Substitution of 2-oxoglutarate alters reaction outcomes of the Pseudomonas savastanoi ethylene-forming enzyme.

In seeding plants, biosynthesis of the phytohormone ethylene, which regulates processes including fruit ripening and senescence, is catalyzed by 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase. The plant pathogen Pseudomonas savastanoi (previously classified as: Pseudomonas syringae) employs a different type of ethylene-forming enzyme (psEFE), though from the same structural superfamily as ACC oxidase, to catalyze ethylene formation from 2-oxoglutarate (2OG) in an arginine dependent manner. psEFE also catalyzes the more typical oxidation of arginine to give L-Δ1-pyrroline-5-carboxylate (P5C), a reaction coupled to oxidative decarboxylation of 2OG giving succinate and CO2. We report on the effects of C3 and/or C4 substituted 2OG derivatives on the reaction modes of psEFE. 1H NMR assays, including using the pure shift method, reveal that, within our limits of detection, none of the tested 2OG derivatives is converted to an alkene; some are converted to the corresponding ß-hydroxypropionate or succinate derivatives, with only the latter being coupled to arginine oxidation. The NMR results reveal that the nature of 2OG derivatization can affect the outcome of the bifurcating reaction, with some 2OG derivatives exclusively favoring the arginine oxidation pathway. Given that some of the tested 2OG derivatives are natural products, the results are of potential biological relevance. There are also opportunities for therapeutic or biocatalytic regulation of the outcomes of reactions catalyzed by 2OG-dependent oxygenases by the use of 2OG derivatives.

Natural and synthetic 2-oxoglutarate derivatives are substrates for oncogenic variants of human isocitrate dehydrogenase 1 and 2.

Variants of isocitrate dehydrogenase (IDH) 1 and 2 (IDH1/2) alter metabolism in cancer cells by catalyzing the NADPH-dependent reduction of 2-oxoglutarate (2OG) to (2R)-hydroxyglutarate. However, it is unclear how derivatives of 2OG can affect cancer cell metabolism. Here, we used synthetic C3- and C4-alkylated 2OG derivatives to investigate the substrate selectivities of the most common cancer-associated IDH1 variant (R132H IDH1), of two cancer-associated IDH2 variants (R172K IDH2, R140Q IDH2), and of WT IDH1/2. Absorbance-based, NMR, and electrochemical assays were employed to monitor WT IDH1/2 and IDH1/2 variant-catalyzed 2OG derivative turnover in the presence and absence of 2OG. Our results reveal that 2OG derivatives can serve as substrates of the investigated IDH1/2 variants, but not of WT IDH1/2, and have the potential to act as 2OG-competitive inhibitors. Kinetic parameters reveal that some 2OG derivatives, including the natural product 3-methyl-2OG, are equally or even more efficient IDH1/2 variant substrates than 2OG. Furthermore, NMR and mass spectrometry studies confirmed IDH1/2 variant-catalyzed production of alcohols in the cases of the 3-methyl-, 3-butyl-, and 3-benzyl-substituted 2OG derivatives; a crystal structure of 3-butyl-2OG with an IDH1 variant (R132C/S280F IDH1) reveals active site binding. The combined results highlight the potential for (i) IDH1/2 variant-catalyzed reduction of 2-oxoacids other than 2OG in cells, (ii) modulation of IDH1/2 variant activity by 2-oxoacid natural products, including some present in common foods, (iii) inhibition of IDH1/2 variants via active site binding rather than the established allosteric mode of inhibition, and (iv) possible use of IDH1/2 variants as biocatalysts.

Combined proteomic and biochemical analyses redefine the consensus sequence requirement for epidermal growth factor-like domain hydroxylation.

Epidermal growth factor-like domains (EGFDs) have important functions in cell-cell signaling. Both secreted and cell surface human EGFDs are subject to extensive modifications, including aspartate and asparagine residue C3-hydroxylations catalyzed by the 2-oxoglutarate oxygenase aspartate/asparagine-ß-hydroxylase (AspH). Although genetic studies show AspH is important in human biology, studies on its physiological roles have been limited by incomplete knowledge of its substrates. Here, we redefine the consensus sequence requirements for AspH-catalyzed EGFD hydroxylation based on combined analysis of proteomic mass spectrometric data and mass spectrometry-based assays with isolated AspH and peptide substrates. We provide cellular and biochemical evidence that the preferred site of EGFD hydroxylation is embedded within a disulfide-bridged macrocycle formed of 10 amino acid residues. This definition enabled the identification of previously unassigned hydroxylation sites in three EGFDs of human fibulins as AspH substrates. A non-EGFD containing protein, lymphocyte antigen-6/plasminogen activator urokinase receptor domain containing protein 6B (LYPD6B) was shown to be a substrate for isolated AspH, but we did not observe evidence for LYPD6B hydroxylation in cells. AspH-catalyzed hydroxylation of fibulins is of particular interest given their important roles in extracellular matrix dynamics. In conclusion, these results lead to a revision of the consensus substrate requirements for AspH and expand the range of observed and potential AspH-catalyzed hydroxylation in cells, which will enable future study of the biological roles of AspH.

Mass spectrometric assays monitoring the deubiquitinase activity of the SARS-CoV-2 papain-like protease inform on the basis of substrate selectivity and have utility for substrate identification.

The SARS-CoV-2 papain-like protease (PLpro) and main protease (Mpro) are nucleophilic cysteine enzymes that catalyze hydrolysis of the viral polyproteins pp1a/1ab. By contrast with Mpro, PLpro is also a deubiquitinase (DUB) that accepts post-translationally modified human proteins as substrates. Here we report studies on the DUB activity of PLpro using synthetic Nε-lysine-branched oligopeptides as substrates that mimic post-translational protein modifications by ubiquitin (Ub) or Ub-like modifiers (UBLs), such as interferon stimulated gene 15 (ISG15). Mass spectrometry (MS)-based assays confirm the DUB activity of isolated recombinant PLpro. They reveal that the sequence of both the peptide fragment derived from the post-translationally modified protein and that derived from the UBL affects PLpro catalysis; the nature of substrate binding in the S sites appears to be more important for catalytic efficiency than binding in the S' sites. Importantly, the results reflect the reported cellular substrate selectivity of PLpro, i.e. human proteins conjugated to ISG15 are better substrates than those conjugated to Ub or other UBLs. The combined experimental and modelling results imply that PLpro catalysis is affected not only by the identity of the substrate residues binding in the S and S' sites, but also by the substrate fold and the conformational dynamics of the blocking loop 2 of the PLpro:substrate complex. Nε-Lysine-branched oligopeptides thus have potential to help the identification of PLpro substrates. More generally, the results imply that MS-based assays with Nε-lysine-branched oligopeptides have potential to monitor catalysis by human DUBs and hence to inform on their substrate preferences.

Kinetic parameters of human aspartate/asparagine-ß-hydroxylase suggest that it has a possible function in oxygen sensing.

Human aspartate/asparagine-ß-hydroxylase (AspH) is a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the post-translational hydroxylation of Asp and Asn residues in epidermal growth factor-like domains (EGFDs). Despite its biomedical significance, studies on AspH have long been limited by a lack of assays for its isolated form. Recent structural work has revealed that AspH accepts substrates with a noncanonical EGFD disulfide connectivity (i.e. the Cys 1-2, 3-4, 5-6 disulfide pattern). We developed stable cyclic thioether analogues of the noncanonical EGFD AspH substrates to avoid disulfide shuffling. We monitored their hydroxylation by solid-phase extraction coupled to MS. The extent of recombinant AspH-catalyzed cyclic peptide hydroxylation appears to reflect levels of EGFD hydroxylation observed in vivo, which vary considerably. We applied the assay to determine the kinetic parameters of human AspH with respect to 2OG, Fe(II), l-ascorbic acid, and substrate and found that these parameters are in the typical ranges for 2OG oxygenases. Of note, a relatively high Km for O2 suggested that O2 availability may regulate AspH activity in a biologically relevant manner. We anticipate that the assay will enable the development of selective small-molecule inhibitors for AspH and other human 2OG oxygenases.

Fluorinated derivatives of pyridine-2,4-dicarboxylate are potent inhibitors of human 2-oxoglutarate dependent oxygenases.

2-Oxoglutarate (2OG) oxygenases have important roles in human biology and are validated medicinal chemistry targets. Improving the selectivity profile of broad-spectrum 2OG oxygenase inhibitors may help enable the identification of selective inhibitors for use in functional assignment work. We report the synthesis of F- and CF3-substituted derivatives of the broad-spectrum 2OG oxygenase inhibitor pyridine-2,4-dicarboxylate (2,4-PDCA). Their inhibition selectivity profile against selected functionally distinct human 2OG oxygenases was determined using mass spectrometry-based assays. F-substituted 2,4-PDCA derivatives efficiently inhibit the 2OG oxygenases aspartate/asparagine-ß-hydroxylase (AspH) and the JmjC lysine-specific N ε-demethylase 4E (KDM4E); The F- and CF3-substituted 2,4-PDCA derivatives were all less efficient inhibitors of the tested 2OG oxygenases than 2,4-PDCA itself, except for the C5 F-substituted 2,4-PDCA derivative which inhibited AspH with a similar efficiency as 2,4-PDCA. Notably, the introduction of a F- or CF3-substituent at the C5 position of 2,4-PDCA results in a substantial increase in selectivity for AspH over KDM4E compared to 2,4-PDCA. Crystallographic studies inform on the structural basis of our observations, which exemplifies how a small change on a 2OG analogue can make a substantial difference in the potency of 2OG oxygenase inhibition.

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.

Small-molecule active pharmaceutical ingredients of approved cancer therapeutics inhibit human aspartate/asparagine-ß-hydroxylase.

Human aspartate/asparagine-ß-hydroxylase (AspH) is a 2-oxoglutarate (2OG) dependent oxygenase that catalyses the hydroxylation of Asp/Asn-residues of epidermal growth factor-like domains (EGFDs). AspH is reported to be upregulated on the cell surface of invasive cancer cells in a manner distinguishing healthy from cancer cells. We report studies on the effect of small-molecule active pharmaceutical ingredients (APIs) of human cancer therapeutics on the catalytic activity of AspH using a high-throughput mass spectrometry (MS)-based inhibition assay. Human B-cell lymphoma-2 (Bcl-2)-protein inhibitors, including the (R)-enantiomer of the natural product gossypol, were observed to efficiently inhibit AspH, as does the antitumor antibiotic bleomycin A2. The results may help in the design of AspH inhibitors with the potential of increased selectivity compared to the previously identified Fe(II)-chelating or 2OG-competitive inhibitors. With regard to the clinical use of bleomycin A2 and of the Bcl-2 inhibitor venetoclax, the results suggest that possible side-effects mediated through the inhibition of AspH and other 2OG oxygenases should be considered.

Achiral Trisubstituted Thioureas as Secondary Ligands to CuI Catalysts: Direct Catalytic Asymmetric Addition of α-Fluoronitriles to Imines.

Thioureas have emerged as effective hydrogen-bonding catalysts over the last two decades, and they are broadly utilized in asymmetric catalysis. We report that achiral trisubstituted thioureas function as beneficial secondary ligands to CuI catalysts, thereby enabling highly diastereo- and enantioselective addition of α-fluoronitriles to imines. The structure of the thiourea significantly affects the reaction outcome, and kinetic experiments indicate that the thioureas enhance the stereocontrol by binding to the CuI complex. The reaction products can be readily transformed into valuable ß-amino acid derivatives bearing a fluorinated tetrasubstituted stereogenic center.

Direct Catalytic Asymmetric Mannich-Type Reaction of α- and ß-Fluorinated Amides.

The last two decades have witnessed the emergence of direct enolization protocols providing atom-economical and operationally simple methods to use enolates for stereoselective C-C bond-forming reactions, eliminating the inherent drawback of the preformation of enolates using stoichiometric amounts of reagents. In its infancy, direct enolization relied heavily on the intrinsic acidity of the latent enolates, and the reaction scope was limited to readily enolizable ketones and aldehydes. Recent advances in this field enabled the exploitation of carboxylic acid derivatives for direct enolization, offering expeditious access to synthetically versatile chiral building blocks. Despite the growing demand for enantioenriched fluorine-containing small molecules, α- and ß-fluorinated carbonyl compounds have been neglected in direct enolization chemistry because of the competing and dominating defluorination pathway. Herein we present a comprehensive study on direct and highly stereoselective Mannich-type reactions of α- and ß-fluorine-functionalized 7-azaindoline amides that rely on a soft Lewis acid/hard Brønsted base cooperative catalytic system to guarantee an efficient enolization while suppressing undesired defluorination. This protocol contributes to provide a series of fluorinated analogs of enantioenriched ß-amino acids for medicinal chemistry.

Catalytic generation of α-CF3 enolate: direct catalytic asymmetric Mannich-type reaction of α-CF3 amide.

The introduction of the CF3 unit is a common strategy for modifying pharmacokinetic properties and slowing metabolic degradation in medicinal chemistry. A catalytic and enantioselective addition of α-CF3 enolates allows for expeditious access to functionalized chiral building blocks with CF3-containing stereogenicity. To date, α-CF3 enolates have been a less explored class of nucleophiles because of rapid defluorination. The present study reveals that a designed α-CF3 amide enables a direct asymmetric Mannich-type reaction in a cooperative catalytic system.

Fixing the Achilles Heel of Pfizer's Paxlovid for COVID-19 Treatment.

Nirmatrelvir (PF-07321332), a first-in-class inhibitor of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) main protease (Mpro), was developed by Pfizer under intense pressure during the pandemic to treat COVID-19. A weakness of nirmatrelvir is its limited metabolic stability, which led to the development of a combination therapy (paxlovid), involving coadministration of nirmatrelvir with the cytochrome P450 inhibitor ritonavir. However, limitations in tolerability of the ritonavir component reduce the scope of paxlovid. In response to these limitations, researchers at Pfizer have now developed the second-generation Mpro inhibitor PF-07817883 (ibuzatrelvir). Structurally related to nirmatrelvir, including with the presence of a trifluoromethyl group, albeit located differently, ibuzatrelvir manifests enhanced oral bioavailability, so it does not require coadministration with ritonavir. The development of ibuzatrelvir is an important milestone, because it is expected to enhance the treatment of COVID-19 without the drawbacks associated with ritonavir. Given the success of paxlovid in treating COVID-19, it is likely that ibuzatrelvir will be granted approval as an improved drug for treatment of COVID-19 infections, so complementing vaccination efforts and improving pandemic preparedness. The development of nirmatrelvir and ibuzatrelvir dramatically highlights the power of appropriately resourced modern medicinal chemistry to very rapidly enable the development of breakthrough medicines. Consideration of how analogous approaches can be used to develop similarly breakthrough medicines for infectious diseases such as tuberculosis and malaria is worthwhile.

Methods for production and assaying catalysis of isolated recombinant human aspartate/asparagine-ß-hydroxylase.

Aspartate/asparagine-ß-hydroxylase (AspH) is a transmembrane 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes the post-translational hydroxylation of aspartate- and asparagine-residues in epidermal growth factor-like domains (EGFDs) of its substrate proteins. Upregulation of ASPH and translocation of AspH from the endoplasmic reticulum membrane to the surface membrane of cancer cells is associated with enhanced cell motility and worsened clinical prognosis. AspH is thus a potential therapeutic and diagnostic target for cancer. This chapter describes methods for the production and purification of soluble constructs of recombinant human AspH suitable for biochemical and crystallographic studies. The chapter also describes efficient methods for performing turnover and inhibition assays which monitor catalysis of isolated recombinant human AspH in vitro using solid phase extraction coupled to mass spectrometry (SPE-MS). The SPE-MS assays employ synthetic disulfide- or thioether-bridged macrocyclic oligopeptides as substrates; a macrocycle is an apparently essential requirement for productive AspH catalysis and mimics an EGFD disulfide isomer that is not typically observed in crystal and NMR structures. SPE-MS assays can be used to monitor catalysis of 2OG oxygenases other than AspH; the methods described herein are representative for 2OG oxygenase SPE-MS assays useful for performing kinetic and/or inhibition studies.

Thiophene-fused γ-lactams inhibit the SARS-CoV-2 main protease via reversible covalent acylation.

Enzyme inhibitors working by O-acylation of nucleophilic serine residues are of immense medicinal importance, as exemplified by the ß-lactam antibiotics. By contrast, inhibition of nucleophilic cysteine enzymes by S-acylation has not been widely exploited for medicinal applications. The SARS-CoV-2 main protease (Mpro) is a nucleophilic cysteine protease and a validated therapeutic target for COVID-19 treatment using small-molecule inhibitors. The clinically used Mpro inhibitors nirmatrelvir and simnotrelvir work via reversible covalent reaction of their electrophilic nitrile with the Mpro nucleophilic cysteine (Cys145). We report combined structure activity relationship and mass spectrometric studies revealing that appropriately functionalized γ-lactams can potently inhibit Mpro by reversible covalent reaction with Cys145 of Mpro. The results suggest that γ-lactams have potential as electrophilic warheads for development of covalently reacting small-molecule inhibitors of Mpro and, by implication, other nucleophilic cysteine enzymes.

Crystallographic and Selectivity Studies on the Approved HIF Prolyl Hydroxylase Inhibitors Desidustat and Enarodustat.

Prolyl hydroxylase domain-containing proteins 1-3 (PHD1-3) are 2-oxoglutarate (2OG)-dependent oxygenases catalysing C-4 hydroxylation of prolyl residues in α-subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF), modifications that promote HIF-α degradation via the ubiquitin-proteasome pathway. Pharmacological inhibition of the PHDs induces HIF-α stabilisation, so promoting HIF target gene transcription. PHD inhibitors are used to treat anaemia caused by chronic kidney disease (CKD) due to their ability to stimulate erythropoietin (EPO) production. We report studies on the effects of the approved PHD inhibitors Desidustat and Enarodustat, and the clinical candidate TP0463518, on activities of a representative set of isolated recombinant human 2OG oxygenases. The three molecules manifest selectivity for PHD inhibition over that of the other 2OG oxygenases evaluated. We obtained crystal structures of Desidustat and Enarodustat in complex with the human 2OG oxygenase factor inhibiting hypoxia-inducible factor-α (FIH), which, together with modelling studies, inform on the binding modes of Desidustat and Enarodustat to active site Fe(II) in 2OG oxygenases, including PHD1-3. The results will help in the design of selective inhibitors of both the PHDs and other 2OG oxygenases, which are of medicinal interest due to their involvement inter alia in metabolic regulation, epigenetic signalling, DNA-damage repair, and agrochemical resistance.

Studies on the selectivity of the SARS-CoV-2 papain-like protease reveal the importance of the P2' proline of the viral polyprotein.

The SARS-CoV-2 papain-like protease (PLpro) is an antiviral drug target that catalyzes the hydrolysis of the viral polyproteins pp1a/1ab, so releasing the non-structural proteins (nsps) 1-3 that are essential for the coronavirus lifecycle. The LXGG↓X motif in pp1a/1ab is crucial for recognition and cleavage by PLpro. We describe molecular dynamics, docking, and quantum mechanics/molecular mechanics (QM/MM) calculations to investigate how oligopeptide substrates derived from the viral polyprotein bind to PLpro. The results reveal how the substrate sequence affects the efficiency of PLpro-catalyzed hydrolysis. In particular, a proline at the P2' position promotes catalysis, as validated by residue substitutions and mass spectrometry-based analyses. Analysis of PLpro catalyzed hydrolysis of LXGG motif-containing oligopeptides derived from human proteins suggests that factors beyond the LXGG motif and the presence of a proline residue at P2' contribute to catalytic efficiency, possibly reflecting the promiscuity of PLpro. The results will help in identifying PLpro substrates and guiding inhibitor design.

Cyclic ß2,3-amino acids improve the serum stability of macrocyclic peptide inhibitors targeting the SARS-CoV-2 main protease.

Due to their constrained conformations, cyclic ß2,3-amino acids (cßAA) are key building blocks that can fold peptides into compact and rigid structures, improving peptidase resistance and binding affinity to target proteins, due to their constrained conformations. Although the translation efficiency of cßAAs is generally low, our engineered tRNA, referred to as tRNAPro1E2, enabled efficient incorporation of cßAAs into peptide libraries using the flexible in vitro translation (FIT) system. Here we report on the design and application of a macrocyclic peptide library incorporating 3 kinds of cßAAs: (1R,2S)-2-aminocyclopentane carboxylic acid (ß1), (1S,2S)-2-aminocyclohexane carboxylic acid (ß2), and (1R,2R)-2-aminocyclopentane carboxylic acid. This library was applied to an in vitro selection against the SARS-CoV-2 main protease (Mpro). The resultant peptides, BM3 and BM7, bearing one ß2 and two ß1, exhibited potent inhibitory activities with IC50 values of 40 and 20 nM, respectively. BM3 and BM7 also showed remarkable serum stability with half-lives of 48 and >168 h, respectively. Notably, BM3A and BM7A, wherein the cßAAs were substituted with alanine, lost their inhibitory activities against Mpro and displayed substantially shorter serum half-lives. This observation underscores the significant contribution of cßAA to the activity and stability of peptides. Overall, our results highlight the potential of cßAA in generating potent and highly stable macrocyclic peptides with drug-like properties.

The selective prolyl hydroxylase inhibitor IOX5 stabilizes HIF-1α and compromises development and progression of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a largely incurable disease, for which new treatments are urgently needed. While leukemogenesis occurs in the hypoxic bone marrow, the therapeutic tractability of the hypoxia-inducible factor (HIF) system remains undefined. Given that inactivation of HIF-1α/HIF-2α promotes AML, a possible clinical strategy is to target the HIF-prolyl hydroxylases (PHDs), which promote HIF-1α/HIF-2α degradation. Here, we reveal that genetic inactivation of Phd1/Phd2 hinders AML initiation and progression, without impacting normal hematopoiesis. We investigated clinically used PHD inhibitors and a new selective PHD inhibitor (IOX5), to stabilize HIF-α in AML cells. PHD inhibition compromises AML in a HIF-1α-dependent manner to disable pro-leukemogenic pathways, re-program metabolism and induce apoptosis, in part via upregulation of BNIP3. Notably, concurrent inhibition of BCL-2 by venetoclax potentiates the anti-leukemic effect of PHD inhibition. Thus, PHD inhibition, with consequent HIF-1α stabilization, is a promising nontoxic strategy for AML, including in combination with venetoclax.

Formal total synthesis of the algal toxin (-)-polycavernoside A.

A concise and largely catalysis-based approach to the potent algal toxin polycavernoside A (1) is described that intercepts a late-stage intermediate of a previous total synthesis; from there on, this challenging target can be reached in a small number of steps. Key to success was a sequence of a molybdenum-catalyzed ring-closing alkyne metathesis (RCAM) reaction to forge the macrocyclic frame, followed by a gold-catalyzed and strictly regioselective transannular hydroalkoxylation of the resulting cycloalkyne that allows the intricate oxygenation pattern of the macrolactone ring of 1 to be properly set. The required cyclization precursor 5 was assembled by the arguably most advanced fragment coupling process based on an Evans-Tishchenko redox esterification known to date, which was optimized to the extent that the precious coupling partners could be used in an almost equimolar ratio (6/7 1:1.3). The preparation of these building blocks features, inter alia, the power of the Sc(OTf)(3)-catalyzed Leighton crotylation as well as the superb selectivities of alkene cross metathesis, asymmetric keto-ester hydrogenation, and the Jacobsen epoxidation/epoxide resolution technologies.

Kinetic and inhibition studies on human Jumonji-C (JmjC) domain-containing protein 5.

Jumonji-C (JmjC) domain-containing protein 5 (JMJD5) is a human 2-oxoglutarate (2OG) and Fe(ii)-dependent oxygenase which catalyses the post-translational C3 hydroxylation of arginyl-residues and which is linked to the circadian rhythm and to cancer biology through as yet unidentified mechanisms. We report robust solid phase extraction coupled to mass spectrometry (SPE-MS)-based JMJD5 assays which enable kinetic and high-throughput inhibition studies. The kinetic studies reveal that some synthetic 2OG derivatives, notably including a 2OG derivative with a cyclic carbon backbone (i.e. (1R)-3-(carboxycarbonyl)cyclopentane-1-carboxylic acid), are efficient alternative cosubstrates of JMJD5 and of factor inhibiting hypoxia-inducible transcription factor HIF-α (FIH), but not of the Jumonji-C (JmjC) histone Nε-methyl lysine demethylase KDM4E, apparently reflecting the closer structural similarity of JMJD5 and FIH. The JMJD5 inhibition assays were validated by investigating the effect of reported 2OG oxygenase inhibitors on JMJD5 catalysis; the results reveal that broad-spectrum 2OG oxygenase inhibitors are also efficient JMJD5 inhibitors (e.g. N-oxalylglycine, pyridine-2,4-dicarboxylic acid, ebselen) whereas most 2OG oxygenase inhibitors that are in clinical use (e.g. roxadustat) do not inhibit JMJD5. The SPE-MS assays will help enable the development of efficient and selective JMJD5 inhibitors for investigating the biochemical functions of JMJD5 in cellular studies.