Deubiquitinases (DUBs), and the dysregulation thereof, are implicated in human disease. The recent inclusion of selective DUB inhibitors in clinical trials has heightened interest in DUB-focused drug discovery. Current DUB screening methods remain constrained, however, as they often rely on recombinant proteins that are truncated or derived from non-human sources, typically necessitating extensive optimisation of initial hits. We introduce a high-throughput, endogenous human DUB-focused activity proteomics workflow designed for the simultaneous screening and profiling of small, targeted libraries of catalytic group-reactive compounds. In a proof-of-concept screen, this innovative platform expanded the repertoire of electrophilic groups targeting DUBs, leading to the discovery of potent and selective inhibitors for USP47, OTUD7B, and USP5. Remarkably, these inhibitors required minimal or no optimisation to confirm the previously reported biological roles of the three DUBs, underscoring the advantages of this methodology for drug discovery applications. ### Competing Interest Statement The authors have declared no competing interest.

The 2-oxoglutarate (2OG)/Fe(II)-dependent γ-butyrobetaine hydroxylase (BBOX) catalyzes the final step in l-carnitine biosynthesis, i.e., stereoselective C-3 oxidation of γ-butyrobetaine (GBB). BBOX inhibition is a validated clinical strategy to modulate l-carnitine levels and to enhance cardiovascular efficiency. Reported BBOX inhibitors, including the clinically used cardioprotective agent Mildronate, manifest moderate inhibitory activity in vitro, limited selectivity, and/or unfavorable physicochemical properties, indicating a need for improved BBOX inhibitors. We report that the clinically used hypoxia-inducible factor-α prolyl residue hydroxylase (PHD) inhibitors Desidustat, Enarodustat, and Vadadustat efficiently inhibit isolated recombinant BBOX, suggesting that BBOX inhibition by clinically used PHD inhibitors should be considered as a possible off-target effect. Structure-activity relationship studies on the Desidustat scaffold enabled development of potent BBOX inhibitors that manifest high levels of selectivity for BBOX inhibition over representative human 2OG oxygenases, including PHD2. The Desidustat derivatives will help to enable investigations into the biological roles of l-carnitine and the therapeutic potential of BBOX inhibition.

The Tet(X) flavin-dependent monooxygenases enable tetracycline antibiotic resistance by catalysing inactivating hydroxylation, so preventing inhibition of bacterial ribosomes. Tet(X) resistance is growing rapidly, threatening the efficacy of important last-resort tetracyclines such as tigecycline. Tet(X) inhibitors have potential to protect tetracyclines in combination therapies, but their discovery has been hampered by lack of high-throughput assays. We report the development of an efficient fluorescence polarisation Tet(X) binding assay employing a tetramethylrhodamine-glycyl-minocycline conjugate that enables inhibitor discovery. The assay was applied to tetracycline substrates and reported inhibitors, providing insight into their binding modes. Screening of a bioactive molecule library identified novel Tet(X) inhibitors, including psychoactive phenothiazine derivatives and the 5-HT4 agonist tegaserod, the activities of which were validated by turnover assays. Crystallographic studies of Tet(X4)-inhibitor complexes reveal two new inhibitor binding modes, importantly providing evidence for active site binding of Tet(X) inhibitors that do not share structural similarity with tetracycline substrates. In some cases, potentiation of tigecycline activity was observed in bacteria expressing Tet(X4). The combined results provide non-tetracycline scaffolds for development of potent Tet(X) inhibitors and highlight the need to evaluate the impact of non-antibiotics on antimicrobial resistance.

The hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD) inhibitors can potentially treat ischemic and hypoxic-related diseases, as demonstrated by their use in anemia treatment. An AlphaScreen assay was utilized to assess the PHD2 inhibition of the 2-( 1H -1,2,3-triazol-1-yl)acetic acid (TA) derivatives ( 1–14 ), which were synthesized using copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) reaction. Most of the TA derivatives did not inhibit PHD2 effectively, with compound 14 demonstrating weak inhibition at 100 µM (>50 %). Docking experiments revealed that 14 forms hydrogen bond interactions with Arg 383 and binds to the PHD2 active site iron in a bidentate manner. Molecular dynamic simulation analysis shows 14 resembles but differs from the positive control inhibitor bicyclic isoquinoline (BIQ). It displays stable dynamic characteristics, with some flexible areas stabilizing upon binding to PHD2. The total binding energies for BIQ-PHD2 and 14-PHD2 were determined to be −42.90 kcal/mol and −36.08 kcal/mol, respectively. These values suggest that 14 and BIQ have similar binding affinities. In conclusion, structural changes of 14 may result in the development of an effective PHD2 inhibitor. However, most TA derivatives showed strong binding in docking studies but did not demonstrate good inhibitory activity against PHD2.

Carbapenemases, β-lactamases hydrolysing carbapenem antibiotics, challenge the treatment of multi-drug resistant bacteria. The OXA-48 carbapenemase is widely disseminated in Enterobacterales, necessitating new treatments for producer strains. Diazabicyclooctane (DBO) inhibitors, including avibactam and nacubactam, act on a wide range of enzymes to overcome β-lactamase-mediated resistance. Here we describe investigations on how avibactam and nacubactam inhibit OXA-48 and two variants, OXA-163 and OXA-405, with deletions in the β5-β6 loop neighbouring the active site that modify activity towards different β-lactam classes. Nacubactam is ∼80-fold less potent than avibactam towards OXA-48, but this difference reduces in OXA-163 and OXA-405. Crystal structures and molecular dynamics simulations reveal electrostatic repulsion between Arg214 on the OXA-48 β5-β6 active-site loop and nacubactam, but not avibactam; effects absent from simulations of OXA-163 and OXA-405, which lack Arg214. Crystallographic and mass spectrometry data demonstrate that all three enzymes support desulfation of the bound DBOs. The results indicate that interactions with Arg214 affect DBO potency, suggesting that sequence variation in OXA-48-like β-lactamases affects reactivity towards inhibitors as well as β-lactam substrates.

β-lactams demonstrate promising in vitro activity against Mycobacterium species and are being explored for tuberculosis treatment; however, evidence of their in vivo efficacy versus Mycobacterium tuberculosis remains limited. To achieve broad clinically relevant potency, optimization of the classical β-lactam scaffolds or development of new or non-β-lactam inhibitors for mycobacterial transpeptidases is likely required. In mycobacteria, potential targets of β-lactams include l,d-transpeptidases (Ldts) and penicillin-binding proteins (PBPs). Reports suggest that dual inhibition of Ldts and PBPs may be necessary to achieve effective anti-mycobacterial activity, yet the specific contributions of Ldt and PBP inhibition to the β-lactam antibacterial mechanisms are poorly understood. We used fluorogenic substrate mimics to investigate the effects of β-lactams and reported LdtMt2 inhibitors on Mycobacterium smegmatis (Msm), assessing their impacts on Ldt and PBP transpeptidase activities in living cells. The results reveal a statistically significant correlation between both Ldt and PBP inhibition and Msm growth suppression; under the tested conditions, a stronger correlation between Ldt inhibition and Msm growth suppression was observed. Notably, apparent inhibition of both PBPs and Ldts was observed with all active inhibitors, though β-lactams manifest increased potency of PBP inhibition. The combination of the β-lactams meropenem and faropenem with selected LdtMt2 inhibitors manifested an additive inhibitory effect against Msm. Our results highlight the importance of further optimizing β-lactam efficacy versus mycobacterial PBPs and Ldt transpeptidases.

N 6-Methyladenosine (m6A) is a prevalent post-transcriptional modification in eukaryotic messenger RNA. Two cancer-linked human Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenases, the fat mass and obesity associated-protein (FTO), and AlkB human homolog 5 (ALKBH5) catalyse m6A methyl group oxidation. While ALKBH5 has consistently been reported to catalyse m6A demethylation, there are conflicting reports concerning the FTO products. We report studies using mass spectrometry and nuclear magnetic resonance comparing products of FTO, ALKBH5, and DNA damage repair demethylases (human ALKBH2 and ALKBH3 and bacterial AlkB, using m1A single-stranded DNA substrates). The results with m6A-containing single-stranded RNA (ssRNA) and N6,2'-O-dimethyladenosine adjacent to the 5' m7G triphosphate cap ssRNA substrates imply that the predominant FTO product is N6-hydroxymethyladenosine, either with or without methylation on the substrate ribose 2'-hydroxyl group. The nascent hemiaminal product undergoes relatively slow non-enzyme catalysed fragmentation giving adenosine/formaldehyde. The other four 2OG-dependent oxygenases tested, including ALKBH5, produce demethylated bases as the predominant products. The results imply that, at least in isolated form, FTO preferentially acts as a hydroxylase, producing a hemiaminal product, rather than a demethylase, distinguishing it from ALKBH5. They highlight a need for investigations into the roles of hemiaminal-type modifications to nucleic acids, in both healthy biology and disease.

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, O2 and BRD substrates. The JMJD6 K app m for O2 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 O2 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.

The catalytic action of enzymes in a cascade trapped within a mesoporous electrode material is simultaneously energized, controlled and observed through the efficient, reversible electrochemical NAD(P)(H) recycling catalyzed by one of the enzymes. In their nanoconfined state, nicotinamide cofactors are tightly channeled current carriers, mediating multi-step reactions in either direction (oxidation or reduction) with a rapid response time. By incorporating a hydrogen-borrowing enzyme pair, the internal action of which opposes the external voltage bias driving oxidation or reduction, a reduction process can be performed under overall oxidizing conditions, and vice versa. The power of the method to control and resolve complex metabolic pathways is demonstrated using a non-linear, three-enzyme cascade extended by incorporating a fourth enzyme, urease. The latter generates in situ ammonia, which is enzymatically consumed in a reductive process, but the immediate current response to each addition of urea is observed, unusually, by applying an oxidizing potential. A practical consequence is that enzyme-catalyzed electrochemical reduction reactions requiring anaerobic conditions (to avoid O2 interference) can readily be studied under ambient aerobic conditions. The results illustrate how a complex enzyme cascade confined within a porous electrode and connected to an electrical power source manifests characteristics associated with electronic circuits.

ALKBH5 is one of only two known human non-heme Fe(II)/2-oxoglutarate-dependent oxygenases that catalyze the demethylation of N6-methyladenine (m6A) in single-stranded mRNA, underscoring its role in diverse cancers. Unlike its homolog, the fat mass and obesity-associated protein (FTO), which oxidizes m6A to a stable N6-hydroxymethyladenine (hm6A) intermediate, ALKBH5 demethylates m6A, yielding adenine and formaldehyde as products. Here, we integrate molecular dynamics simulations and quantum mechanics/molecular mechanics methods to elucidate ALKBH5's complete catalytic mechanism. Two post-hydroxylation pathways were evaluated: a proton transfer pathway and a Schiff base formation pathway, with the former emerging as the favored mechanism. We identify second-sphere residues Lys132 and Tyr139 as essential contributors to catalysis and demonstrate how Val191 and Tyr133 modulate activity. Dynamic analyses reveal that correlated motions of structural elements such as nucleotide recognition lids NRL1 and NRL2 and increased flexibility of the NRL2 loop in the hm6A intermediate may be critical for efficient demethylation.

Nirmatrelvir is a substrate-related inhibitor of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) main protease (Mpro) that is clinically used in combination with ritonavir to treat COVID-19. Derivatives of nirmatrelvir, modified at the substrate P2-equivalent position, have been developed to fine-tune inhibitor properties and are now in clinical use. We report the synthesis of nirmatrelvir derivatives with a (R)-4,4-dimethyl-4-silaproline (silaproline) group at the P2-equivalent position. Mass spectrometry (MS)-based assays demonstrate that silaproline-bearing nirmatrelvir derivatives efficiently inhibit isolated recombinant Mpro, albeit with reduced potency compared to nirmatrelvir. Investigations with SARS-CoV-2 infected VeroE6 cells reveal that the silaproline-bearing inhibitors with a CF3 group at the P4-equivalent position inhibit viral progression, implying that incorporating silicon atoms into Mpro inhibitors can yield in vivo active inhibitors with appropriate optimization. MS and crystallographic studies show that the nucleophilic active site cysteine residue of Mpro (Cys145) reacts with the nitrile group of the silaproline-bearing inhibitors. Substituting the electrophilic nitrile group for a non-activated terminal alkyne shifts the inhibition mode from reversible covalent inhibition to irreversible covalent inhibition. One of the two prochiral silaproline methyl groups occupies space in the S2 pocket that is unoccupied in Mpro:nirmatrelvir complex structures, highlighting the value of sila-derivatives in structure-activity-relationship (SAR) studies. The combined results highlight the potential of silicon-containing molecules for inhibition of Mpro and, by implication, other nucleophilic cysteine enzymes.

The ribosomal synthesis of a helical peptide library containing structurally constrained cyclic β-amino acid at every third position and its application for the RaPID selection against SARS-CoV-2 main protease (M pro ) inhibitors.

Members of the Jumonji C (JmjC) subfamily of non-heme Fe(ii) and 2-oxoglutarate (2OG) dependent N ε-lysine demethylases have established roles in catalysing demethylation of N ε-methylated lysine residues in core histones; their roles in accepting linker H1 histones as substrates have been less well explored. We report studies on the H1 substrate specificity of human JmjC lysine demethylases (KDMs), specifically KDM3A-C, KDM4A, KDM4D, KDM4E, KDM5D, and KDM6B, for mono-, di- and trimethylated N ε-lysine residues in peptide fragments of the N-terminal tail of human linker histone H1 isoforms (H1.2, H1.3, H1.4 and H1.5). The KDM4s, but not the other tested JmjC KDMs, catalysed demethylation of tri- and dimethylated H1 peptide isoforms with activities: KDM4E > KDM4D > KDM4A. The order of substrate preference for KDM4E was H1.2K26me3 > H1.5K26me3 ≈ H1.3K24me3 > H1.2K25me3 ≈ H1.4K25me3. For KDM4D, the most efficient tested substrate was H1.5K26me3. Among the dimethylated H1 peptide isoforms, H1.3K24me2 appeared to be the most efficient KDM4E substrate, with comparable activity to the core histone H3K9me2 substrate. The results demonstrate that JmjC KDM4s can accept the N-terminal H1 tails as substrates, further highlighting the potential for flexibility in substrate and product selectivity of the JmjC KDMs, in particular, within the KDM4 subfamily. Molecular and cellular investigations on JmjC KDM-catalysed H1 demethylation are of molecular and biomedical interest.

Factor inhibiting hypoxia-inducible factor (FIH) is a JmjC domain 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase that catalyzes protein hydroxylations, including of specific asparagines in the C-terminal transcriptional activation domains of hypoxia-inducible factor alpha (HIF-α) isoforms. FIH is of medicinal interest due to its ability to alter metabolism and modulate the course of the HIF-mediated hypoxic response. We report the development of a light-induced, lysine (Lys106)-targeting irreversible covalent inhibitor of FIH. The approach is complementary to optogenetic methods for regulation of transcription. The covalently reacting inhibitor NBA-ZG-2291 was the result of structure-guided modification of the reported active site binding FIH inhibitor ZG-2291 with an appropriately positioned o-nitrobenzyl alcohol (o-NBA) group. The results demonstrate that NBA-ZG-2291 forms a stable covalent bond in a light-dependent process with Lys106 of FIH, inactivating its hydroxylation activity and resulting in sustained upregulation of FIH-dependent HIF target genes. The light-controlled inhibitors targeting a lysine residue enable light and spatiotemporal control of FIH activity in a manner useful for dissecting the context-dependent physiological roles of FIH.

The biomechanical properties and responses of tissues underpin a variety important of physiological functions and pathologies. In striated muscle, the actin-binding protein filamin C (FLNC) is a key protein whose variants causative for a wide range of cardiomyopathies and musculoskeletal pathologies. FLNC is a multi-functional protein that interacts with a variety of partners, however, how it is regulated at the molecular level is not well understood. Here we investigate its interaction with HSPB7, a cardiac-specific molecular chaperone whose absence is embryonically lethal. We find that FLNC and HSPB7 interact in cardiac tissue under biomechanical stress, forming a strong hetero-dimer whose structure we solve by X-ray crystallography. Our quantitative analyses show that the hetero-dimer out-competes the FLNC homo-dimer interface, potentially acting to abrogate the ability of the protein to cross-link the actin cytoskeleton, and to enhance its diffusive mobility. We show that phosphorylation of FLNC at threonine 2677, located at the dimer interface and associated with cardiac stress, acts to favour the homo-dimer. Conversely, phosphorylation at tyrosine 2683, also at the dimer interface, has the opposite effect and shifts the equilibrium towards the hetero-dimer. Evolutionary analysis and ancestral sequence reconstruction reveals this interaction and its mechanisms of regulation to date around the time primitive hearts evolved in chordates. Our work therefore shows, structurally, how HSPB7 acts as a specific molecular chaperone that regulates FLNC dimerisation.

Abstract2-Oxoglutarate (2OG) dependentNε-methyl lysine demethylases (JmjC-KDMs) regulate eukaryotic transcription. We report studies showing that isolated forms of all human KDM4 and KDM5 JmjC enzymes catalyse demethylation ofN-methylated Arg-3 of histone H2a. Unexpectedly, the results reveal that KDM4E and, less efficiently, KDM4D catalyse C-4 hydroxylation of Arg-20 of H2a on peptides, recombinant H2a, and calf histone extracts, including when the Arg-20 guanidino group isN-methylated. Combined with previous observations, our biochemical results highlight the importance of sequence context in determining the relative efficiencies of lysine and arginine demethylation reactions catalysed by KDM4s and KDM5s. At least in some cases changes in sequence can also enable a different JmjC reaction mode, such as C-4 arginine hydroxylation instead of demethylation. Further work is thus required to define the full scope of JmjC catalysed reactions in cells.

Cancer cellular heterogeneity and therapy resistance arise substantially from metabolic and transcriptional adaptations, but how these are interconnected is poorly understood. Here, we show that, in melanoma, the cancer stem cell marker aldehyde dehydrogenase 1A3 (ALDH1A3) forms an enzymatic partnership with acetyl-coenzyme A (CoA) synthetase 2 (ACSS2) in the nucleus to couple high glucose metabolic flux with acetyl-histone H3 modification of neural crest (NC) lineage and glucose metabolism genes. Importantly, we show that acetaldehyde is a metabolite source for acetyl-histone H3 modification in an ALDH1A3-dependent manner, providing a physiologic function for this highly volatile and toxic metabolite. In a zebrafish melanoma residual disease model, an ALDH1-high subpopulation emerges following BRAF inhibitor treatment, and targeting these with an ALDH1 suicide inhibitor, nifuroxazide, delays or prevents BRAF inhibitor drug-resistant relapse. Our work reveals that the ALDH1A3-ACSS2 couple directly coordinates nuclear acetaldehyde-acetyl-CoA metabolism with specific chromatin-based gene regulation and represents a potential therapeutic vulnerability in melanoma.

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.