Structural basis for binding of the renal carcinoma target hypoxia-inducible factor 2α to prolyl hydroxylase domain 2.

The hypoxia-inducible factor (HIF) prolyl-hydroxylases (human PHD1-3) catalyze prolyl hydroxylation in oxygen-dependent degradation (ODD) domains of HIFα isoforms, modifications that signal for HIFα proteasomal degradation in an oxygen-dependent manner. PHD inhibitors are used for treatment of anemia in kidney disease. Increased erythropoietin (EPO) in patients with familial/idiopathic erythrocytosis and pulmonary hypertension is associated with mutations in EGLN1 (PHD2) and EPAS1 (HIF2α); a drug inhibiting HIF2α activity is used for clear cell renal cell carcinoma (ccRCC) treatment. We report crystal structures of PHD2 complexed with the C-terminal HIF2α-ODD in the presence of its 2-oxoglutarate cosubstrate or N-oxalylglycine inhibitor. Combined with the reported PHD2.HIFα-ODD structures and biochemical studies, the results inform on the different PHD.HIFα-ODD binding modes and the potential effects of clinically observed mutations in HIFα and PHD2 genes. They may help enable new therapeutic avenues, including PHD isoform-selective inhibitors and sequestration of HIF2α by the PHDs for ccRCC treatment.

Structural analysis of the 2-oxoglutarate binding site of the circadian rhythm linked oxygenase JMJD5.

JmjC (Jumonji-C) domain-containing 5 (JMJD5) plays important roles in circadian regulation in plants and humans and is involved in embryonic development and cell proliferation. JMJD5 is a 2-oxoglutarate (2OG) and Fe(II) dependent oxygenase of the JmjC subfamily, which includes histone Nε-methyl lysine-demethylases (KDMs) and hydroxylases catalysing formation of stable alcohol products. JMJD5 is reported to have KDM activity, but has been shown to catalyse C-3 hydroxylation of arginine residues in sequences from human regulator of chromosome condensation domain-containing protein 1 (RCCD1) and ribosomal protein S6 (RPS6) in vitro. We report crystallographic analyses of human JMJD5 complexed with 2OG analogues, including the widely used hypoxia mimic pyridine-2,4-dicarboxylate, both D- and L-enantiomers of the oncometabolite 2-hydroxyglutarate, and a cyclic N-hydroxyimide. The results support the assignment of JMJD5 as a protein hydroxylase and reveal JMJD5 has an unusually compact 2OG binding pocket suitable for exploitation in development of selective inhibitors. They will be useful in the development of chemical probes to investigate the physiologically relevant roles of JMJD5 in circadian rhythm and development and explore its potential as a medicinal chemistry target.

Novel Small Molecule Tyrosine Kinase 2 Pseudokinase Ligands Block Cytokine-Induced TYK2-Mediated Signaling Pathways.

A member of the Janus kinase (JAK) family, Tyrosine Kinase 2 (TYK2), is crucial in mediating various cytokine-signaling pathways such as interleukin-23 (IL23), interleukin-12 (IL12) and type I Interferons (IFN) which contribute to autoimmune disorders (e.g., psoriasis, lupus, and inflammatory bowel disease). Thus, TYK2 represents an attractive target to develop small-molecule therapeutics for the treatment of cytokine-driven inflammatory diseases. Selective inhibition of TYK2 over other JAK isoforms is critical to achieve a favorable therapeutic index in the development of TYK2 inhibitors. However, designing small molecule inhibitors to target the adenosine triphosphate (ATP) binding site of TYK2 kinase has been challenging due to the substantial structural homology of the JAK family catalytic domains. Here, we employed an approach to target the JAK homology 2 (JH2) pseudokinase regulatory domain of the TYK2 protein. We developed a series of small-molecule TYK2 pseudokinase ligands, which suppress the TYK2 catalytic activity through allosteric regulation. The TYK2 pseudokinase-binding small molecules in this study simultaneously achieve high affinity-binding for the TYK2 JH2 domain while also affording significantly reduced affinity for the TYK2 JAK homology 1 (JH1) kinase domain. These TYK2 JH2 selective molecules, although possessing little effect on suppressing the catalytic activity of the isolated TYK2 JH1 catalytic domain in the kinase assays, can still significantly block the TYK2-mediated receptor-stimulated pathways by binding to the TYK2 JH2 domain and allosterically regulating the TYK2 JH1 kinase. These compounds are potent towards human T-cell lines and primary immune cells as well as in human whole-blood specimens. Moreover, TYK2 JH2-binding ligands exhibit remarkable selectivity of TYK2 over JAK isoforms not only biochemically but also in a panel of receptor-stimulated JAK1/JAK2/JAK3-driven cellular functional assays. In addition, the TYK2 JH2-targeting ligands also demonstrate high selectivity in a multi-kinase screening panel. The data in the current study underscores that the TYK2 JH2 pseudokinase is a promising therapeutic target for achieving a high degree of biological selectivity. Meanwhile, targeting the JH2 domain represents an appealing strategy for the development of clinically well-tolerated TYK2 inhibitors that would have superior efficacy and a favorable safety profile compared to the existing Janus kinase inhibitors against autoimmune diseases.

Factor inhibiting HIF can catalyze two asparaginyl hydroxylations in VNVN motifs of ankyrin fold proteins.

The aspariginyl hydroxylase human factor inhibiting hypoxia-inducible factor (FIH) is an important regulator of the transcriptional activity of hypoxia-inducible factor. FIH also catalyzes the hydroxylation of asparaginyl and other residues in ankyrin repeat domain-containing proteins, including apoptosis stimulating of p53 protein (ASPP) family members. ASPP2 is reported to undergo a single FIH-catalyzed hydroxylation at Asn-986. We report biochemical and crystallographic evidence showing that FIH catalyzes the unprecedented post-translational hydroxylation of both asparaginyl residues in "VNVN" and related motifs of ankyrin repeat domains in ASPPs (i.e., ASPP1, ASPP2, and iASPP) and the related ASB11 and p18-INK4C proteins. Our biochemical results extend the substrate scope of FIH catalysis and may have implications for its biological roles, including in the hypoxic response and ASPP family function.

SHR1032, a novel STING agonist, stimulates anti-tumor immunity and directly induces AML apoptosis.

Stimulator of interferon genes (STING) activation induces type I interferons and pro-inflammatory cytokines which stimulate tumor antigen cross presentation and the adaptive immune responses against tumor. The first-generation of STING agonists, cyclic di-nucleotide (CDN), mimicked the endogenous STING ligand cyclic guanosine monophosphate adenosine monophosphate, and displayed limited clinical efficacy. Here we report the discovery of SHR1032, a novel small molecule non-CDN STING agonist. Compared to the clinical CDN STING agonist ADU-S100, SHR1032 has much higher activity in human cells with different STING haplotypes and robustly induces interferon ß (IFNß) production. When dosed intratumorally, SHR1032 induced strong anti-tumor effects in the MC38 murine syngeneic tumor model. Pharmacodynamic studies showed induction of IFNß, tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) in the tumors and, to a lower extent, in the plasma. More importantly, we found SHR1032 directly causes cell death in acute myeloid leukemia (AML) cells. In conclusion, our findings demonstrate that in addition to their established ability to boost anti-tumor immune responses, STING agonists can directly eradicate AML cells, and SHR1032 may present a new and promising therapeutic agent for cancer patients.

Conservation of the unusual dimeric JmjC fold of JMJD7 from Drosophila melanogaster to humans.

The JmjC family of 2-oxoglutarate dependent oxygenases catalyse a range of hydroxylation and demethylation reactions in humans and other animals. Jumonji domain-containing 7 (JMJD7) is a JmjC (3S)-lysyl-hydroxylase that catalyses the modification of Developmentally Regulated GTP Binding Proteins 1 and 2 (DRG1 and 2); JMJD7 has also been reported to have histone endopeptidase activity. Here we report biophysical and biochemical studies on JMJD7 from Drosophila melanogaster (dmJMJD7). Notably, crystallographic analyses reveal that the unusual dimerization mode of JMJD7, which involves interactions between both the N- and C-terminal regions of both dmJMJD7 monomers and disulfide formation, is conserved in human JMJD7 (hsJMJD7). The results further support the assignment of JMJD7 as a lysyl hydroxylase and will help enable the development of selective inhibitors for it and other JmjC oxygenases.

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.

Human Oxygenase Variants Employing a Single Protein FeII Ligand Are Catalytically Active.

Aspartate/asparagine-ß-hydroxylase (AspH) is a human 2-oxoglutarate (2OG) and FeII oxygenase that catalyses C3 hydroxylations of aspartate/asparagine residues of epidermal growth factor-like domains (EGFDs). Unusually, AspH employs two histidine residues to chelate FeII rather than the typical triad of two histidine and one glutamate/aspartate residue. We report kinetic, inhibition, and crystallographic studies concerning human AspH variants in which either of its FeII binding histidine residues are substituted for alanine. Both the H725A and, in particular, the H679A AspH variants retain substantial catalytic activity. Crystal structures clearly reveal metal-ligation by only a single protein histidine ligand. The results have implications for the functional assignment of 2OG oxygenases and for the design of non-protein biomimetic catalysts.

Management of COVID-19: current status and future prospects.

COVID-19, a highly transmissible pandemic disease, is affecting millions of lives around the world. Severely infected patients show acute respiratory distress symptoms. Sustainable management strategies are required to save lives of the infected people and further preventing spread of the virus. Diagnosis, treatment, and vaccination development initiatives are already exhibited from the scientific community to fight against this virus. In this review, we primarily discuss the management strategies including prevention of spread, prophylaxis, vaccinations, and treatment for COVID-19. Further, analysis of vaccine development status and performance are also briefly discussed. Global socioeconomic impact of COVID-19 is also analyzed as part of this review.

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.

Diagnosis for COVID-19: current status and future prospects.

Introduction: Coronavirus disease 2019 (COVID-19), a respiratory illness caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had its first detection in December 2019 in Wuhan (China) and spread across the world. In March 2020, the World Health Organization (WHO) declared COVID-19 a pandemic disease. The utilization of prompt and accurate molecular diagnosis of SARS-CoV-2 virus, isolating the infected patients, and treating them are the keys to managing this unprecedented pandemic. International travel acted as a catalyst for the widespread transmission of the virus.Areas covered: This review discusses phenotype, structural, and molecular evolution of recognition elements and primers, its detection in the laboratory, and at point of care. Further, market analysis of commercial products and their performance are also evaluated, providing new ways to confront the ongoing global public health emergency.Expert commentary: The outbreak for COVID-19 created mammoth chaos in the healthcare sector, and still, day by day, new epicenters for the outbreak are being reported. Emphasis should be placed on developing more effective, rapid, and early diagnostic devices. The testing laboratories should invest more in clinically relevant multiplexed and scalable detection tools to fight against a pandemic like this where massive demand for testing exists.

Human Oxygenase Variants Employing a Single Protein FeII Ligand Are Catalytically Active.

Aspartate/asparagine-ß-hydroxylase (AspH) is a human 2-oxoglutarate (2OG) and FeII oxygenase that catalyses C3 hydroxylations of aspartate/asparagine residues of epidermal growth factor-like domains (EGFDs). Unusually, AspH employs two histidine residues to chelate FeII rather than the typical triad of two histidine and one glutamate/aspartate residue. We report kinetic, inhibition, and crystallographic studies concerning human AspH variants in which either of its FeII binding histidine residues are substituted for alanine. Both the H725A and, in particular, the H679A AspH variants retain substantial catalytic activity. Crystal structures clearly reveal metal-ligation by only a single protein histidine ligand. The results have implications for the functional assignment of 2OG oxygenases and for the design of non-protein biomimetic catalysts.

Use of cyclic peptides to induce crystallization: case study with prolyl hydroxylase domain 2.

Crystallization is the bottleneck in macromolecular crystallography; even when a protein crystallises, crystal packing often influences ligand-binding and protein-protein interaction interfaces, which are the key points of interest for functional and drug discovery studies. The human hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) readily crystallises as a homotrimer, but with a sterically blocked active site. We explored strategies aimed at altering PHD2 crystal packing by protein modification and molecules that bind at its active site and elsewhere. Following the observation that, despite weak inhibition/binding in solution, succinamic acid derivatives readily enable PHD2 crystallization, we explored methods to induce crystallization without active site binding. Cyclic peptides obtained via mRNA display bind PHD2 tightly away from the active site. They efficiently enable PHD2 crystallization in different forms, both with/without substrates, apparently by promoting oligomerization involving binding to the C-terminal region. Although our work involves a specific case study, together with those of others, the results suggest that mRNA display-derived cyclic peptides may be useful in challenging protein crystallization cases.

Biochemical and biophysical analyses of hypoxia sensing prolyl hydroxylases from Dictyostelium discoideum and Toxoplasma gondii.

In animals, the response to chronic hypoxia is mediated by prolyl hydroxylases (PHDs) that regulate the levels of hypoxia-inducible transcription factor α (HIFα). PHD homologues exist in other types of eukaryotes and prokaryotes where they act on non HIF substrates. To gain insight into the factors underlying different PHD substrates and properties, we carried out biochemical and biophysical studies on PHD homologues from the cellular slime mold, Dictyostelium discoideum, and the protozoan parasite, Toxoplasma gondii, both lacking HIF. The respective prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation of S-phase kinase-associated protein 1 (Skp1), a reaction enabling adaptation to different dioxygen availability. Assays with full-length Skp1 substrates reveal substantial differences in the kinetic properties of DdPhyA and TgPhyA, both with respect to each other and compared with human PHD2; consistent with cellular studies, TgPhyA is more active at low dioxygen concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate. No cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. The human Skp1 E147P variant is a DdPhyA and TgPhyA substrate, suggesting some retention of ancestral interactions. Crystallographic analysis of DdPhyA enables comparisons with homologues from humans, Trichoplax adhaerens, and prokaryotes, informing on differences in mobile elements involved in substrate binding and catalysis. In DdPhyA, two mobile loops that enclose substrates in the PHDs are conserved, but the C-terminal helix of the PHDs is strikingly absent. The combined results support the proposal that PHD homologues have evolved kinetic and structural features suited to their specific sensing roles.

A human protein hydroxylase that accepts D-residues.

Factor inhibiting hypoxia-inducible factor (FIH) is a 2-oxoglutarate-dependent protein hydroxylase that catalyses C3 hydroxylations of protein residues. We report FIH can accept (D)- and (L)-residues for hydroxylation. The substrate selectivity of FIH differs for (D) and (L) epimers, e.g., (D)- but not (L)-allylglycine, and conversely (L)- but not (D)-aspartate, undergo monohydroxylation, in the tested sequence context. The (L)-Leu-containing substrate undergoes FIH-catalysed monohydroxylation, whereas (D)-Leu unexpectedly undergoes dihydroxylation. Crystallographic, mass spectrometric, and DFT studies provide insights into the selectivity of FIH towards (L)- and (D)-residues. The results of this work expand the potential range of known substrates hydroxylated by isolated FIH and imply that it will be possible to generate FIH variants with altered selectivities.

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.

Biochemical and structural investigations clarify the substrate selectivity of the 2-oxoglutarate oxygenase JMJD6.

JmjC domain-containing protein 6 (JMJD6) is a 2-oxoglutarate (2OG)-dependent oxygenase linked to various cellular processes, including splicing regulation, histone modification, transcriptional pause release, hypoxia sensing, and cancer. JMJD6 is reported to catalyze hydroxylation of lysine residue(s) of histones, the tumor-suppressor protein p53, and splicing regulatory proteins, including u2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65). JMJD6 is also reported to catalyze N-demethylation of N-methylated (both mono- and di-methylated) arginine residues of histones and other proteins, including HSP70 (heat-shock protein 70), estrogen receptor α, and RNA helicase A. Here, we report MS- and NMR-based kinetic assays employing purified JMJD6 and multiple substrate fragment sequences, the results of which support the assignment of purified JMJD6 as a lysyl hydroxylase. By contrast, we did not observe N-methyl arginyl N-demethylation with purified JMJD6. Biophysical analyses, including crystallographic analyses of JMJD6Δ344-403 in complex with iron and 2OG, supported its assignment as a lysyl hydroxylase rather than an N-methyl arginyl-demethylase. The screening results supported some, but not all, of the assigned JMJD6 substrates and identified other potential JMJD6 substrates. We envision these results will be useful in cellular and biological work on the substrates and functions of JMJD6 and in the development of selective inhibitors of human 2OG oxygenases.

Studies on spiro[4.5]decanone prolyl hydroxylase domain inhibitors.

The 2-oxoglutarate (2OG) dependent hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are targets for treatment of anaemia and other ischaemia related diseases. PHD inhibitors are in clinical trials; however, the number of reported templates for PHD inhibition is limited. We report structure-activity relationship and crystallographic studies on spiro[4.5]decanone containing PHD inhibitors. Together with other studies, our results reveal spiro[4.5]decanones as useful templates for generation of potent and selective 2OG oxygenase inhibitors.

Small-molecules that covalently react with a human prolyl hydroxylase - towards activity modulation and substrate capture.

We describe covalently binding modulators of the activity of human prolyl hydroxylase domain 2 (PHD2) and studies towards a strategy for photocapture of PHD2 substrates. Reversible active site binding of electrophile bearing compounds enables susbsequent covalent reaction with a lysine residue (K408) in the flexible C-terminal region of PHD2 to give a modified protein that retains catalytic activity.

Born to sense: biophysical analyses of the oxygen sensing prolyl hydroxylase from the simplest animal Trichoplax adhaerens.

BACKGROUND: In humans and other animals, the chronic hypoxic response is mediated by hypoxia inducible transcription factors (HIFs) which regulate the expression of genes that counteract the effects of limiting oxygen. Prolyl hydroxylases (PHDs) act as hypoxia sensors for the HIF system in organisms ranging from humans to the simplest animal Trichoplax adhaerens. METHODS: We report structural and biochemical studies on the T. adhaerens HIF prolyl hydroxylase (TaPHD) that inform about the evolution of hypoxia sensing in animals. RESULTS: High resolution crystal structures (≤1.3 Å) of TaPHD, with and without its HIFα substrate, reveal remarkable conservation of key active site elements between T. adhaerens and human PHDs, which also manifest in kinetic comparisons. CONCLUSION: Conserved structural features of TaPHD and human PHDs include those apparently enabling the slow binding/reaction of oxygen with the active site Fe(II), the formation of a stable 2-oxoglutarate complex, and a stereoelectronically promoted change in conformation of the hydroxylated proline-residue. Comparison of substrate selectivity between the human PHDs and TaPHD provides insights into the selectivity determinants of HIF binding by the PHDs, and into the evolution of the multiple HIFs and PHDs present in higher animals.