Metal Ions as Activators of Hypoxia Inducible Factor

Activation of antihypoxic program under the action of a number of transition and heavy metals has been studied using cell-based HIF1 ODD-luc and HRE-luc reporters. It has been demonstrated that Au³⁺, Pb²⁺, Sn²⁺, Hg²⁺ are weak HIF1 ODD-luc activators, likely reflecting their weak competition for the ironbinding site in the active center of HIF prolyl hydroxylase. Metals capable of replacing iron–Mn²⁺, Zn²⁺, Cu²⁺ и Ni²⁺–activate at high submillimolar concentrations, which indicates low permeability of the cell membrane for transition metals. The highest activation is observed for Co²⁺ and Cd²⁺, however, Cd²⁺ is highly toxic even at 10 μM, in contrast to Co²⁺, which activates both reporters without toxicity signs up to 25 μM for 24 h. A significant activation by Co²⁺ is observed already in low micromolar range of concentrations, which can be recommended for use in hypoxia mimicking.     

Structural optimization of adaptaquin,a HIF prolyl hydroxylase inhibitor

Russian Chemical Bulletin - The structural optimization of adaptaquin, viz., 7-(4-chlorophenyl)[(3-hydroxypyridin-2-yl)amino]methylquinolin-8-ol, a HIF (hypoxia inducible factor) prolyl hydroxylase...     

Reactivation of HIF prolyl hydroxylase 2 from E.coli inclusion bodies

Russian Chemical Bulletin - Hypoxia inducible factor (HIF) prolyl hydroxylases (PHD) belong to α-ketoglutarate-dependent non-heme iron dioxygenases catalyzing hydroxylation of HIF prolines....     

Histone demethylase KDM4A plays an oncogenic role in nasopharyngeal carcinoma by promoting cell migration and invasion

Compelling evidence has indicated the vital role of lysine-specific demethylase 4 A (KDM4A), hypoxia-inducible factor-1α (HIF1α) and the mechanistic target of rapamycin (mTOR) signaling pathway in nasopharyngeal carcinoma (NPC). Therefore, we aimed to investigate whether KDM4A affects NPC progression by regulating the HIF1α/DDIT4/mTOR signaling pathway. First, NPC and adjacent tissue samples were collected, and KDM4A protein expression was examined by immunohistochemistry. Then, the interactions among KDM4A, HIF1α and DDIT4 were assessed. Gain- and loss-of-function approaches were used to alter KDM4A, HIF1α and DDIT4 expression in NPC cells. The mechanism of KDM4A in NPC was evaluated both in vivo and in vitro via RT-qPCR, Western blot analysis, MTT assay, Transwell assay, flow cytometry and tumor formation experiments. KDM4A, HIF1α, and DDIT4 were highly expressed in NPC tissues and cells. Mechanistically, KDM4A inhibited the enrichment of histone H3 lysine 9 trimethylation (H3K9me3) in the HIF1α promoter region and thus inhibited the methylation of HIF1α to promote HIF1α expression, thus upregulating DDIT4 and activating the mTOR signaling pathway. Overexpression of KDM4A, HIF1α, or DDIT4 or activation of the mTOR signaling pathway promoted SUNE1 cell proliferation, migration, and invasion but inhibited apoptosis. KDM4A silencing blocked the mTOR signaling pathway by inhibiting the HIF1α/DDIT4 axis to inhibit the growth of SUNE1 cells in vivo. Collectively, KDM4A silencing could inhibit NPC progression by blocking the activation of the HIF1α/DDIT4/mTOR signaling pathway by increasing H3K9me3, highlighting a promising therapeutic target for NPC.Subject terms: Oncogenes, Cancer     

Pyruvate dehydrogenase kinase 1 and 2 deficiency reduces high-fat diet-induced hypertrophic obesity and inhibits the differentiation of preadipocytes into mature adipocytes

Obesity is now recognized as a disease. This study revealed a novel role for pyruvate dehydrogenase kinase (PDK) in diet-induced hypertrophic obesity. Mice with global or adipose tissue-specific PDK2 deficiency were protected against diet-induced obesity. The weight of adipose tissues and the size of adipocytes were reduced. Adipocyte-specific PDK2 deficiency slightly increased insulin sensitivity in HFD-fed mice. In studies with 3T3-L1 preadipocytes, PDK2 and PDK1 expression was strongly increased during adipogenesis. Evidence was found for epigenetic induction of both PDK1 and PDK2. Gain- and loss-of-function studies with 3T3-L1 cells revealed a critical role for PDK1/2 in adipocyte differentiation and lipid accumulation. PDK1/2 induction during differentiation was also accompanied by increased expression of hypoxia-inducible factor-1α (HIF1α) and enhanced lactate production, both of which were absent in the context of PDK1/2 deficiency. Exogenous lactate supplementation increased the stability of HIF1α and promoted adipogenesis. PDK1/2 overexpression-mediated adipogenesis was abolished by HIF1α inhibition, suggesting a role for the PDK-lactate-HIF1α axis during adipogenesis. In human adipose tissue, the expression of PDK1/2 was positively correlated with that of the adipogenic marker PPARγ and inversely correlated with obesity. Similarly, PDK1/2 expression in mouse adipose tissue was decreased by chronic high-fat diet feeding. We conclude that PDK1 and 2 are novel regulators of adipogenesis that play critical roles in obesity.Subject terms: Mechanisms of disease, Obesity     

Antihypoxic activity of adaptaquin enantiomers

Russian Chemical Bulletin - Adaptaquin, 7-{(4-chlorophenyl)[(3-hydroxypyridin-2-yl)amino]methyl}quinolin-8-ol (1), was identified in our earlier study as a hypoxia-inducible factor (HIF) prolyl...     

Unfolding mechanism of PHD2 as a vital protein: all-atom simulation approach

Prolyl hydroxylase domain 2 containing protein (PHD2) is a central protein in regulation of cellular response to hypoxia. This protein controls the responses of cell to oxygen level via the regulation of hypoxia inducible factor (HIF) stability. HIF induces the expression of many genes, especially ones orchestrate angiogenesis. There are some reports that mentioned in some tumor types the level of HIF is high in spite of the presence of wild-type PHD2 and normoxic environment. Therefore, the possibility of PHD2 misfolding in some cancer cells arises. Studying such important protein unfolding pathway is insightful for possible therapeutic approaches. In this study, the unfolding pathway of PHD2 illustrates utilizing molecular dynamics simulation of protein thermal denaturation. Based on current study results, we represent the possible mechanisms of PHD2 unfolding in detail. The possible intermediates of PHD2 thermal unfolding are characterized, and the most venomous state of its unfolding pathway is introduced.     

The disubstituted adamantyl derivative LW1564 inhibits the growth of cancer cells by targeting mitochondrial respiration and reducing hypoxia-inducible factor (HIF)-1α accumulation

Targeting cancer metabolism has emerged as an important cancer therapeutic strategy. Here, we describe the synthesis and biological evaluation of a novel class of hypoxia-inducible factor (HIF)-1α inhibitors, disubstituted adamantyl derivatives. One such compound, LW1564, significantly suppressed HIF-1α accumulation and inhibited the growth of various cancer cell lines, including HepG2, A549, and HCT116. Measurements of the oxygen consumption rate (OCR) and ATP production rate revealed that LW1564 suppressed mitochondrial respiration, thereby increasing the intracellular oxygen concentration to stimulate HIF-1α degradation. LW1564 also significantly decreased overall ATP levels by inhibiting mitochondrial electron transport chain (ETC) complex I and downregulated mammalian target of rapamycin (mTOR) signaling by increasing the AMP/ATP ratio, which increased AMP-activated protein kinase (AMPK) phosphorylation. Consequently, LW1564 promoted the phosphorylation of acetyl-CoA carboxylase, which inhibited lipid synthesis. In addition, LW1564 significantly inhibited tumor growth in a HepG2 mouse xenograft model. Taken together, the results indicate that LW1564 inhibits the growth of cancer cells by targeting mitochondrial ETC complex I and impairing cancer cell metabolism. We, therefore, suggest that LW1564 may be a potent therapeutic agent for a subset of cancers that rely on oxidative phosphorylation for ATP generation.Subject terms: Cancer metabolism, Cancer metabolism, Drug development     

Synthesis of the HIF-2α translation inhibitor compound 76 via a Japp-Klingemann coupling

The Iliopoulos laboratory previously reported the discovery of compound 76, a small molecule HIF2α inhibitor. HIF2α activation is known to play a critical role in both von Hippel-Lindau (VHL) disease-related tumors and sporadic renal cell carcinoma (RCC). We required a facile and scalable synthetic route to confirm the structure, to investigate the biological properties of compound 76, and to perform structure activity relationship optimization studies. Herein, we report the straightforward synthesis of compound 76 and a preliminary investigation into the scope of the methods developed.     

Targeting tumour angiogenesis with small molecule inhibitors of hypoxia inducible factor

The adaptation of tumours to hypoxia is critical for their survival and growth. The high proliferation rate of solid tumours causes the continuous outstripping of the oxygen supply provided by the local vasculature, resulting in hypoxic regions within the tumour. Hypoxia inducible factor (HIF) is the key mediator of cellular response to hypoxia, activating the expression of multiple genes that participate in angiogenesis, iron metabolism, glycolysis, glucose transport and cell proliferation and survival. The critical role of the hypoxia response network and HIF in cancer has resulted in it being viewed as an ideal target for small molecule intervention. In this tutorial review we discuss the central role of HIF in the adaptation of tumours to a hypoxic environment, going on to describe recent attempts at developing small molecules that disrupt this pathway and their potential for use as the next generation anticancer therapeutics.     

1,3-取代吲唑类低氧诱导因子l抑制剂的设计合成及其抗肝癌活性

低氧诱导因子l(HIF-1)与肿瘤细胞的生长、侵袭和耐药密切相关,在肿瘤细胞内HIF-1高度表达,因此新型的HIF-1抑制剂可作为潜在的抗肿瘤药物。 本文合成了9个1,3-取代吲唑衍生物。 通过蛋白质印迹(Western Blot)法及实时定量荧光PCR(Real time-PCR)等方法检测了其对HIF-1及其靶基因血管内皮生长因子(VEGF)表达水平的影响,并以3-(5'-羟甲基-2'-呋喃基)-1-苯甲基吲唑(YC-1)为阳性对照药物初步评价了其体外抗肝癌细胞增殖的生物活性。 实验发现化合物7b可显著抑制HIF-1及其下游靶基因VEGF的表达,且体外抗肝癌增殖生物活性优于YC-1,半抑制浓度(IC₅₀)值为10.37 μmol/L。 研究结果表明,3-(5'-羟甲基-2'-呋喃)-1-(1″-对甲苯磺酰基)吲唑具有靶向抑制HIF及良好的抗肝癌活性作用。     

Screening prolyl hydroxylase domain 2 inhibitory activity of traditional Chinese medicine by CZE-UV

Prolyl hydroxylase domain 2 (PHD2) is a key enzyme regulating the expression of hypoxia inducible factor (HIF). Its inhibitors can improve the expression of HIF and downstream genes, which can treat hypoxia-related diseases. Therefore, the establishment of a reliable PHD2 inhibitors screening method is of great significance for the drug development of hypoxia-related diseases. In this work, an accurate, rapid, and simple screening method for PHD2 inhibitors was introduced by capillary zone electrophoresis (CZE). In order to improve the detection sensitivity, the derivative reaction of α-ketoglutaric acid (α-OG) and 1,2-diaminobenzene (OPD) was used to enhance the UV absorption of α-OG (the substrate in the enzymatic reaction). The CZE method selected 20 mM Na₂B₄O₇ buffer (pH 9.0) as the separation buffer, +25 kV as the separation voltage, 25°C as the cartridge temperature, and 210 nm as the detection wavelength. Under this condition, the analysis of a single sample can be realized within 9 min. Compared with the existing reported methods, the present work can directly screen the PHD2 inhibitory activity of traditional Chinese medicine (TCM) extracts, which is of significance for the target-purification of bioactive individual compounds from TCMs. Under the optimal conditions, the PHD2 inhibitor screening platform was successfully established, and it was found that 70% methanol/water extracts of Astragali Radix and Codonopsis pilosula had good PHD2 inhibitory activity. Furthermore, the present work provides a novel approach for screening the PHD2 inhibitory activity of TCM extracts and the discovery of anti-hypoxia bioactive compounds.     

Effects of Angiotensin II on Erythropoietin Production in the Kidney and Liver

The kidney is a main site of erythropoietin production in the body. We developed a new method for the detection of Epo protein by deglycosylation-coupled Western blotting. Detection of deglycosylated Epo enables the examination of small changes in Epo production. Using this method, we investigated the effects of angiotensin II (ATII) on Epo production in the kidney. ATII stimulated the plasma Epo concentration; Epo, HIF2α, and PHD2 mRNA expression in nephron segments in the renal cortex and outer medulla; and Epo protein expression in the renal cortex. In situ hybridization and immunohistochemistry revealed that ATII stimulates Epo mRNA and protein expression not only in proximal tubules but also in collecting ducts, especially in intercalated cells. These data support the regulation of Epo production in the kidney by the renin–angiotensin–aldosterone system (RAS).     

Metabolite Profiling and Characterization of LW6, a Novel HIF-1α Inhibitor,as an Antitumor Drug Candidate in Mice

A novel HIF (hypoxia-inducible factor)-1α inhibitor, the (aryloxyacetylamino)benzoic acid derivative LW6, is an anticancer agent that inhibits the accumulation of HIF-1α. The aim of this study was to characterize and determine the structures of the metabolites of LW6 in ICR mice. Metabolite identification was performed using a predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (pMRM-IDA-EPI) method in negative ion mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (QTRAP). A total of 12 metabolites were characterized based on their MS/MS spectra, and the retention times were compared with those of the parent compound. The metabolites were divided into five structural classes based on biotransformation reactions: amide hydrolysis, ester hydrolysis, mono-oxidation, glucuronidation, and a combination of these reactions. From this study, 2-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)acetic acid (APA, M7), the metabolite produced via amide hydrolysis, was found to be a major circulating metabolite of LW6 in mice. The results of this study can be used to improve the pharmacokinetic profile by lowering the clearance and increasing the exposure relative to LW6.