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Background: Hidradenitis suppurativa (HS) is a chronic inflammatory skin condition of the terminal hair follicle, which can present in sporadic, familial, or syndromic forms. The exact pathogenesis of HS remains elusive, posing a challenge for the development of effective treatments. Among the various immunological mechanisms, the NLRP3 inflammasome is thought to contribute to the pathogenesis of HS, releasing cytokines such as IL-1β and IL-18 which initiates and exacerbates inflammation. Consequently, targeting NLRP3 offers a potential strategy for mitigating inflammation in HS-affected skin., Methods: In this study we used the docking, molecular dynamics simulation and binding free energy approaches to identify the potent inhibitor of NLRP3 by screening the African phytocompounds and traditional Chinese medicine databases., Results: Our virtual drug screening analysis identified two lead compounds from each database, characterized by high docking scores such as SA-21676268 (-8.135 kcal/mol), SA-167673 (-10.251 kcal/mol), EA-45360194 (-10.376 kcal/mol), EA-46881231 (-10.011 kcal/mol), NEA-44258150 (-9.856 kcal/mol), NEA-135926572 (-7.662 kcal/mol), NA-163089376 (-9.237 kcal/mol), NA-440735 (-8.826 kcal/mol), TCM-392442 (-10.438 kcal/mol), and TCM-10043097 (-9.046 kcal/mol) which highlighted the strong binding affinity as compared to the control NP3-146 drug (-5.09 kcal/mol). Moreover, the values of dissociation constant further validated the strong binding affinity between the identified lead compounds and NLRP3. The dynamic stability and strong bonding energies of the lead compounds-NLRP3 complexes were confirmed by the molecular dynamic simulation and binding free energy calculation. The analysis of ADMET properties for all compounds indicated high intestinal absorption, water solubility, absence of hepatotoxicity, and skin sensitivity., Conclusion: In conclusion, our molecular simulations and binding free energy calculations confirmed the strong affinity of these lead compounds for NLRP3 as compared to the control drug, highlighting their potential as part of a combinatorial therapeutic strategy for HS to effectively reduce disease-related inflammation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Introduction: Ebola virus (EBOV) is a highly lethal RNA virus that causes severe hemorrhagic fever in humans and non-human primates. The lack of effective treatment or vaccine for this pathogen poses a serious threat to a global pandemic. Therefore, it is imperative to explore new drugs and therapies to combat this life-threatening infection., Materials and Methods: In this study, we employed in silico methods to assess the inhibitory activity of natural products from traditional Chinese medicine (TCM) against four EBOV proteins that are crucial for viral replication and assembly: VP40, VP35, VP30, and VP24. We performed molecular docking of TCM compounds with the EBOV proteins and screened them based on their docking scores, binding free energies, and pharmacokinetic properties., Results: Our results pinpointed eight TCM compounds (TCM1797, TCM2872, TCM250, TCM2837, TCM2644, TCM4697, TCM2322, and TCM277) that exhibited superior efficacy in inhibiting all the EBOV proteins compared to the controls. These compounds interacted with key residues of the EBOV proteins through various types of bonds, such as hydrogen bonds, salt bridges, and π-π interactions, forming stable complexes that could disrupt the function of the EBOV proteins. These compounds were found to possess known antiviral activity, acceptable pharmacokinetic properties, and human usage history, which make them promising candidates for anti-EBOV drug development. Moreover, the molecular simulation analysis confirmed the binding stability, structural compactness, and residue flexibility properties of these compounds. Furthermore, the binding free energy results revealed that VP30-TCM2644, VP30-TCM4697, VP35-TCM2837, VP24-TCM250, and VP24-TCM277 complexes exhibit significant binding free energy values compared to the control ligands. Principal Component Analysis (PCA) and Free Energy Landscape (FEL) results revealed the trajectories' motion and conformational energy states., Conclusions: Our findings provide valuable insights into the molecular mechanisms driving the efficacy of TCM drugs against EBOV and suggest novel approaches for the development of anti-EBOV therapies., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024. Published by Elsevier Ltd.)

Hypoxia-inducible factors (HIFs) are transcription factors that regulate erythropoietin (EPO) synthesis and red blood cell (RBC) production. Prolyl-4-hydroxylase domain (PHD) enzymes are key regulators of HIF's stability and activity. Inhibiting PHD enzymes can enhance HIF-mediated responses and have therapeutic potential for diseases such as anemia, cancer, stroke, ischemia, neurodegeneration, and inflammation. In this study, we searched for novel PHD inhibitors from four databases of natural products and synthetic compounds: AfroDb Natural Products, AnalytiCon Discovery Natural Product (NP), HIM-Herbal Ingredients In-Vivo Metabolism, and Herbal Ingredients' Targets, with a total number of 13,597 compounds. We screened the candidate compounds by molecular docking and validated them by molecular dynamics simulations and free energy calculations. We identified four target hits (ZINC36378940, ZINC2005305, ZINC31164438, and ZINC67910437) that showed stronger binding affinity to PHD2 compared to the positive control, Vadadustat (AKB-6548), with docking scores of - 13.34 kcal/mol, - 12.76 kcal/mol, - 11.96 kcal/mol, - 11.41 kcal/mol, and - 9.04 kcal/mol, respectively. The target ligands chelated the active site iron and interacted with key residues (Arg 383, Tyr329, Tyr303) of PHD2, in a similar manner as Vadadustat. Moreover, the dynamic stability-based assessment revealed that they also exhibited stable dynamics and compact trajectories. Then the total binding free energy was calculated for each complex which revealed that the control has a TBE of - 31.26 ± 0.30 kcal/mol, ZINC36378940 reported a TBE of - 38.65 ± 0.51 kcal/mol, for the ZINC31164438 the TBE was - 26.16 ± 0.30 kcal/mol while the ZINC2005305 complex reported electrostatic energy of - 32.75 ± 0.58 kcal/mol. This shows that ZINC36378940 is the best hit than the other and therefore further investigation should be performed for the clinical usage. Our results suggest that these target hits are promising candidates that reserve further in vitro and in vivo validations as potential PHD inhibitors for the treatment of renal anemia, cancer, stroke, ischemia, neurodegeneration, and inflammation., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Because of the essential role of PLpro in the regulation of replication and dysregulation of the host immune sensing, it is considered a therapeutic target for novel drug development. To reduce the risk of immune evasion and vaccine effectiveness, small molecular therapeutics are the best complementary approach. Hence, we used a structure-based drug-designing approach to identify potential small molecular inhibitors for PLpro of SARS-CoV-2. Initial scoring and re-scoring of the best hits revealed that three compounds NPC320891 (2,2-Dihydroxyindene-1,3-Dione), NPC474594 (Isonarciclasine), and NPC474595 (7-Deoxyisonarciclasine) exhibit higher docking scores than the control GRL0617. Investigation of the binding modes revealed that alongside the essential contacts, i.e., Asp164, Glu167, Tyr264, and Gln269, these molecules also target Lys157 and Tyr268 residues in the active site. Moreover, molecular simulation demonstrated that the reported top hits also possess stable dynamics and structural packing. Furthermore, the residues' flexibility revealed that all the complexes demonstrated higher flexibility in the regions 120-140, 160-180, and 205-215. The 120-140 and 160-180 lie in the finger region of PLpro, which may open/close during the simulation to cover the active site and push the ligand inside. In addition, the total binding free energy was reported to be - 32.65 ± 0.17 kcal/mol for the GRL0617-PLpro, for the NPC320891-PLpro complex, the TBE was - 35.58 ± 0.14 kcal/mol, for the NPC474594-PLpro, the TBE was - 43.72 ± 0.22 kcal/mol, while for NPC474595-PLpro complex, the TBE was calculated to be - 41.61 ± 0.20 kcal/mol, respectively. Clustering of the protein's motion and FEL further revealed that in NPC474594 and NPC474595 complexes, the drug was seen to have moved inside the binding cavity along with the loop in the palm region harboring the catalytic triad, thus justifying the higher binding of these two molecules particularly. In conclusion, the overall results reflect favorable binding of the identified hits strongly than the control drug, thus demanding in vitro and in vivo validation for clinical purposes., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Previous empirical studies have shown that both spiritual well-being (SWB) and death anxiety (DA) significantly affect the mental health of patients with acute diseases. In this regard, our paper contributes to the extant literature by scrutinizing the conditional relationship between SWB and DA as well as the various mechanisms underpinning such a relationship in patients with acute myocardial infraction (AMI). A descriptive, correlational methodology was utilized. Our main sample consisted of 300 patients with acute myocardial infraction who were hospitalized in a specialized medical institution in Iran throughout a two-month period (i.e. August-October 2015). Patients completed Spiritual Well-Being Scale (SWBS) and Templer's Death Anxiety Scale (TDAS). Even though our study showed that the relationship between SWB and DA in patients with AMI is non-significant, we found that (1) single patients with higher SWB have lower DA, (2) single patients with higher SWB as well as social support have significantly lower DA, and (3) for single men/men without social support, there is a negative relationship between SWB and DA. The relationship between SWB and DA is influenced by factors such as sex, marital status and social support. In addition, the specific nature of this relationship (i.e. strength and sign) is dependent upon the sociodemographic characteristics of patients as well as other contextual influences. Result revealed that although relationship between SWB and DA is non-significant, this is influenced by factors such as sex and social support. In addition, the specific nature of this relationship (i.e. strength and sign) is dependent upon the sociodemographic characteristics of patients as well as other contextual influences.

Nonpharmacological methods of pain relief such as acupressure are becoming increasingly popular worldwide. Practitioners often claim that such methods are highly effective and less invasive than analgesic drugs, but available empirical evidence is largely inconclusive. This study aimed to test the hypothesis that, postacupressure intervention, the amount of pain experienced by sampled leukemia patients, who received acupressure in addition to standard care, will reduce substantially compared to patients who received only routine treatment. This controlled trial was undertaken with hospitalized leukemia patients between February and June 2015. Our primary sample consisted of 100 participants who were randomly allocated to 2 groups. One (intervention) group underwent 12 acupressure sessions in addition to standard treatment while the control group received no intervention apart from being given the standard treatment for leukemia. The visual analogue scale was used to measure the levels of pain experienced. No significant differences were found between the 2 groups across 12 interventions. However, each group reported significantly different pain level changes before and after each intervention, suggested that the acupressure method was effective in reducing pain in the short term. Nurses are able to apply this complementary therapy alongside other procedures to manage these patients' pain. It is recommended that further studies be conducted to better understand the specific conditions under which acupressure can provide effective pain relief., (© 2017 John Wiley & Sons Australia, Ltd.)

The role of peroxisome proliferator-activated receptor α (PPARα)-mediated metabolic remodeling in cardiac adaptation to hypoxia has yet to be defined. Here, mice were housed in hypoxia for 3 wk before in vivo contractile function was measured using cine MRI. In isolated, perfused hearts, energetics were measured using (31)P magnetic resonance spectroscopy (MRS), and glycolysis and fatty acid oxidation were measured using [(3)H] labeling. Compared with a normoxic, chow-fed control mouse heart, hypoxia decreased PPARα expression, fatty acid oxidation, and mitochondrial uncoupling protein 3 (UCP3) levels, while increasing glycolysis, all of which served to maintain normal ATP concentrations ([ATP]) and thereby, ejection fractions. A high-fat diet increased cardiac PPARα expression, fatty acid oxidation, and UCP3 levels with decreased glycolysis. Hypoxia was unable to alter the high PPARα expression or reverse the metabolic changes caused by the high-fat diet, with the result that [ATP] and contractile function decreased significantly. The adaptive metabolic changes caused by hypoxia in control mouse hearts were found to have occurred already in PPARα-deficient (PPARα(-/-)) mouse hearts and sustained function in hypoxia despite an inability for further metabolic remodeling. We conclude that decreased cardiac PPARα expression is essential for adaptive metabolic remodeling in hypoxia, but is prevented by dietary fat.-Cole, M. A., Abd Jamil, A. H., Heather, L. C., Murray, A. J., Sutton, E. R., Slingo, M., Sebag-Montefiore, L., Tan, S. C., Aksentijević, D., Gildea, O. S., Stuckey, D. J., Yeoh, K. K., Carr, C. A., Evans, R. D., Aasum, E., Schofield, C. J., Ratcliffe, P. J., Neubauer, S., Robbins, P. A., Clarke, K. On the pivotal role of PPARα in adaptation of the heart to hypoxia and why fat in the diet increases hypoxic injury., (© FASEB.)

Cardiosphere-derived cells (CDCs), which can be isolated from heart explants, are a promising candidate cell source for infarcted myocardium regeneration. However, current protocols used to expand CDCs require at least 1 month in vitro to obtain sufficient cells for transplantation. We report that CDC culture can be optimized by preconditioning the cells under hypoxia (2% oxygen), which may reflect the physiological oxygen level of the stem cell niche. Under hypoxia, the CDC proliferation rate increased by 1.4-fold, generating 6 × 10(6) CDCs with higher expression of cardiac stem cell and pluripotency gene markers compared to normoxia. Furthermore, telomerase (TERT), cytokines/ligands involved in stem cell trafficking (SDF/CXCR-4), erythropoiesis (EPO), and angiogenesis (VEGF) were increased under hypoxia. Hypoxic preconditioning was mimicked by treatment with two types of hypoxia-inducible factor (HIF) prolyl-4-hydroxylase inhibitors (PHDIs): dimethyloxaloylglycine (DMOG) and 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetic acid (BIC). Despite the difference in specificity, both PHDIs significantly increased c-Kit expression and activated HIF, EPO, and CXCR-4. Furthermore, treatment with PHDIs for 24 h increased cell proliferation. Notably, all hypoxic and PHDI-preconditioned CDCs had decreased oxygen consumption and increased glycolytic metabolism. In conclusion, cells cultured under hypoxia could have potentially enhanced therapeutic potential, which can be mimicked, in part, by PHDIs.

This study investigated the effects of 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetic acid (IOX3), a selective small molecule inhibitor of hypoxia-inducible factor (HIF) prolyl hydroxylases, on mouse brains subject to transient focal cerebral ischaemia. Male, 8- to 12-week-old C57/B6 mice were subjected to 45 min of middle cerebral artery occlusion (MCAO) either immediately or 24 h after receiving IOX3. Mice receiving IOX3 at 20 mg/kg 24 h prior to the MCAO had better neuroscores and smaller blood-brain barrier (BBB) disruption and infarct volumes than mice receiving the vehicle, whereas those having IOX3 at 60 mg/kg showed no significant changes. IOX3 treatment immediately before MCAO was not neuroprotective. IOX3 up-regulated HIF-1α, and increased EPO expression in mouse brains. In an in vitro BBB model (RBE4 cell line), IOX3 up-regulated HIF-1α and delocalized ZO-1. Pre-treating IOX3 on RBE4 cells 24 h before oxygen-glucose deprivation had a protective effect on endothelial barrier preservation with ZO-1 being better localized, while immediate IOX3 treatment did not. Our study suggests that HIF stabilization with IOX3 before cerebral ischaemia is neuroprotective partially because of BBB protection, while immediate application could be detrimental. These results provide information for studies aimed at the therapeutic activation of HIF pathway for neurovascular protection from cerebral ischaemia. We show that IOX3, a selective small molecule (280.66 Da) HIF prolyl hydroxylase inhibitor, could up-regulate HIF-1α and increase erythropoietin expression in mice. We further demonstrate that HIF stabilization with IOX3 before cerebral ischaemia is neuroprotective partially because of blood-brain barrier (BBB) protection, while immediate application is detrimental both in vivo and in vitro. These findings provide new insights into the role of HIF stabilization in ischaemic stroke., (© 2014 International Society for Neurochemistry.)

2-Oxoglutarate and iron dependent oxygenases are therapeutic targets for human diseases. Using a representative 2OG oxygenase panel, we compare the inhibitory activities of 5-carboxy-8-hydroxyquinoline (IOX1) and 4-carboxy-8-hydroxyquinoline (4C8HQ) with that of two other commonly used 2OG oxygenase inhibitors, N -oxalylglycine (NOG) and 2,4-pyridinedicarboxylic acid (2,4-PDCA). The results reveal that IOX1 has a broad spectrum of activity, as demonstrated by the inhibition of transcription factor hydroxylases, representatives of all 2OG dependent histone demethylase subfamilies, nucleic acid demethylases and γ-butyrobetaine hydroxylase. Cellular assays show that, unlike NOG and 2,4-PDCA, IOX1 is active against both cytosolic and nuclear 2OG oxygenases without ester derivatisation. Unexpectedly, crystallographic studies on these oxygenases demonstrate that IOX1, but not 4C8HQ, can cause translocation of the active site metal, revealing a rare example of protein ligand-induced metal movement.

The hypoxia inducible factor (HIF) system is central to the signaling of low oxygen (hypoxia) in animals. The levels of HIF-α isoforms are regulated in an oxygen-dependent manner by the activity of the HIF prolyl-hydroxylases (PHD or EGLN enzymes), which are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. Here, we describe biochemical, crystallographic, cellular profiling, and animal studies on PHD inhibitors including selectivity studies using a representative set of human 2OG oxygenases. We identify suitable probe compounds for use in studies on the functional effects of PHD inhibition in cells and in animals.

The fat mass and obesity associated protein (FTO) is a potential target for anti-obesity medicines. FTO is a 2-oxoglutarate (2OG)-dependent N-methyl nucleic acid demethylase that acts on substrates including 3-methylthymidine, 3-methyluracil, and 6-methyladenine. To identify FTO inhibitors, we screened a set of 2OG analogues and related compounds using differential scanning fluorometry- and liquid chromatography-based assays. The results revealed sets of both cyclic and acyclic 2OG analogues that are FTO inhibitors. Identified inhibitors include small molecules that have been used in clinical studies for the inhibition of other 2OG oxygenases. Crystallographic analyses reveal inhibition by 2OG cosubstrate or primary substrate competitors as well as compounds that bind across both cosubstrate and primary substrate binding sites. The results will aid the development of more potent and selective FTO inhibitors.

Inhibition of the hypoxia-inducible factor (HIF) prolyl hydroxylases (PHD or EGLN enzymes) is of interest for the treatment of anemia and ischemia-related diseases. Most PHD inhibitors work by binding to the single ferrous ion and competing with 2-oxoglutarate (2OG) co-substrate for binding at the PHD active site. Non-specific iron chelators also inhibit the PHDs, both in vitro and in cells. We report the identification of dual action PHD inhibitors, which bind to the active site iron and also induce the binding of a second iron ion at the active site. Following analysis of small-molecule iron complexes and application of non-denaturing protein mass spectrometry to assess PHD2·iron·inhibitor stoichiometry, selected diacylhydrazines were identified as PHD2 inhibitors that induce the binding of a second iron ion. Some compounds were shown to inhibit the HIF hydroxylases in human hepatoma and renal carcinoma cell lines.

The JmjC oxygenases catalyze the N-demethylation of N(ε)-methyl lysine residues in histones and are current therapeutic targets. A set of human 2-oxoglutarate analogues were screened using a unified assay platform for JmjC demethylases and related oxygenases. Results led to the finding that daminozide (N-(dimethylamino)succinamic acid, 160 Da), a plant growth regulator, selectively inhibits the KDM2/7 JmjC subfamily. Kinetic and crystallographic studies reveal that daminozide chelates the active site metal via its hydrazide carbonyl and dimethylamino groups.

Chronic hypoxia decreases cardiomyocyte respiration, yet the mitochondrial mechanisms remain largely unknown. We investigated the mitochondrial metabolic pathways and enzymes that were decreased following in vivo hypoxia, and questioned whether hypoxic adaptation was protective for the mitochondria. Wistar rats were housed in hypoxia (7 days acclimatisation and 14 days at 11% oxygen), while control rats were housed in normoxia. Chronic exposure to physiological hypoxia increased haematocrit and cardiac vascular endothelial growth factor, in the absence of weight loss and changes in cardiac mass. In both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria isolated from hypoxic hearts, state 3 respiration rates with fatty acid were decreased by 17-18%, and with pyruvate were decreased by 29-15%, respectively. State 3 respiration rates with electron transport chain (ETC) substrates were decreased only in hypoxic SSM, not in hypoxic IFM. SSM from hypoxic hearts had decreased activities of ETC complexes I, II and IV, which were associated with decreased reactive oxygen species generation and protection against mitochondrial permeability transition pore (MPTP) opening. In contrast, IFM from hypoxic hearts had decreased activity of the Krebs cycle enzyme, aconitase, which did not modify ROS production or MPTP opening. In conclusion, cardiac mitochondrial respiration was decreased following chronic hypoxia, associated with downregulation of different pathways in the two mitochondrial populations, determined by their subcellular location. Hypoxic adaptation was not deleterious for the mitochondria, in fact, SSM acquired increased protection against oxidative damage under the oxygen-limited conditions.

Infarction irreversibly damages the heart, with formation of an akinetic scar that may lead to heart failure. Endogenous cardiac stem cells (CSCs) are a promising candidate cell source for restoring lost tissue and thereby preventing heart failure. CSCs may be isolated in vitro, via the formation of cardiospheres, to give cardiosphere-derived cells (CDCs). Although qRT-PCR analyses of CDCs have been performed, no justification for the selection of the housekeeping gene has been published. Here, we evaluated the most suitable housekeeping gene for RNA expression analysis in CDCs cultured under normoxia, hypoxia or with prolyl-4-hydroxylase inhibitors (PHDIs), from both neonatal and adult rats, to determine the effects of ageing and different culture conditions on the stability of the housekeeping gene for CDCs. Six candidate housekeeping genes, [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (Actb), hypoxanthine phosphoribosyltransferase 1 (HPRT-1), beta-2-microtubulin (β2M), 60S acidic ribosomal protein large P1 (RPLP-1) and TATA box binding protein (Tbp)] were evaluated in this study. Analysis using geNorm and NormFinder revealed that GAPDH was the most constant housekeeping gene among all genes tested under normoxia for both neonatal and adult CDCs, whereas Actb was the most stable housekeeping gene under hypoxia. For the PHDI-treated CDCs, overall, GADPH, Actb and β2M were more consistently expressed, whereas HPRT-1, RPLP-1 and Tbp showed unstable expression. The ranking for β2M, HPRT-1 and RPLP-1 stability was different for neonatal and adult cells, indicating that expression of these genes was age-dependent. Lastly, independent of age or culture conditions, Tbp was the least stable housekeeping gene. In conclusion, a combination of Actb and GADPH gave the most reliable normalization for comparative analyses of gene transcription in neonatal and adult rat CDCs preconditioned by hypoxia or PHDIs.

Mutations in isocitrate dehydrogenases (IDHs) have a gain-of-function effect leading to R(-)-2-hydroxyglutarate (R-2HG) accumulation. By using biochemical, structural and cellular assays, we show that either or both R- and S-2HG inhibit 2-oxoglutarate (2OG)-dependent oxygenases with varying potencies. Half-maximal inhibitory concentration (IC(50)) values for the R-form of 2HG varied from approximately 25 μM for the histone N(ɛ)-lysine demethylase JMJD2A to more than 5 mM for the hypoxia-inducible factor (HIF) prolyl hydroxylase. The results indicate that candidate oncogenic pathways in IDH-associated malignancy should include those that are regulated by other 2OG oxygenases than HIF hydroxylases, in particular those involving the regulation of histone methylation.

Hypoxia inducible factor (HIF) is regulated by dual pathways involving oxygen-dependent prolyl and asparaginyl hydroxylation of its α-subunits. Prolyl hydroxylation at two sites within a central degradation domain promotes association of HIF-α with the von Hippel-Lindau ubiquitin E3 ligase and destruction by the ubiquitin-proteasome pathways. Asparaginyl hydroxylation blocks the recruitment of p300/CBP co-activators to a C-terminal activation domain in HIF-α. These hydroxylations are catalyzed by members of the Fe(II) and 2-oxoglutarate (2-OG) oxygenase family. Activity of the enzymes is suppressed by hypoxia, increasing both the abundance and activity of the HIF transcriptional complex. We have used hydroxy residue-specific antibodies to compare and contrast the regulation of each site of prolyl hydroxylation (Pro(402), Pro(564)) with that of asparaginyl hydroxylation (Asn(803)) in human HIF-1α. Our findings reveal striking differences in the sensitivity of these hydroxylations to hypoxia and to different inhibitor types of 2-OG oxygenases. Hydroxylation at the three sites in endogenous human HIF-1α proteins was suppressed by hypoxia in the order Pro(402) > Pro(564) > Asn(803). In contrast to some predictions from in vitro studies, prolyl hydroxylation was substantially more sensitive than asparaginyl hydroxylation to inhibition by iron chelators and transition metal ions; studies of a range of different small molecule 2-OG analogues demonstrated the feasibility of selectively inhibiting either prolyl or asparaginyl hydroxylation within cells.

The HIF (hypoxia-inducible factor) hydroxylases [PHDs or EGLNs (prolyl hydroxylases), which in humans are PHD isoforms 1-3, and FIH (factor inhibiting HIF)] regulate HIF levels and activity. These enzymes are Fe(II)/2-oxoglutarate-dependent oxygenases, many of which are stimulated by ascorbate. We have investigated the ascorbate dependence of PHD2-catalysed hydroxylation of two prolyl hydroxylation sites in human HIF-1alpha, and of FIH-catalysed hydroxylation of asparaginyl hydroxylation sites in HIF-1alpha and in a consensus ankyrin repeat domain peptide. The initial rate and extent of hydroxylation was increased in the presence of ascorbate for each of these reactions. When ascorbate was replaced with structural analogues, the results revealed that the ascorbate side chain was not important in its contribution to HIF hydroxylase catalysis, whereas modifications to the ene-diol portion of the molecule negated the ability to promote hydroxylation. We investigated whether alternative reducing agents (glutathione and dithiothreitol) could be used to promote HIF hydroxylase activity, and found partial stimulation of hydroxylation in an apparently enzyme- and substrate-specific manner. The results raise the possibility of developing reducing agents targeted to specific HIF hydroxylase-catalysed reactions.

This report demonstrates that solvent water relaxation measurements can be used for quantitative screening of ligand binding and for mechanistic investigations of enzymes containing paramagnetic metal centers by using conventional NMR instrumentation at high field. The method was exemplified using prolyl hydroxylase domain containing enzyme 2 (PHD2), a human enzyme involved in hypoxic sensing, with Mn(II) substituting for Fe(II) at the active site. K(D) values were determined for inhibitors that hinder access of water to the paramagnetic center. This technique is also useful for investigating the mechanism of suitable metalloenzymes, including order of ligand binding and modes of inhibition.

Limited proteolysis coupled to matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analyses can be used to screen for compounds that alter protein structure by monitoring stabilizing/destabilizing effects with respect to the rate and nature of proteolysis. When applied to prolyl hydroxylase 2, a key enzyme involved in human oxygen sensing, the method efficiently revealed differential effects on proteolytic stability for structurally similar compounds and for different substrates.

A series of potential UDP-sugar mimics were readily synthesised by copper(I) catalysed modified Huisgen cycloaddition of the corresponding alpha-propargyl glycosides with 5-azido uridine in aqueous solution. None of the compounds accessed displayed significant inhibitory activity at concentrations of up to 4.5mM in an assay against bovine milk beta-1,4-galactosyltransferase.