The bicyclic boronate VNRX-5133 (taniborbactam) is a new type of β-lactamase inhibitor in clinical development. We report that VNRX-5133 inhibits serine-β-lactamases (SBLs) and some clinically ...important metallo-β-lactamases (MBLs), including NDM-1 and VIM-1/2. VNRX-5133 activity against IMP-1 and tested B2/B3 MBLs was lower/not observed. Crystallography reveals how VNRX-5133 binds to the class D SBL OXA-10 and MBL NDM-1. The crystallographic results highlight the ability of bicyclic boronates to inhibit SBLs and MBLs via binding of a tetrahedral (sp3) boron species. The structures imply conserved binding of the bicyclic core with SBLs/MBLs. With NDM-1, by crystallography, we observed an unanticipated VNRX-5133 binding mode involving cyclization of its acylamino oxygen onto the boron of the bicyclic core. Different side-chain binding modes for bicyclic boronates for SBLs and MBLs imply scope for side-chain optimization. The results further support the “high-energy-intermediate” analogue approach for broad-spectrum β-lactamase inhibitor development and highlight the ability of boron inhibitors to interchange between different hybridization states/binding modes.
Group VII ethylene response factors (ERF-VIIs) regulate transcriptional adaptation to flooding-induced hypoxia in plants. ERF-VII stability is controlled in an O2-dependent manner by the Cys/Arg ...branch of the N-end rule pathway whereby oxidation of a conserved N-terminal cysteine residue initiates target degradation. This oxidation is catalyzed by plant cysteine oxidases (PCOs), which use O2 as cosubstrate to generate Cys-sulfinic acid. The PCOs directly link O2 availability to ERF-VII stability and anaerobic adaptation, leading to the suggestion that they act as plant O2 sensors. However, their ability to respond to fluctuations in O2 concentration has not been established. Here, we investigated the steady-state kinetics of Arabidopsis thaliana PCOs 1–5 to ascertain whether their activities are sensitive to O2 levels. We found that the most catalytically competent isoform is AtPCO4, both in terms of responding to O2 and oxidizing AtRAP2.2/2,12 (two of the most prominent ERF-VIIs responsible for promoting the hypoxic response), which suggests that AtPCO4 plays a central role in ERF-VII regulation. Furthermore, we found that AtPCO activity is susceptible to decreases in pH and that the hypoxia-inducible AtPCOs 1/2 and the noninducible AtPCOs 4/5 have discrete AtERF-VII substrate preferences. Pertinently, the AtPCOs had Km(O2)app values in a physiologically relevant range, which should enable them to sensitively react to changes in O2 availability. This work validates an O2-sensing role for the PCOs and suggests that differences in expression pattern, ERF-VII selectivity, and catalytic capability may enable the different isoforms to have distinct biological functions. Individual PCOs could therefore be targeted to manipulate ERF-VII levels and improve stress tolerance in plants.
Epigenetics is currently the focus of intense research interest across a broad range of disciplines due to its importance in a multitude of biological processes and disease states. Epigenetic ...functions result partly from modification of the nucleobases in DNA and RNA, and/or post‐translational modifications of histone proteins. These modifications are dynamic, with cellular machinery identified to modulate and interpret the marks. Our focus is on bromodomains, which bind to acetylated lysine residues. Progress in the study of bromodomains, and the development of bromodomain ligands, has been rapid. These advances have been underpinned by many disciplines, but chemistry and chemical biology have undoubtedly played a significant role. Herein, we review the key chemistry and chemical biology approaches that have furthered our study of bromodomains, enabled the development of bromodomain ligands, and played a critical role in the validation of bromodomains as therapeutic targets.
Progress in the study of bromodomains, and the development of their ligands, has been underpinned by many disciplines, but chemistry and chemical biology have undoubtedly played a very significant role. Here we review the key chemistry and chemical biology techniques that have furthered our study of bromodomains, enabled the development of bromodomain ligands, and played a critical role in the validation of bromodomains as therapeutic targets.
Covering: up to 2017
2-Oxoglutarate (2OG) dependent oxygenases and the homologous oxidase isopenicillin N synthase (IPNS) play crucial roles in the biosynthesis of β-lactam ring containing natural ...products. IPNS catalyses formation of the bicyclic penicillin nucleus from a tripeptide. 2OG oxygenases catalyse reactions that diversify the chemistry of β-lactams formed by both IPNS and non-oxidative enzymes. Reactions catalysed by the 2OG oxygenases of β-lactam biosynthesis not only involve their typical hydroxylation reactions, but also desaturation, epimerisation, rearrangement, and ring-forming reactions. Some of the enzymes involved in β-lactam biosynthesis exhibit remarkable substrate and product selectivities. We review the roles of 2OG oxygenases and IPNS in β-lactam biosynthesis, highlighting opportunities for application of knowledge of their roles, structures, and mechanisms.
The 2OG oxygenases and IPNS contribute to the great structural diversity of β-lactam natural products, employing some remarkable mechanisms.
The two SARS‐CoV‐2 proteases, i. e. the main protease (Mpro) and the papain‐like protease (PLpro), which hydrolyze the viral polypeptide chain giving functional non‐structural proteins, are essential ...for viral replication and are medicinal chemistry targets. We report a high‐throughput mass spectrometry (MS)‐based assay which directly monitors PLpro catalysis in vitro. The assay was applied to investigate the effect of reported small‐molecule PLpro inhibitors and selected Mpro inhibitors on PLpro catalysis. The results reveal that some, but not all, PLpro inhibitor potencies differ substantially from those obtained using fluorescence‐based assays. Some substrate‐competing Mpro inhibitors, notably PF‐07321332 (nirmatrelvir) which is in clinical development, do not inhibit PLpro. Less selective Mpro inhibitors, e. g. auranofin, inhibit PLpro, highlighting the potential for dual PLpro/Mpro inhibition. MS‐based PLpro assays, which are orthogonal to widely employed fluorescence‐based assays, are of utility in validating inhibitor potencies, especially for inhibitors operating by non‐covalent mechanisms.
Double check: A mass spectrometry‐based direct SARS‐CoV‐2 PLpro assay reveals discrepancies in inhibition profiles for non‐covalent inhibitors. Use of assays orthogonal to the established fluorescence‐based assays is important for development of efficient small‐molecule PLpro inhibitors.
A large number of structurally diverse epigenetic reader proteins specifically recognize methylated lysine residues on histone proteins. Here we describe comparative thermodynamic, structural and ...computational studies on recognition of the positively charged natural trimethyllysine and its neutral analogues by reader proteins. This work provides experimental and theoretical evidence that reader proteins predominantly recognize trimethyllysine via a combination of favourable cation-π interactions and the release of the high-energy water molecules that occupy the aromatic cage of reader proteins on the association with the trimethyllysine side chain. These results have implications in rational drug design by specifically targeting the aromatic cage of readers of trimethyllysine.
Rhodanines have been characterised as 'difficult to progress' compounds for medicinal use, though one rhodanine is used for diabetes mellitus treatment and others are in clinical development. ...Rhodanines can undergo hydrolysis to enethiols which are inhibitors of metallo-enzymes, such as metallo β-lactamases. We report that in DMSO, rhodanine derived enethiols undergo dimerisations to give 1,3-dithiolanes and mixed disulfides. The results highlight the potential of rhodanines and enethiols to give multiple products. They suggest that where possible DMSO should be avoided as a storage solvent for rhodanines/enethiols and highlight the need for further research on biologically relevant enethiols/mixed disulfides.
Unexpected reaction of enethiols to form 1,3-dithiolanes and mixed disulfides in DMSO occur through a common thioketone intermediate.
How formaldehyde reacts with amino acids Kamps, Jos J. A. G.; Hopkinson, Richard J.; Schofield, Christopher J. ...
Communications chemistry,
11/2019, Letnik:
2, Številka:
1
Journal Article
Recenzirano
Odprti dostop
Abstract
Formaldehyde is a biological electrophile produced via processes including enzymatic demethylation. Despite its apparent simplicity, the reactions of formaldehyde with even basic biological ...components are incompletely defined. Here we report NMR-based studies on the reactions of formaldehyde with common proteinogenic and other nucleophilic amino acids. The results reveal formaldehyde reacts at different rates, forming hydroxymethylated, cyclised, cross-linked, or disproportionated products of varying stabilities. Of the tested common amino acids, cysteine reacts most efficiently, forming a stable thiazolidine. The reaction with lysine is less efficient; low levels of an N
ε
-methylated product are observed, raising the possibility of non-enzymatic lysine methylation by formaldehyde. Reactions with formaldehyde are faster than reactions with other tested biological carbonyl compounds, and the adducts are also more stable. The results reveal reactions of formaldehyde with amino acids, and by extension peptides and proteins, have potential roles in healthy and diseased biology, as well as in evolution.
Site-specific incorporation of post-translationally modified amino acids into proteins, including histones, has been a subject of great interest for chemical and biochemical communities. Here, we ...describe a site-specific incorporation of structurally simplest trimethyllysine analogs into position 4 of the intact histone H3 protein. An efficient alkylation of cysteine 4 of the recombinantly expressed histone H3 provides a panel of trimethyllysine analogs that differ in charge, charge density, sterics, and chain length. We demonstrate that H3 histone that bears trimethyllysine analogs can be further assembled into the octameric histone complex that constitutes the nucleosome. Binding studies showed that H3 histone that possesses trimethyllysine analogs is well recognized by a PHD3 reader domain of human JARID1A. This work provides important (bio)chemical tools for fundamental biomolecular studies aimed at unravelling the molecular basis of the higher order nucleosome and chromatin assemblies.
Enzymes often use nucleophilic serine, threonine, and cysteine residues to achieve the same type of reaction; the underlying reasons for this are not understood. While bacterial d,d‐transpeptidases ...(penicillin‐binding proteins) employ a nucleophilic serine, l,d‐transpeptidases use a nucleophilic cysteine. The covalent complexes formed by l,d‐transpeptidases with some β‐lactam antibiotics undergo non‐hydrolytic fragmentation. This is not usually observed for penicillin‐binding proteins, or for the related serine β‐lactamases. Replacement of the nucleophilic serine of serine β‐lactamases with cysteine yields enzymes which fragment β‐lactams via a similar mechanism as the l,d‐transpeptidases, implying the different reaction outcomes are principally due to the formation of thioester versus ester intermediates. The results highlight fundamental differences in the reactivity of nucleophilic serine and cysteine enzymes, and imply new possibilities for the inhibition of nucleophilic enzymes.
Weapon of choice: Penicillin‐binding proteins and serine β‐lactamases, which employ nucleophilic serines, hydrolyze β‐lactam antibiotics. However, the l,d‐transpeptidases, which employ nucleophilic cysteines, also fragment some β‐lactams. Serine β‐lactamases where the nucleophilic serine is substituted with cysteine also catalyze β‐lactam fragmentation, consistent with a mechanism involving the formation of thioester‐enolate intermediates.
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