Inhibition of prolyl 4-hydroxylase by hydroxyanthraquinones.
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ABSTRACT:
SUBMITTER: Cunliffe CJ
PROVIDER: S-EPMC1147282 | biostudies-other | 1986 Oct
REPOSITORIES: biostudies-other
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Project description: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 β2-β3, which are involved in dynamic substrate binding/product release.
| S-EPMC8359944 | biostudies-literature
Project description:Collagen is the most abundant protein in animals. Its overproduction is associated with fibrosis and cancer metastasis. The stability of collagen relies on post-translational modifications, the most prevalent being the hydroxylation of collagen strands by collagen prolyl 4-hydroxylases (CP4Hs). Catalysis by CP4Hs enlists an iron cofactor to convert proline residues to 4-hydroxyproline residues, which are essential for the conformational stability of mature collagen. Ethyl 3,4-dihydroxybenzoate (EDHB) is commonly used as a "P4H" inhibitor in cells, but suffers from low potency, poor selectivity, and off-target effects that cause iron deficiency. Dicarboxylates of 2,2'-bipyridine are among the most potent known CP4H inhibitors but suffer from a high affinity for free iron. A screen of biheteroaryl compounds revealed that replacing one pyridyl group with a thiazole moiety retains potency and enhances selectivity. A diester of 2-(5-carboxythiazol-2-yl)pyridine-5-carboxylic acid is bioavailable to human cells and inhibits collagen biosynthesis at concentrations that neither cause general toxicity nor disrupt iron homeostasis. These data anoint a potent and selective probe for CP4H and a potential lead for the development of a new class of antifibrotic and antimetastatic agents.
| S-EPMC4798942 | biostudies-literature
Project description:Posttranslational modifications can cause profound changes in protein function. Typically, these modifications are reversible, and thus provide a biochemical on-off switch. In contrast, proline residues are the substrates for an irreversible reaction that is the most common posttranslational modification in humans. This reaction, which is catalyzed by prolyl 4-hydroxylase (P4H), yields (2S,4R)-4-hydroxyproline (Hyp). The protein substrates for P4Hs are diverse. Likewise, the biological consequences of prolyl hydroxylation vary widely, and include altering protein conformation and protein-protein interactions, and enabling further modification. The best known role for Hyp is in stabilizing the collagen triple helix. Hyp is also found in proteins with collagen-like domains, as well as elastin, conotoxins, and argonaute 2. A prolyl hydroxylase domain protein acts on the hypoxia inducible factor alpha, which plays a key role in sensing molecular oxygen, and could act on inhibitory kappaB kinase and RNA polymerase II. P4Hs are not unique to animals, being found in plants and microbes as well. Here, we review the enzymic catalysts of prolyl hydroxylation, along with the chemical and biochemical consequences of this subtle but abundant posttranslational modification.
| S-EPMC2841224 | biostudies-literature
Project description:Skeletal muscle has an impressive ability to repair itself after a damaging insult and this response is essential to the process of muscle adaptation. In conditions such as muscular dystrophy and the sarcopenia of old age, repair is compromised leading to fibrosis and fatty tissue accumulation. Hypoxia-inducible factors (HIFs) are highly conserved regulators of gene transcription under conditions of low oxygen tension and HIF target genes such as EPO and VEGF have been associated with muscle protection and repair. We sought to interrogate the importance of HIF activation to skeletal muscle repair through the use of prolyl hydroxylase inhibitors (PHI) that stabilize HIF and activate target gene transcription in a mouse eccentric exercise limb damage model. We used microarrays to detail the global effects of prolyl hydroxylase inhibitors (PHI) in a mouse eccentric exercise limb damage model.
2017-08-23 | GSE95244 | GEO
Project description:The identity of the peritubular population of cells with mesenchymal phenotype thought responsible for producing erythropoietin in humans remains unclear. Here, renal CD133(+)/CD73(+) progenitor cells, isolated from the human renal inner medulla and described as a population of mesenchymal progenitors, released erythropoietin under hypoxic conditions. CD133(-) cells did not synthesize erythropoietin, and CD133(+) progenitor cells stopped producing erythropoietin when they differentiated and acquired an epithelial phenotype. Inhibition of prolyl hydroxylases, using either dimethyloxalylglycine or a small hairpin RNA against prolyl hydroxylase-2, increased both hypoxia-inducible factor-2? (HIF-2?) expression and erythropoietin transcription. Moreover, under hypoxic conditions, inhibition of prolyl hydroxylase significantly increased erythropoietin release by CD133(+) progenitors. Finally, blockade of HIF-2? impaired erythropoietin synthesis by CD133(+) progenitors. Taken together, these results suggest that it is the renal CD133(+) progenitor cells that synthesize and release erythropoietin under hypoxia, via the prolyl hydroxylase-HIF-2? axis, in the human kidney. In addition, this study provides rationale for the therapeutic use of prolyl hydroxylase inhibitors in the setting of acute or chronic renal injury.
| S-EPMC3736703 | biostudies-literature
Project description:BACKGROUND:In muscular dystrophy and old age, skeletal muscle repair is compromised leading to fibrosis and fatty tissue accumulation. Therefore, therapies that protect skeletal muscle or enhance repair would be valuable medical treatments. Hypoxia-inducible factors (HIFs) regulate gene transcription under conditions of low oxygen, and HIF target genes EPO and VEGF have been associated with muscle protection and repair. We tested the importance of HIF activation following skeletal muscle injury, in both a murine model and human volunteers, using prolyl hydroxylase inhibitors that stabilize and activate HIF. METHODS:Using a mouse eccentric limb injury model, we characterized the protective effects of prolyl hydroxylase inhibitor, GSK1120360A. We then extended these studies to examine the impact of EPO modulation and infiltrating immune cell populations on muscle protection. Finally, we extended this study with an experimental medicine approach using eccentric arm exercise in untrained volunteers to measure the muscle-protective effects of a clinical prolyl hydroxylase inhibitor, daprodustat. RESULTS:GSK1120360A dramatically prevented functional deficits and histological damage, while accelerating recovery after eccentric limb injury in mice. Surprisingly, this effect was independent of EPO, but required myeloid HIF1?-mediated iNOS activity. Treatment of healthy human volunteers with high-dose daprodustat reduced accumulation of circulating damage markers following eccentric arm exercise, although we did not observe any diminution of functional deficits with compound treatment. CONCLUSION:The results of these experiments highlight a novel skeletal muscle protective effect of prolyl hydroxylase inhibition via HIF-mediated expression of iNOS in macrophages. Partial recapitulation of these findings in healthy volunteers suggests elements of consistent pharmacology compared to responses in mice although there are clear differences between these two systems.
| S-EPMC6234580 | biostudies-literature
Project description:The topical application of minoxidil may achieve millimolar concentrations in the skin. We investigated whether millimolar minoxidil could induce vascular endothelial growth factor (VEGF), a possible effector for minoxidil-mediated hair growth, and how it occurred at the molecular level. Cell-based experiments were performed to investigate a molecular mechanism underlying the millimolar minoxidil induction of VEGF. The inhibitory effect of minoxidil on hypoxia-inducible factor (HIF) prolyl hydroxylase-2 (PHD-2) was tested by an in vitro von Hippel-Lindau protein (VHL) binding assay. To examine the angiogenic potential of millimolar minoxidil, a chorioallantoic membrane (CAM) assay was used. In human keratinocytes and dermal papilla cells, millimolar minoxidil increased the secretion of VEGF, which was not attenuated by a specific adenosine receptor antagonist that inhibits the micromolar minoxidil induction of VEGF. Millimolar minoxidil induced hypoxia-inducible factor-1? (HIF-1?), and the induction of VEGF was dependent on HIF-1. Moreover, minoxidil applied to the dorsal area of mice increased HIF-1? and VEGF in the skin. In an in vitro VHL binding assay, minoxidil directly inhibited PHD-2, thus preventing the hydroxylation of cellular HIF-1? and VHL-dependent proteasome degradation and resulting in the stabilization of HIF-1? protein. Minoxidil inhibition of PHD-2 was reversed by ascorbate, a cofactor of PHD-2, and the minoxidil induction of cellular HIF-1? was abrogated by the cofactor. Millimolar minoxidil promoted angiogenesis in the CAM assay, an in vivo angiogenic test, and this was nullified by the specific inhibition of VEGF. Our data demonstrate that PHD may be the molecular target for millimolar minoxidil-mediated VEGF induction via HIF-1.
| S-EPMC5796003 | biostudies-literature
Project description:Complement C1q is part of the C1 macromolecular complex that mediates the classical complement activation pathway: a major arm of innate immune defense. C1q is composed of A, B, and C chains that require post-translational prolyl 4-hydroxylation of their N-terminal collagen-like domain to enable the formation of the functional triple helical multimers. The prolyl 4-hydroxylase(s) that hydroxylate C1q have not previously been identified. Recognized prolyl 4-hydroxylases include collagen prolyl-4-hydroxylases (CP4H) and the more recently described prolyl hydroxylase domain (PHD) enzymes that act as oxygen sensors regulating hypoxia-inducible factor (HIF). We show that several small-molecule prolyl hydroxylase inhibitors that activate HIF also potently suppress C1q secretion by human macrophages. However, reducing oxygenation to a level that activates HIF does not compromise C1q hydroxylation. In vitro studies showed that a C1q A chain peptide is not a substrate for PHD2 but is a substrate for CP4H1. Circulating levels of C1q did not differ between wild-type mice or mice with genetic deficits in PHD enzymes, but were reduced by prolyl hydroxylase inhibitors. Thus, C1q is hydroxylated by CP4H, but not the structurally related PHD hydroxylases. Hence, reduction of C1q levels may be an important off-target side effect of small molecule PHD inhibitors developed as treatments for renal anemia.
| S-EPMC5612014 | biostudies-literature
Project description:OBJECTIVES:Rotator cuff tears are among the most frequent upper extremity injuries. Current treatment strategies do not address the poor quality of the muscle and tendon following chronic rotator cuff tears. Hypoxia-inducible factor-1 alpha (HIF-1?) is a transcription factor that activates many genes that are important in skeletal muscle regeneration. HIF-1? is inhibited under normal physiological conditions by the HIF prolyl 4-hydroxylases (PHDs). In this study, we used a pharmacological PHD inhibitor, GSK1120360A, to enhance the activity of HIF-1? following the repair of a chronic cuff tear, and measured muscle fibre contractility, fibrosis, gene expression, and enthesis mechanics. METHODS:Chronic supraspinatus tears were induced in adult rats, and repaired 28 days later. Rats received 0 mg/kg, 3 mg/kg, or 10 mg/kg GSK1120360A daily. Collagen content, contractility, fibre type distribution and size, the expression of genes involved in fibrosis, lipid accumulation, atrophy and inflammation, and the mechanical properties of the enthesis were then assessed two weeks following surgical repair. RESULTS:At two weeks following repair, treatment groups showed increased muscle mass but there was a 15% decrease in force production in the 10 mg/kg group from controls, and no difference between the 0 mg/kg and the 3 mg/kg groups. There was a decrease in the expression of several gene transcripts related to matrix accumulation and fibrosis, and a 50% decrease in collagen content in both treated groups compared with controls. Additionally, the expression of inflammatory genes was reduced in the treated groups compared with controls. Finally, PHD inhibition improved the maximum stress and displacement to failure in repaired tendons. CONCLUSIONS:GSK1120360A resulted in improved enthesis mechanics with variable effects on muscle function. PHD inhibition may be beneficial for connective tissue injuries in which muscle atrophy has not occurred.Cite this article: J. P. Gumucio, M. D. Flood, A. Bedi, H. F. Kramer, A. J. Russell, C. L. Mendias. Inhibition of prolyl 4-hydroxylase decreases muscle fibrosis following chronic rotator cuff tear. Bone Joint Res 2017;6:57-65. DOI: 10.1302/2046-3758.61.BJR-2016-0232.R1.
| S-EPMC5301902 | biostudies-literature
Project description:Background and purposeSmall-molecule inhibitors of prolyl hydroxylase (PHD) enzymes are a novel target for the treatment of anaemia and functional iron deficiency (FID). Other than being orally bioavailable, the differentiation of PHD inhibitors from recombinant human erythropoietin (rhEPO) has not been demonstrated.Experimental approachJNJ-42905343 was identified and characterized as a novel inhibitor of PHD and its action was compared with rhEPO in healthy rats and in a rat model of inflammation-induced anaemia and FID [peptidoglycan-polysaccharide (PGPS) model].Key resultsOral administration of JNJ-42905343 to healthy rats increased the gene expression of cytochrome b (DcytB) and divalent metal-ion transporter 1 (DMT1) in the duodenum, and increased plasma EPO. Repeated administration of JNJ-42905343 for 28 days increased blood haemoglobin, mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV). The serum iron concentration was increased with low doses (0.3 mg·kg(-1) ) but reduced at high doses (6 mg·kg(-1) ). In PGPS-treated rats, administration of JNJ-42905343 for 28 days corrected FID and anaemia, as reflected by increased blood haemoglobin, MCH and MCV. Increased expression of DcytB and DMT1 genes in the duodenum resulting in increased iron availability was defined as the mechanism for these effects. rhEPO did not affect DcytB and DMT1 and was not effective in PGPS-treated rats.Conclusions and implicationsPHD inhibition has a beneficial effect on iron metabolism in addition to stimulating the release of EPO. Small-molecule inhibitors of PHD such as JNJ-42905343 represent a mechanism distinct from rhEPO to treat anaemia and FID.
| S-EPMC4543614 | biostudies-literature