early evaluation of toxicity and metabolism is desired right at the step of lead selection. OBJECTIVE: The aim of the study is to characterize the toxicity and metabolism of adaptaquin and its new improved analogs. METHOD: Liver-on-a-chip technology with differentiated HepaRG cells followed by LC-MS detection of the studied compounds and metabolites of the P450 substrate-inhibitor panel for CYP2B6, CYP2C9, CYP2C19, and CYP3A4. RESULTS: The optimized adaptaquin analogs show no toxicity up to a 100-fold increased range over EC50. The drugs are metabolized by CYP3A4 and CYP2B6 as shown with the use of the cytochrome P450 substrate-inhibitor panel designed and optimized for preclinical evaluation of drugs' in vitro biotransformation on a 3D human histotypical cell model using "liver-on-a-chip" technology. Activation of CYP2B6 with the drugs tested has been observed. A scheme for adaptaquin oxidative conversion is proposed. CONCLUSION: The optimized adaptaquin analogs are suitable for further preclinical trials. Activation of CYP2B6 with adaptaquin and its variants points to a potential increase in Tylenol toxicity if administered together.

A Fluorescent Benzo[g]isoquinoline-Based HIF Prolyl Hydroxylase Inhibitor for Cellular Imaging.

Prolyl hydroxylation domain (PHD) enzymes catalyze the hydroxylation of the transcription factor hypoxia-inducible factor (HIF) and serve as cellular oxygen sensors. HIF and the PHD enzymes regulate numerous potentially tissue-protective target genes which can adapt cells to metabolic and ischemic stress. We describe a fluorescent PHD inhibitor (1-chloro-4-hydroxybenzo[g]isoquinoline-3-carbonyl)glycine which is suited to fluorescence-based detection assays and for monitoring PHD inhibitors in biological systems. In cell-based assays, application of the fluorescent PHD inhibitor allowed co-localization with a cellular PHD enzyme and led to live cell imaging of processes involved in cellular oxygen sensing.

Selective Inhibitors of a Human Prolyl Hydroxylase (OGFOD1) Involved in Ribosomal Decoding.

Human prolyl hydroxylases are involved in the modification of transcription factors, procollagen, and ribosomal proteins, and are current medicinal chemistry targets. To date, there are few reports on inhibitors selective for the different types of prolyl hydroxylases. We report a structurally informed template-based strategy for the development of inhibitors selective for the human ribosomal prolyl hydroxylase OGFOD1. These inhibitors did not target the other human oxygenases tested, including the structurally similar hypoxia-inducible transcription factor prolyl hydroxylase, PHD2.

Click Chemistry-Based Discovery of [3-Hydroxy-5-(1 H-1,2,3-triazol-4-yl)picolinoyl]glycines as Orally Active Hypoxia-Inducing Factor Prolyl Hydroxylase Inhibitors with Favorable Safety Profiles for the Treatment of Anemia.

As a gene associated with anemia, the erythropoiesis gene is physiologically expressed under hypoxia regulated by †hypoxia-inducing factor-α (HIF-α). Thus, stabilizing HIF-α is a potent strategy to stimulate the expression and secretion of erythropoiesis. In this study, we applied click chemistry to the discovery of HIF prolyl hydroxylase 2 (HIF-PHD2) inhibitors for the first time, and a series of triazole compounds showed preferable inhibitory activity in fluorescence polarization assays. Of particular note was the orally active HIF-PHD inhibitor 15i (IC50 = 62.23 nM), which was almost ten times more active than the phase III drug FG-4592 (IC50 = 591.4 nM). Furthermore, it can upregulate the hemoglobin of cisplatin-induced anemia mice (120 g/L) to normal levels (160 g/L) with no apparent toxicity observed in vivo. These results confirm that triazole compound 15i is a promising candidate for the treatment of renal anemia.

Prolyl Hydroxylase Inhibitors: A Breakthrough in the Therapy of Anemia Associated with Chronic Diseases.

Chronic kidney disease, cancer, chronic inflammatory disorders, nutritional, and genetic deficiency can cause anemia. Hypoxia causes induction of hypoxia-inducible factor (HIF), which stimulates erythropoietin (EPO) synthesis. Prolyl hydroxylase domain (PHD) enzyme inhibition can stabilize hypoxia-inducible factor (HIF). HIF stabilization also decreases hepcidin, a hormone of hepatic origin, which regulates iron homeostasis. PHD inhibitors represent a novel pharmacological treatment of anemia associated with chronic diseases. Many orally active PHD inhibitors like roxadustat, molidustat, vadadustat, and desidustat are in late phase clinical trials. This review discusses the role of PHD inhibitors in the treatment of anemia associated with chronic diseases.

Discovery of novel 2-[(4-hydroxy-6-oxo-2,3-dihydro-1H-pyridine-5-carbonyl)amino]acetic acid derivatives as HIF prolyl hydroxylase inhibitors for treatment of renal anemia.

Prolyl hydroxylase domain-containing protein (PHD) inhibitors are useful as orally administered agents for the treatment of renal anemia. Based on the common structures of known PHD inhibitors, we found novel PHD inhibitor 1 with a 2-[(4-hydroxy-6-oxo-2,3-dihydro-1H-pyridine-5-carbonyl)amino]acetic acid motif. The PHD2-inhibitory activity, lipophilicity (CLogP), and PK profiles (hepatocyte metabolism, protein binding, and/or elimination half-life) of this inhibitor were used as the evaluation index to optimize the structure and eventually discovered clinical candidate 42 as the suitable compound. Compound 42 was demonstrated to promote the production of erythropoietin (EPO) following oral administration in mice and rats. The predicted half-life of this compound in humans was 1.3-5.6 h, therefore, this drug may be expected to administer once daily with few adverse effects caused by excessive EPO production.

Drug delivery and epimorphic salamander-type mouse regeneration: A full parts and labor plan.

The capacity to regenerate entire body parts, tissues, and organs had generally been thought to be lost in evolution with very few exceptions (e.g. the liver) surviving in mammals. The discovery of the MRL mouse and the elucidation of the underlying molecular pathway centering around hypoxia inducible factor, HIF-1α, has allowed a drug and materials approach to regeneration in mice and hopefully humans. The HIF-1α pathway is ancient and permitted the transition from unicellular to multicellular organisms. Furthermore, HIF-1α and its regulation by PHDs, important oxygen sensors in the cell, provides a perfect drug target. We review the historical background of regeneration biology, the discovery of the MRL mouse, and its underlying biology, and novel approaches to drugs, targets, and delivery systems (see Fig. 1).