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Jumonji-C domain-containing protein 6 (JMJD6) is a human 2-oxoglutarate (2OG)/Fe(ii)-dependent oxygenase catalysing post-translational C5 hydroxylation of multiple lysine residues, including in the bromodomain-containing proteins BRD2, BRD3 and BRD4. The role(s) of JMJD6-catalysed substrate hydroxylation are unclear. JMJD6 is important in development and JMJD6 catalysis may promote cancer. We report solid-phase extraction coupled to mass spectrometry assays monitoring JMJD6-catalysed hydroxylation of BRD2-4 derived oligopeptides containing multiple lysyl residues. The assays enabled determination of apparent steady-state kinetic parameters for 2OG, Fe(ii), l-ascorbate, O 2 and BRD substrates. The JMJD6 K app m for O 2 was comparable to that reported for the structurally related 2OG oxygenase factor inhibiting hypoxia-inducible factor-α (FIH), suggesting potential for limitation of JMJD6 activity by O 2 availability in cells, as proposed for FIH and some other 2OG oxygenases. The new assays will help development of small-molecule JMJD6 inhibitors for functional assignment studies and as potential cancer therapeutics., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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., (© 2024 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Hypoxia inducible transcription factors (HIFs) mediate the hypoxic response in metazoans. When sufficient O 2 is present, Fe(II)/2-oxoglutarate (2OG)-dependent oxygenases (human PHD1-3) promote HIFα degradation via prolyl-hydroxylation. We report crystallographic, spectroscopic, and biochemical characterization of stable and inactive PHD2.Fe(III).2OG complexes. Aerobic incubation of PHD2 with Fe(II) and 2OG enables formation of PHD2.Fe(III).2OG complexes which bind HIF1-2α to give inactive PHD2.Fe(III).2OG.HIF1-2α complexes. The Fe(III) oxidation state in the inactive complexes was shown by EPR spectroscopy. L-Ascorbate hinders formation of the PHD2.Fe(III).2OG.(+/-HIFα) complexes and slowly regenerates them to give the catalytically active PHD2.Fe(II).2OG complex. Crystallographic comparison of the PHD2.Fe(III).2OG.HIF2α complex with the analogous anaerobic Fe(II) complex reveals near identical structures. Exposure of the anaerobic PHD2.Fe(II).2OG.HIF2α crystals to O 2 enables in crystallo hydroxylation. The resulting PHD2.product structure, manifests conformational changes compared to the substrate structures. The results have implications for the role of the PHDs in hypoxia sensing and open new opportunities for inhibition of the PHDs and other 2OG dependent oxygenases by promoting formation of stable Fe(III) complexes., (© 2024. The Author(s).)

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-Δ 1 -pyrroline-5-carboxylate (P5C), a reaction coupled to oxidative decarboxylation of 2OG giving succinate and CO 2 . We report on the effects of C3 and/or C4 substituted 2OG derivatives on the reaction modes of psEFE. 1 H 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 β-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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Nirmatrelvir (PF-07321332), a first-in-class inhibitor of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) main protease (M pro ), 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 M pro 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.

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., (© 2024. The Author(s).)

Enzyme inhibitors working by O -acylation of nucleophilic serine residues are of immense medicinal importance, as exemplified by the β-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 (M pro ) is a nucleophilic cysteine protease and a validated therapeutic target for COVID-19 treatment using small-molecule inhibitors. The clinically used M pro inhibitors nirmatrelvir and simnotrelvir work via reversible covalent reaction of their electrophilic nitrile with the M pro nucleophilic cysteine (Cys145). We report combined structure activity relationship and mass spectrometric studies revealing that appropriately functionalized γ-lactams can potently inhibit M pro by reversible covalent reaction with Cys145 of M pro . The results suggest that γ-lactams have potential as electrophilic warheads for development of covalently reacting small-molecule inhibitors of M pro and, by implication, other nucleophilic cysteine enzymes., Competing Interests: The authors declare no competing interests., (This journal is © The Royal Society of Chemistry.)

Due to their constrained conformations, cyclic β 2,3 -amino acids (cβAA) 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 cβAAs is generally low, our engineered tRNA, referred to as tRNA Pro1E2 , enabled efficient incorporation of cβAAs 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 cβAAs: (1 R ,2 S )-2-aminocyclopentane carboxylic acid (β 1 ), (1 S ,2 S )-2-aminocyclohexane carboxylic acid (β 2 ), and (1 R ,2 R )-2-aminocyclopentane carboxylic acid. This library was applied to an in vitro selection against the SARS-CoV-2 main protease (M pro ). The resultant peptides, BM3 and BM7, bearing one β 2 and two β 1 , exhibited potent inhibitory activities with IC 50 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 cβAAs were substituted with alanine, lost their inhibitory activities against M pro and displayed substantially shorter serum half-lives. This observation underscores the significant contribution of cβAA to the activity and stability of peptides. Overall, our results highlight the potential of cβAA in generating potent and highly stable macrocyclic peptides with drug-like properties., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Chemical Society of Japan.)

Aspartate/asparagine-β-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., (Copyright © 2024. Published by Elsevier Inc.)

The SARS-CoV-2 papain-like protease (PL pro ) and main protease (M pro ) are nucleophilic cysteine enzymes that catalyze hydrolysis of the viral polyproteins pp1a/1ab. By contrast with M pro , PL pro is also a deubiquitinase (DUB) that accepts post-translationally modified human proteins as substrates. Here we report studies on the DUB activity of PL pro using synthetic N ε -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 PL pro . They reveal that the sequence of both the peptide fragment derived from the post-translationally modified protein and that derived from the UBL affects PL pro 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 PL pro , 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 PL pro 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 PL pro :substrate complex. N ε -Lysine-branched oligopeptides thus have potential to help the identification of PL pro substrates. More generally, the results imply that MS-based assays with N ε -lysine-branched oligopeptides have potential to monitor catalysis by human DUBs and hence to inform on their substrate preferences., 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 © 2023. Published by Elsevier Ltd.)

We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.