The catalytic domains of all human KDM5 JmjC demethylases catalyse N-methyl arginine demethylation.

The demethylation of Nε -methyllysine residues on histones by Jumonji-C lysine demethylases (JmjC-KDMs) has been established. A subset of JmjC-KDMs has also been reported to have Nω -methylarginine residue demethylase (RDM) activity. Here, we describe biochemical screening studies, showing that the catalytic domains of all human KDM5s (KDM5A-KDM5D), KDM4E and, to a lesser extent, KDM4A/D, have both KDM and RDM activities with histone peptides. Ras GTPase-activating protein-binding protein 1 peptides were shown to be RDM substrates for KDM5C/D. No RDM activity was observed with KDM1A and the other JmjC-KDMs tested. The results highlight the potential of JmjC-KDMs to catalyse reactions other than Nε -methyllysine demethylation. Although our study is limited to peptide fragments, the results should help guide biological studies investigating JmjC functions

Substrate selectivity and inhibition of histidine JmjC hydroxylases MINA53 and NO66.

Non-haem Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases catalyse oxidation of multiple proteins in organisms ranging from bacteria to humans. We describe studies on the substrate selectivity and inhibition of the human ribosomal oxygenases (ROX) MINA53 and NO66, members of the JmjC 2OG oxygenase subfamily, which catalyse C-3 hydroxylation of histidine residues in Rpl27a and Rpl8, respectively. Assays with natural and unnatural histidine analogues incorporated into Rpl peptides provide evidence that MINA53 and NO66 have narrow substrate selectivities compared to some other human JmjC hydroxylases, including factor inhibiting HIF and JMJD6. Notably, the results of inhibition assays with Rpl peptides containing histidine analogues with acyclic side chains, including Asn, Gln and homoGln, suggest the activities of MINA53/NO66, and by implication related 2OG dependent protein hydroxylases/demethylases, might be regulated in vivo by competition with non-oxidised proteins/peptides. The inhibition results also provide avenues for development of inhibitors selective for MINA53 and NO66

Structure of the CaMKIIδ/Calmodulin Complex Reveals the Molecular Mechanism of CaMKII Kinase Activation

Structural and biophysical studies reveal how CaMKII kinases, which are important for cellular learning and memory, are switched on by binding of Ca2+/calmodulin

Focused screening identifies different sensitivities of human TET oxygenases to the oncometabolite 2-hydroxyglutarate

Ten-eleven translocation enzymes (TETs) are Fe(II)/2-oxoglutarate (2OG) oxygenases that catalyze the sequential oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine in eukaryotic DNA. Despite their roles in epigenetic regulation, there is a lack of reported TET inhibitors. The extent to which 2OG oxygenase inhibitors, including clinically used inhibitors and oncometabolites, modulate DNA modifications via TETs has been unclear. Here, we report studies on human TET1–3 inhibition by a set of 2OG oxygenase-focused inhibitors, employing both enzyme-based and cellular assays. Most inhibitors manifested similar potencies for TET1–3 and caused increases in cellular 5hmC levels. (R)-2-Hydroxyglutarate, an oncometabolite elevated in isocitrate dehydrogenase mutant cancer cells, showed different degrees of inhibition, with TET1 being less potently inhibited than TET3 and TET2, potentially reflecting the proposed role of TET2 mutations in tumorigenesis. The results highlight the tractability of TETs as drug targets and provide starting points for selective inhibitor design

Structure-guided optimisation of N-hydroxythiazole-derived inhibitors of factor inhibiting hypoxia-inducible factor-α

The human 2-oxoglutarate (2OG)- and Fe(II)-dependent oxygenases factor inhibiting hypoxia-inducible factor-α (FIH) and HIF-α prolyl residue hydroxylases 1–3 (PHD1–3) regulate the response to hypoxia in humans via catalysing hydroxylation of the α-subunits of the hypoxia-inducible factors (HIFs). Small-molecule PHD inhibitors are used for anaemia treatment; by contrast, few selective inhibitors of FIH have been reported, despite their potential to regulate the hypoxic response, either alone or in combination with PHD inhibition. We report molecular, biophysical, and cellular evidence that the N-hydroxythiazole scaffold, reported to inhibit PHD2, is a useful broad spectrum 2OG oxygenase inhibitor scaffold, the inhibition potential of which can be tuned to achieve selective FIH inhibition. Structure-guided optimisation resulted in the discovery of N-hydroxythiazole derivatives that manifest substantially improved selectivity for FIH inhibition over PHD2 and other 2OG oxygenases, including Jumonji-C domain-containing protein 5 (∼25-fold), aspartate/asparagine-β-hydroxylase (>100-fold) and histone Nε-lysine demethylase 4A (>300-fold). The optimised N-hydroxythiazole-based FIH inhibitors modulate the expression of FIH-dependent HIF target genes and, consistent with reports that FIH regulates cellular metabolism, suppressed lipid accumulation in adipocytes. Crystallographic studies reveal that the N-hydroxythiazole derivatives compete with both 2OG and the substrate for binding to the FIH active site. Derivatisation of the N-hydroxythiazole scaffold has the potential to afford selective inhibitors for 2OG oxygenases other than FIH

5‑Substituted Pyridine-2,4-dicarboxylate Derivatives Have Potential for Selective Inhibition of Human Jumonji‑C Domain-Containing Protein 5

Jumonji-C domain-containing protein 5 (JMJD5) is a 2-oxoglutarate (2OG)-dependent oxygenase that plays important roles in development, circadian rhythm, and cancer through unclear mechanisms. JMJD5 has been reported to have activity as a histone protease, as an Nε-methyl lysine demethylase, and as an arginine residue hydroxylase. Small-molecule JMJD5-selective inhibitors will be useful for investigating its (patho)physiological roles. Following the observation that the broad-spectrum 2OG oxygenase inhibitor pyridine-2,4-dicarboxylic acid (2,4-PDCA) is a 2OG-competing JMJD5 inhibitor, we report that 5-aminoalkyl-substituted 2,4-PDCA derivatives are potent JMJD5 inhibitors manifesting selectivity for JMJD5 over other human 2OG oxygenases. Crystallographic analyses with five inhibitors imply induced fit binding and reveal that the 2,4-PDCA C5 substituent orients into the JMJD5 substrate-binding pocket. Cellular studies indicate that the lead compounds display similar phenotypes as reported for clinically observed JMJD5 variants, which have a reduced catalytic activity compared to wild-type JMJD5

Alkyne derivatives of SARS-CoV-2 main protease inhibitors including nirmatrelvir inhibit by reacting covalently with the nucleophilic cysteine

Nirmatrelvir (PF-07321332) is a nitrile-bearing small-molecule inhibitor that, in combination with ritonavir, is used to treat infections by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Nirmatrelvir interrupts the viral life cycle by inhibiting the SARS-CoV-2 main protease (Mpro), which is essential for processing viral polyproteins into functional nonstructural proteins. We report studies which reveal that derivatives of nirmatrelvir and other Mpro inhibitors with a nonactivated terminal alkyne group positioned similarly to the electrophilic nitrile of nirmatrelvir can efficiently inhibit isolated Mpro and SARS-CoV-2 replication in cells. Mass spectrometric and crystallographic evidence shows that the alkyne derivatives inhibit Mpro by apparent irreversible covalent reactions with the active site cysteine (Cys145), while the analogous nitriles react reversibly. The results highlight the potential for irreversible covalent inhibition of Mpro and other nucleophilic cysteine proteases by alkynes, which, in contrast to nitriles, can be functionalized at their terminal position to optimize inhibition and selectivity, as well as pharmacodynamic and pharmacokinetic 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