Automated sample preparation for the detection and confirmation of hypoxia-inducible factor stabilizers in urine.

As hypoxia-inducible factor stabilizers (HIFs) can artificially enhance an athlete's erythropoiesis, the World Anti-Doping Agency prohibits their use at all times. Every urine sample for doping control analysis has to be evaluated for the presence of HIFs and therefore sensitive methods that allow high sample throughput are needed. Samples suspicious for the presence of HIFs need to be confirmed following the identification criteria established by the World Anti-Doping Agency. Previous work has shown the advantages of using turbulent flow online solid-phase extraction (SPE) procedures to reduce matrix effects and retention time shifts. Furthermore, the use of online SPE allows for automation and high sample throughput. Both an initial testing procedure (ITP) and a confirmation method were developed and validated, using online SPE liquid chromatography-tandem mass spectrometry (LC-MS/MS), with limits of detection between 0.1 ng/ml (or possibly lower) and 4 ng/ml (or higher for GSK360a) and limits of identification between 0.1 ng/ml (or possibly lower) and 1.17 ng/ml. The ITP only takes 6.5 min per sample. To the best of our knowledge, these are the first ITP and confirmation methods that include more than three HIFs without the need for manual sample preparation.

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.

Effects of curcumin on hypoxia-inducible factor as a new therapeutic target.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that consists of two subunits, the HIF-1α and HIF-1ß (ARNT). Under hypoxic conditions, HIF-1 is an adaptive system that regulates the transcription of multiple genes associated with growth, angiogenesis, proliferation, glucose transport, metabolism, pH regulation and cell death. However, aberrant HIF-1 activation contributes to the pathophysiology of several human diseases such as cancer, ischemic cardiovascular disorders, and pulmonary and kidney diseases. A growing body of evidence indicates that curcumin, a natural bioactive compound of turmeric root, significantly targets both HIF-1 subunits, but is more potent against HIF-1α. In this review, we have summarized the knowledge about the pharmacological effects of curcumin on HIF-1 and the related molecular mechanisms that may be effective candidates for the development of multi-targeted therapy for several human diseases.

The effect of within-instar development on tracheal diameter and hypoxia-inducible factors α and ß in the tobacco hornworm, Manduca sexta.

As insects grow within an instar, body mass increases, often more than doubling. The increase in mass causes an increase in metabolic rate and hence oxygen demand. However, the insect tracheal system is hypothesized to increase only after molting and may be compressed as tissues grow within an instar. The increase in oxygen demand in the face of a potentially fixed or decreasing supply could result in hypoxia as insects near the end of an instar. To test these hypotheses, we first used synchrotron X-ray imaging to determine how diameters of large tracheae change within an instar and after molting to the next instar in the tobacco hornworm, Manduca sexta. Large tracheae did not increase in diameter within the first, second, third, and fourth instars, but increased upon molting. To determine if insects are hypoxic at the end of instars, we used the presence of hypoxia-inducible factors (HIFs) as an index. HIF-α and HIF-ß dimerize in hypoxia and act as a transcription factor that turns on genes that will increase oxygen delivery. We sequenced both of these genes and measured their mRNA levels at the beginning and end of each larval instar. Finally, we obtained an antibody to HIF-α and measured protein expression during the same time. Both mRNA and protein levels of HIFs were increased at the end of most instars. These data support the hypothesis that some insects may experience hypoxia at the end of an instar, which could be a signal for molting. SUMMARY STATEMENT: As caterpillars grow within an instar, major tracheae do not increase in size, while metabolic demand increases. At the same life stages, caterpillars increased expression of hypoxia inducible factors, suggesting that they become hypoxic near the end of an instar.

Investigations on the role of a solvent tunnel in the α-ketoglutarate dependent oxygenase factor inhibiting HIF (FIH).

Non-heme Fe(II)/α-ketoglutarate (αKG)-dependent oxygenases catalyze a wide array of reactions through coupling oxidative decarboxylation of αKG to substrate oxygenation. This class of enzymes follows a sequential mechanism in which O2 reacts only after binding primary substrate, raising questions over how protein structure tailors molecular access to the Fe(II) cofactor. The enzyme "factor inhibiting hypoxia inducible factor" (FIH) senses pO2 in human cells by hydroxylating the C-terminal transactivation domain (CTAD), suggesting that structural elements limiting molecular access to the active site may limit the pO2 response. In this study, we tested the impact of a solvent-accessible tunnel in FIH on molecular access to the active site in FIH. The size of the tunnel was increased through alanine point mutagenesis (Y93A, E105A, and Q147A), followed by a suite of mechanistic and spectroscopic probes. Steady-state kinetics varying O2 or CTAD indicated that O2 passage through the tunnel was not affected by Ala substitutions, allowing us to conclude that this narrow tunnel did not impact pO2 sensing by FIH. Steady-state kinetics with varied αKG concentrations revealed increased substrate inhibition for the Ala variants, suggesting that a second αKG molecule may bind near the active site of FIH. If this solvent-accessible tunnel is the O2 entry tunnel, it may be narrow in order to permit O2 access while preventing metabolic intermediates, such as αKG, from inhibiting FIH under physiological conditions.

Radiolabeled probes targeting hypoxia-inducible factor-1-active tumor microenvironments.

Because tumor cells grow rapidly and randomly, hypoxic regions arise from the lack of oxygen supply in solid tumors. Hypoxic regions in tumors are known to be resistant to chemotherapy and radiotherapy. Hypoxia-inducible factor-1 (HIF-1) expressed in hypoxic regions regulates the expression of genes related to tumor growth, angiogenesis, metastasis, and therapy resistance. Thus, imaging of HIF-1-active regions in tumors is of great interest. HIF-1 activity is regulated by the expression and degradation of its α subunit (HIF-1α), which is degraded in the proteasome under normoxic conditions, but escapes degradation under hypoxic conditions, allowing it to activate transcription of HIF-1-target genes. Therefore, to image HIF-1-active regions, HIF-1-dependent reporter systems and injectable probes that are degraded in a manner similar to HIF-1α have been recently developed and used in preclinical studies. However, no probe currently used in clinical practice directly assesses HIF-1 activity. Whether the accumulation of (18)F-FDG or (18)F-FMISO can be utilized as an index of HIF-1 activity has been investigated in clinical studies. In this review, the current status of HIF-1 imaging in preclinical and clinical studies is discussed.

Synthesis and biological evaluation of quinaldopeptin.

The second-generation total synthesis of quinaldopeptin (1) was established via a Staudinger/aza-Wittig/diastereoselective Ugi three-component reaction sequence and a racemization-free [5 + 5] coupling and macrolactamization. A single-crystal X-ray structure of the chromophore analogue 26 confirmed the structural and stereochemical assignments of the macrocycle. Synthetic 1 successfully unwound supercoiled DNA to form a relaxed DNA in a dose-dependent manner, the binding affinity of 1 to four dsODNs was within a similar range (K(b) = 1.45-2.53 × 10(7) M(-1)), and the sequence selectivity was subtle. It was suggested that 1 possesses biological behaviors similar to those of sandramycin (2) in terms of cytotoxic activity against human cancer cell lines (IC50 = 3.2-12 nM) and HIF-1 inhibitory activity.

Transcriptional regulation by hypoxia inducible factors.

The cellular response to oxygen deprivation is governed largely by a family of transcription factors known as Hypoxia Inducible Factors (HIFs). This review focuses on the molecular mechanisms by which HIFs regulate the transcriptional apparatus to enable the cellular and organismal response to hypoxia. We discuss here how the various HIF polypeptides, their posttranslational modifications, binding partners and transcriptional cofactors affect RNA polymerase II activity to drive context-dependent transcriptional programs during hypoxia.

Protein domain mimetics as in vivo modulators of hypoxia-inducible factor signaling.

Selective blockade of gene expression by designed small molecules is a fundamental challenge at the interface of chemistry, biology, and medicine. Transcription factors have been among the most elusive targets in genetics and drug discovery, but the fields of chemical biology and genetics have evolved to a point where this task can be addressed. Herein we report the design, synthesis, and in vivo efficacy evaluation of a protein domain mimetic targeting the interaction of the p300/CBP coactivator with the transcription factor hypoxia-inducible factor-1α. Our results indicate that disrupting this interaction results in a rapid down-regulation of hypoxia-inducible genes critical for cancer progression. The observed effects were compound-specific and dose-dependent. Gene expression profiling with oligonucleotide microarrays revealed effective inhibition of hypoxia-inducible genes with relatively minimal perturbation of nontargeted signaling pathways. We observed remarkable efficacy of the compound HBS 1 in suppressing tumor growth in the fully established murine xenograft models of renal cell carcinoma of the clear cell type. Our results suggest that rationally designed synthetic mimics of protein subdomains that target the transcription factor-coactivator interfaces represent a unique approach for in vivo modulation of oncogenic signaling and arresting tumor growth.

Hypoxia-inducible factor-1 (HIF-1): a potential target for intervention in ocular neovascular diseases.

Constant oxygen supply is essential for proper tissue development, homeostasis and function of all eukaryotic organisms. Cellular response to reduced oxygen levels is mediated by the transcriptional regulator hypoxia-inducible factor-1 (HIF-1). It is a heterodimeric complex protein consisting of an oxygen dependent subunit (HIF-1α) and a constitutively expressed nuclear subunit (HIF-1ß). In normoxic conditions, de novo synthesized cytoplasmic HIF-1α is degraded by 26S proteasome. Under hypoxic conditions, HIF-1α is stabilized, binds with HIF-1ß and activates transcription of various target genes. These genes play a key role in regulating angiogenesis, cell survival, proliferation, chemotherapy, radiation resistance, invasion, metastasis, genetic instability, immortalization, immune evasion, metabolism and stem cell maintenance. This review highlights the importance of hypoxia signaling in development and progression of various vision threatening pathologies such as diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration and glaucoma. Further, various inhibitors of HIF-1 pathway that may have a viable potential in the treatment of oxygen-dependent ocular diseases are also discussed.

Development of an oxygen-sensitive degradable peptide probe for the imaging of hypoxia-inducible factor-1-active regions in tumors.

PURPOSE: We aimed to develop a radiolabeled peptide probe for the imaging of hypoxia-inducible factor-1 (HIF-1)-active tumors. PROCEDURES: We synthesized the peptide probes that contain or lack an essential sequence of the oxygen-dependent degradation of HIF-1α in proteasomes ((123/125)I-DKOP30 or (125)I-mDKOP, respectively). The degradation of probes was evaluated in vitro using cell lysates containing proteasomes. In vivo biodistribution study, planar imaging, autoradiography, and comparison between probe accumulation and HIF-1 transcriptional activity were also performed. RESULTS: The (125)I-DKOP30 underwent degradation in a proteasome-dependent manner, while (125)I-mDKOP was not degraded. Biodistribution analysis showed (125)I-DKOP30 accumulation in tumors. The tumors were clearly visualized by in vivo imaging, and intratumoral distribution of (125)I-DKOP30 coincided with the HIF-1α-positive hypoxic regions. Tumoral accumulation of (125)I-DKOP30 was significantly correlated with HIF-1-dependent luciferase bioluminescence, while that of (125)I-mDKOP was not. CONCLUSION: (123)I-DKOP30 is a useful peptide probe for the imaging of HIF-1-active tumors.

Enhanceosomes as integrators of hypoxia inducible factor (HIF) and other transcription factors in the hypoxic transcriptional response.

Hypoxia is a prevalent attribute of the solid tumor microenvironment that promotes the expression of genes through posttranslational modifications and stabilization of alpha subunits (HIF1α and HIF2α) of hypoxia-inducible factors (HIFs). Despite significant similarities, HIF1 (HIF1α/ARNT) and HIF2 (HIF2α/ARNT) activate common as well as unique target genes and exhibit different functions in cancer biology. More surprisingly, accumulating data indicates that the HIF1- and/or HIF2-mediated hypoxia responses can be oncogenic as well as tumor suppressive. While the role of HIF in the hypoxia response is well established, recent data support the concept that HIF is necessary, but not sufficient for the hypoxic response. Other transcription factors that are activated by hypoxia are also required for the HIF-mediated hypoxia response. HIFs, other transcription factors, co-factors and RNA poll II recruited by HIF and other transcription factors form multifactorial enhanceosome complexes on the promoters of HIF target genes to activate hypoxia inducible genes. Importantly, HIF1 or HIF2 requires distinct partners in activating HIF1 or HIF2 target genes. Because HIF enhanceosome formation is required for the gene activation and distinct functions of HIF1 and HIF2 in tumor biology, disruption of the HIF1 or HIF2 specific enhanceosome complex may prove to be a beneficial strategy in tumor treatment in which tumor growth is specifically dependent upon HIF1 or HIF2 activity.

HIFs, angiogenesis, and cancer.

Tumor hypoxia was first described in the 1950s by radiation oncologists as a frequent cause of failure to radiotherapy in solid tumors. Today, it is evident that tumor hypoxia is a common feature of many cancers and the master regulator of hypoxia, hypoxia-inducible factor-1 (HIF-1), regulates multiple aspects of tumorigenesis, including angiogenesis, proliferation, metabolism, metastasis, differentiation, and response to radiation therapy. Although the tumor hypoxia response mechanism leads to a multitude of downstream effects, it is angiogenesis that is most crucial and also most susceptible to molecular manipulation. The delineation of molecular mechanisms of angiogenesis has revealed a critical role for HIF-1 in the regulation of angiogenic growth factors. In this article, we review what has been described about HIF-1: its structure, its regulation, and its implication for cancer therapy and we focus on its role in angiogenesis and cancer.

Catalytic mechanism and substrate specificity of HIF prolyl hydroxylases.

This review describes the catalytic mechanism, substrate specificity, and structural peculiarities of alpha-ketoglutarate dependent nonheme iron dioxygenases catalyzing prolyl hydroxylation of hypoxia-inducible factor (HIF). Distinct localization and regulation of three isoforms of HIF prolyl hydroxylases suggest their different roles in cells. The recent identification of novel substrates other than HIF, namely ß2-adrenergic receptor and the large subunit of RNA polymerase II, places these enzymes in the focus of drug development efforts aimed at development of isoform-specific inhibitors. The challenges and prospects of designing isoform-specific inhibitors are discussed.

Recent advances in hypoxia-inducible factor (HIF)-1 inhibitors.

Tumor hypoxia has been recognized as a common feature of solid tumors and a negative prognostic factor for response to treatment and survival of cancer patients. The discovery of hypoxia-inducible factor-1 (HIF-1), a molecular determinant of responses to hypoxia in mammalian cells, has renewed enthusiasm for discovery and development of targeted therapies exploiting the hypoxic tumor microenvironment. HIF-1 activity in tumors depends on availability of the HIF-1α subunit, the levels of which increase under hypoxic conditions and through activation of oncogenes and/or inactivation of tumor suppressor genes. Increased HIF-1 has been correlated with increased angiogenesis, aggressive tumor growth, and poor patient prognosis, leading to current interest in HIF-1 as promising anticancer drug target. In spite of an ever increasing number of putative small molecule inhibitors of HIF-1, only a few are progressing through preclinical and early clinical development. In this review, we will discuss recent advances in discovery and development of small molecule inhibitors that target the HIF-1 pathway as potential anticancer agents.

Identification of novel potential HIF-prolyl hydroxylase inhibitors by in silico screening.

Suppression of HIF-prolyl hydroxylase (PHD) activity by small-molecule inhibitors leads to the stabilization of hypoxia inducible factor and has been recognized as promising drug target for the treatment of ischemic diseases. In this study, pharmacophore-based virtual screening and molecular docking approaches were concurrently used with suitable modifications to identify target-specific PHD inhibitors with better absorption, distribution, metabolism, and excretion properties and to readily minimize false positives and false negatives. A customized method based on the active site information of the enzyme was used to generate a pharmacophore hypothesis (AAANR). The hypothesis was validated and utilized for chemical database screening and the retrieved hit compounds were subjected to molecular docking for further refinement. AAANR hypothesis comprised three H-bond acceptor, one negative ionizable group and one aromatic ring feature. The hypothesis was validated using decoy set with a goodness of fit score of 2 and was used as a 3D query for database screening. After manual selection, molecular docking and further refinement based on the molecular interactions of inhibitors with the essential amino acid residues, 18 hits with good absorption, distribution, metabolism, and excretion (ADME) properties were selected as excellent PHD inhibitors. The best pharmacophore hypothesis, AAANR, contains chemical features required for the effective inhibition of PHD. Using AAANR, we have identified 18 potential and diverse virtual leads, which can be readily evaluated for their potency as novel inhibitors of PHD. It can be concluded that the combination of pharmacophore, molecular docking, and manual interpretation approaches can be more successful than the traditional approach alone for discovering more effective inhibitors.

HIF-1α ODD polypeptides increased the expression of HIF1 and VEGF in hypoxic rat cortical neuron.

To investigate the effect of HIF-1α ODD polypeptides on the expression of HIF-1α and VEGF in primary rat cortical neuron in hypoxia. Primary cultured neurons were exposed to hypoxia for 2, 4, and 8 h. Tat-ODD fusion polypeptide was added before hypoxia in the experimental groups. The expression of HIF-1α and VEGF was detected by Western blot and real time PCR. The levels of HIF-1α and VEGF peaked at 4 h then declined in hypoxia. The expression of HIF-1α and VEGF was increased by tat-ODD fusion polypeptide. tat-ODD fusion polypeptide might improve HIF1 adaptation to hypoxia by increased expression of HIF-1α and VEGF.

Fentanyl activates hypoxia-inducible factor 1 in neuronal SH-SY5Y cells and mice under non-hypoxic conditions in a µ-opioid receptor-dependent manner.

Hypoxia-inducible factor 1 (HIF-1) is the main transcription factor responsible for hypoxia-induced gene expression. Perioperative drugs including anesthetics have been reported to affect HIF-1 activity. However, the effect of fentanyl on HIF-1 activity is not well documented. In this study, we investigated the effect of fentanyl and other opioids on HIF-1 activity in human SH-SY5Y neuroblastoma cells, hepatoma Hep3B cells, lung adenocarcinoma A549 cells and mice. Cells were exposed to fentanyl, and HIF-1 protein expression was examined by Western blot analysis using anti-HIF-1α and ß antibodies. HIF-1-dependent gene expression was investigated by semi-quantitative real-time reverse transcriptase (RT)-PCR (qRT-PCR) and luciferase assay. Furthermore, fentanyl was administered intraperitoneally and HIF-1-dependent gene expression was investigated by qRT-PCR in the brains and kidneys of mice. A 10-µM concentration of fentanyl and other opioids, including 1 µM morphine and 4 µM remifentanil, induced HIF-1α protein expression and HIF-1 target gene expression in an opioid receptor-dependent manner in SH-SY5Y cells with activity peaking at 24h. Fentanyl did not augment HIF-1α expression during hypoxia-induced induction. HIF-1α stabilization assays and experiments with cycloheximide revealed that fentanyl increased translation from HIF-1α mRNA but did not stabilize the HIF-1α protein. Furthermore, fentanyl induced HIF-1 target gene expression in the brains of mice but not in their kidneys in a naloxone-sensitive manner. In this report, we describe for the first time that fentanyl, both in vitro and in vivo, induces HIF-1 activation under non-hypoxic conditions, leading to increases in expression of genes associated with adaptation to hypoxia.