Publications by authors named "Martine I Abboud"

32 Publications

A Diverse View of Science to Catalyse Change.

Angew Chem Int Ed Engl 2020 10 17;59(42):18306-18310. Epub 2020 Aug 17.

State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, International Joint Research Laboratory of Nano-Micro Architecture Chemistry (NMAC), College of Chemistry, Jilin University, Changchun, 130012, People's Republic of China.

Valuing diversity leads to scientific excellence, the progress of science and most importantly, it is simply the right thing to do. We can value diversity not only in words, but also in actions.
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http://dx.doi.org/10.1002/anie.202009834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7590070PMC
October 2020

Use of cyclic peptides to induce crystallization: case study with prolyl hydroxylase domain 2.

Sci Rep 2020 12 15;10(1):21964. Epub 2020 Dec 15.

Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, OX1 3TA, UK.

Crystallization is the bottleneck in macromolecular crystallography; even when a protein crystallises, crystal packing often influences ligand-binding and protein-protein interaction interfaces, which are the key points of interest for functional and drug discovery studies. The human hypoxia-inducible factor prolyl hydroxylase 2 (PHD2) readily crystallises as a homotrimer, but with a sterically blocked active site. We explored strategies aimed at altering PHD2 crystal packing by protein modification and molecules that bind at its active site and elsewhere. Following the observation that, despite weak inhibition/binding in solution, succinamic acid derivatives readily enable PHD2 crystallization, we explored methods to induce crystallization without active site binding. Cyclic peptides obtained via mRNA display bind PHD2 tightly away from the active site. They efficiently enable PHD2 crystallization in different forms, both with/without substrates, apparently by promoting oligomerization involving binding to the C-terminal region. Although our work involves a specific case study, together with those of others, the results suggest that mRNA display-derived cyclic peptides may be useful in challenging protein crystallization cases.
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http://dx.doi.org/10.1038/s41598-020-76307-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7738489PMC
December 2020

Biochemical and biophysical analyses of hypoxia sensing prolyl hydroxylases from and .

J Biol Chem 2020 12 15;295(49):16545-16561. Epub 2020 Sep 15.

Chemistry Research Laboratory, University of Oxford, Oxford, United Kingdom. Electronic address:

In animals, the response to chronic hypoxia is mediated by prolyl hydroxylases (PHDs) that regulate the levels of hypoxia-inducible transcription factor α (HIFα). PHD homologues exist in other types of eukaryotes and prokaryotes where they act on non HIF substrates. To gain insight into the factors underlying different PHD substrates and properties, we carried out biochemical and biophysical studies on PHD homologues from the cellular slime mold, and the protozoan parasite, , both lacking HIF. The respective prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation of S-phase kinase-associated protein 1 (Skp1), a reaction enabling adaptation to different dioxygen availability. Assays with full-length Skp1 substrates reveal substantial differences in the kinetic properties of DdPhyA and TgPhyA, both with respect to each other and compared with human PHD2; consistent with cellular studies, TgPhyA is more active at low dioxygen concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate. No cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. The human Skp1 E147P variant is a DdPhyA and TgPhyA substrate, suggesting some retention of ancestral interactions. Crystallographic analysis of DdPhyA enables comparisons with homologues from humans, , and prokaryotes, informing on differences in mobile elements involved in substrate binding and catalysis. In DdPhyA, two mobile loops that enclose substrates in the PHDs are conserved, but the C-terminal helix of the PHDs is strikingly absent. The combined results support the proposal that PHD homologues have evolved kinetic and structural features suited to their specific sensing roles.
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http://dx.doi.org/10.1074/jbc.RA120.013998DOI Listing
December 2020

Anion-exchange chromatography mass spectrometry provides extensive coverage of primary metabolic pathways revealing altered metabolism in IDH1 mutant cells.

Commun Biol 2020 May 20;3(1):247. Epub 2020 May 20.

Department of Chemistry, University of Oxford, Mansfield Road, Oxford, OX1 3TA, UK.

Altered central carbon metabolism is a hallmark of many diseases including diabetes, obesity, heart disease and cancer. Identifying metabolic changes will open opportunities for better understanding aetiological processes and identifying new diagnostic, prognostic, and therapeutic targets. Comprehensive and robust analysis of primary metabolic pathways in cells, tissues and bio-fluids, remains technically challenging. We report on the development and validation of a highly reproducible and robust untargeted method using anion-exchange tandem mass spectrometry (IC-MS) that enables analysis of 431 metabolites, providing detailed coverage of central carbon metabolism. We apply the method in an untargeted, discovery-driven workflow to investigate the metabolic effects of isocitrate dehydrogenase 1 (IDH1) mutations in glioblastoma cells. IC-MS provides comprehensive coverage of central metabolic pathways revealing significant elevation of 2-hydroxyglutarate and depletion of 2-oxoglutarate. Further analysis of the data reveals depletion in additional metabolites including previously unrecognised changes in lysine and tryptophan metabolism.
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http://dx.doi.org/10.1038/s42003-020-0957-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239943PMC
May 2020

Structure-Activity Relationship and Crystallographic Studies on 4-Hydroxypyrimidine HIF Prolyl Hydroxylase Domain Inhibitors.

ChemMedChem 2020 02 3;15(3):270-273. Epub 2019 Dec 3.

Department of Chemistry, University of Oxford Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA, UK.

The 2-oxoglutarate-dependent hypoxia inducible factor prolyl hydroxylases (PHDs) are targets for treatment of a variety of diseases including anaemia. One PHD inhibitor is approved for use for the treatment of renal anaemia and others are in late stage clinical trials. The number of reported templates for PHD inhibition is limited. We report structure-activity relationship and crystallographic studies on a promising class of 4-hydroxypyrimidine-containing PHD inhibitors.
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http://dx.doi.org/10.1002/cmdc.201900557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496690PMC
February 2020

Lack of activity of recombinant HIF prolyl hydroxylases (PHDs) on reported non-HIF substrates.

Elife 2019 09 10;8. Epub 2019 Sep 10.

The Francis Crick Institute, London, United Kingdom.

Human and other animal cells deploy three closely related dioxygenases (PHD 1, 2 and 3) to signal oxygen levels by catalysing oxygen regulated prolyl hydroxylation of the transcription factor HIF. The discovery of the HIF prolyl-hydroxylase (PHD) enzymes as oxygen sensors raises a key question as to the existence and nature of non-HIF substrates, potentially transducing other biological responses to hypoxia. Over 20 such substrates are reported. We therefore sought to characterise their reactivity with recombinant PHD enzymes. Unexpectedly, we did not detect prolyl-hydroxylase activity on any reported non-HIF protein or peptide, using conditions supporting robust HIF-α hydroxylation. We cannot exclude PHD-catalysed prolyl hydroxylation occurring under conditions other than those we have examined. However, our findings using recombinant enzymes provide no support for the wide range of non-HIF PHD substrates that have been reported.
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http://dx.doi.org/10.7554/eLife.46490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739866PMC
September 2019

The Clinically Used Iron Chelator Deferasirox Is an Inhibitor of Epigenetic JumonjiC Domain-Containing Histone Demethylases.

ACS Chem Biol 2019 08 19;14(8):1737-1750. Epub 2019 Jul 19.

Institute of Pharmaceutical Sciences , Albert-Ludwigs-Universität Freiburg , Albertstraße 25 , 79104 Freiburg i.Br. , Germany.

Fe(II)- and 2-oxoglutarate (2OG)-dependent JumonjiC domain-containing histone demethylases (JmjC KDMs) are "epigenetic eraser" enzymes involved in the regulation of gene expression and are emerging drug targets in oncology. We screened a set of clinically used iron chelators and report that they potently inhibit JMJD2A (KDM4A) . Mode of action investigations revealed that one compound, deferasirox, is a active site-binding inhibitor as shown by kinetic and spectroscopic studies. Synthesis of derivatives with improved cell permeability resulted in significant upregulation of histone trimethylation and potent cancer cell growth inhibition. Deferasirox was also found to inhibit human 2OG-dependent hypoxia inducible factor prolyl hydroxylase activity. Therapeutic effects of clinically used deferasirox may thus involve transcriptional regulation through 2OG oxygenase inhibition. Deferasirox might provide a useful starting point for the development of novel anticancer drugs targeting 2OG oxygenases and a valuable tool compound for investigations of KDM function.
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http://dx.doi.org/10.1021/acschembio.9b00289DOI Listing
August 2019

Inhibition of a viral prolyl hydroxylase.

Bioorg Med Chem 2019 06 23;27(12):2405-2412. Epub 2019 Jan 23.

Chemistry Research Laboratory, Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom. Electronic address:

The hydroxylation of prolyl-residues in eukaryotes is important in collagen biosynthesis and in hypoxic signalling. The hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are drug targets for the treatment of anaemia, while the procollagen prolyl hydroxylases and other 2-oxoglutarate dependent oxygenases are potential therapeutic targets for treatment of cancer, fibrotic disease, and infection. We describe assay development and inhibition studies for a procollagen prolyl hydroxylase from Paramecium bursaria chlorella virus 1 (vCPH). The results reveal HIF PHD inhibitors in clinical trials also inhibit vCPH. Implications for the targeting of the human PHDs and microbial prolyl hydroxylases are discussed.
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http://dx.doi.org/10.1016/j.bmc.2019.01.018DOI Listing
June 2019

Born to sense: biophysical analyses of the oxygen sensing prolyl hydroxylase from the simplest animal .

Hypoxia (Auckl) 2018 9;6:57-71. Epub 2018 Nov 9.

Chemistry Research Laboratory, University of Oxford, Oxford, UK,

Background: In humans and other animals, the chronic hypoxic response is mediated by hypoxia inducible transcription factors (HIFs) which regulate the expression of genes that counteract the effects of limiting oxygen. Prolyl hydroxylases (PHDs) act as hypoxia sensors for the HIF system in organisms ranging from humans to the simplest animal .

Methods: We report structural and biochemical studies on the HIF prolyl hydroxylase (PHD) that inform about the evolution of hypoxia sensing in animals.

Results: High resolution crystal structures (≤1.3 Å) of PHD, with and without its HIFα substrate, reveal remarkable conservation of key active site elements between and human PHDs, which also manifest in kinetic comparisons.

Conclusion: Conserved structural features of PHD and human PHDs include those apparently enabling the slow binding/reaction of oxygen with the active site Fe(II), the formation of a stable 2-oxoglutarate complex, and a stereoelectronically promoted change in conformation of the hydroxylated proline-residue. Comparison of substrate selectivity between the human PHDs and PHD provides insights into the selectivity determinants of HIF binding by the PHDs, and into the evolution of the multiple HIFs and PHDs present in higher animals.
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http://dx.doi.org/10.2147/HP.S174655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235002PMC
November 2018

Selective Inhibitors of a Human Prolyl Hydroxylase (OGFOD1) Involved in Ribosomal Decoding.

Chemistry 2019 Feb 8;25(8):2019-2024. Epub 2019 Jan 8.

Department of Chemistry, University of Oxford, Oxford, OX1 3TA, UK.

Human prolyl hydroxylases are involved in the modification of transcription factors, procollagen, and ribosomal proteins, and are current medicinal chemistry targets. To date, there are few reports on inhibitors selective for the different types of prolyl hydroxylases. We report a structurally informed template-based strategy for the development of inhibitors selective for the human ribosomal prolyl hydroxylase OGFOD1. These inhibitors did not target the other human oxygenases tested, including the structurally similar hypoxia-inducible transcription factor prolyl hydroxylase, PHD2.
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http://dx.doi.org/10.1002/chem.201804790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471485PMC
February 2019

A Fluorescent Benzo[g]isoquinoline-Based HIF Prolyl Hydroxylase Inhibitor for Cellular Imaging.

ChemMedChem 2019 01 21;14(1):94-99. Epub 2018 Dec 21.

Department of Chemistry and Pharmacy, Inorganic and Organometallic Chemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, Egerlandstraße 1, 91058, Erlangen, Germany.

Prolyl hydroxylation domain (PHD) enzymes catalyze the hydroxylation of the transcription factor hypoxia-inducible factor (HIF) and serve as cellular oxygen sensors. HIF and the PHD enzymes regulate numerous potentially tissue-protective target genes which can adapt cells to metabolic and ischemic stress. We describe a fluorescent PHD inhibitor (1-chloro-4-hydroxybenzo[g]isoquinoline-3-carbonyl)glycine which is suited to fluorescence-based detection assays and for monitoring PHD inhibitors in biological systems. In cell-based assays, application of the fluorescent PHD inhibitor allowed co-localization with a cellular PHD enzyme and led to live cell imaging of processes involved in cellular oxygen sensing.
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http://dx.doi.org/10.1002/cmdc.201800483DOI Listing
January 2019

Studies on the Substrate Selectivity of the Hypoxia-Inducible Factor Prolyl Hydroxylase 2 Catalytic Domain.

Chembiochem 2018 11 26;19(21):2262-2267. Epub 2018 Sep 26.

Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford, OX1 3TA, UK.

In animals, the response to chronic hypoxia is mediated by upregulation of the α,β-heterodimeric hypoxia-inducible factors (HIFs). Levels of HIFα isoforms, but not HIFβ, are regulated by their post-translational modification as catalysed by prolyl hydroxylase domain enzymes (PHDs). Different roles for the human HIF-1/2α isoforms and their two oxygen-dependent degradation domains (ODDs) are proposed. We report kinetic and NMR analyses of the ODD selectivity of the catalytic domain of wild-type PHD2 (which is conserved in nearly all animals) and clinically observed variants. Studies using Ala scanning and "hybrid" ODD peptides imply that the relatively rigid conformation of the (hydroxylated) proline plays an important role in ODD binding. They also reveal differential roles in binding for the residues on the N- and C-terminal sides of the substrate proline. The overall results indicate how the PHDs achieve selectivity for HIFα ODDs and might be of use in identifying substrate-selective PHD inhibitors.
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http://dx.doi.org/10.1002/cbic.201800246DOI Listing
November 2018

Publisher Correction: The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases.

Nat Chem Biol 2018 Oct;14(10):988

Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, UK.

In the version of this article initially published, authors Sarah E. Wilkins, Charlotte D. Eaton, Martine I. Abboud and Maximiliano J. Katz were incorrectly included in the equal contributions footnote in the affiliations list. Footnote number seven linking to the equal contributions statement should be present only for Suzana Markolovic and Qinqin Zhuang, and the statement should read "These authors contributed equally: Suzana Markolovic, Qinqin Zhuang." The error has been corrected in the HTML and PDF versions of the article.
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http://dx.doi.org/10.1038/s41589-018-0104-6DOI Listing
October 2018

The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases.

Nat Chem Biol 2018 07 18;14(7):688-695. Epub 2018 Jun 18.

Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, UK.

Biochemical, structural and cellular studies reveal Jumonji-C (JmjC) domain-containing 7 (JMJD7) to be a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes (3S)-lysyl hydroxylation. Crystallographic analyses reveal JMJD7 to be more closely related to the JmjC hydroxylases than to the JmjC demethylases. Biophysical and mutation studies show that JMJD7 has a unique dimerization mode, with interactions between monomers involving both N- and C-terminal regions and disulfide bond formation. A proteomic approach identifies two related members of the translation factor (TRAFAC) family of GTPases, developmentally regulated GTP-binding proteins 1 and 2 (DRG1/2), as activity-dependent JMJD7 interactors. Mass spectrometric analyses demonstrate that JMJD7 catalyzes Fe(II)- and 2OG-dependent hydroxylation of a highly conserved lysine residue in DRG1/2; amino-acid analyses reveal that JMJD7 catalyzes (3S)-lysyl hydroxylation. The functional assignment of JMJD7 will enable future studies to define the role of DRG hydroxylation in cell growth and disease.
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http://dx.doi.org/10.1038/s41589-018-0071-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6027965PMC
July 2018

Carcinogenic and neurotoxic risks of acrylamide consumed through caffeinated beverages among the lebanese population.

Chemosphere 2018 Oct 6;208:352-357. Epub 2018 Jun 6.

School of Arts and Sciences, Department of Natural Sciences, Lebanese American University, Byblos, Lebanon. Electronic address:

The present study aims to quantify acrylamide in caffeinated beverages including American coffee, Lebanese coffee, espresso, instant coffee and hot chocolate, and to determine their carcinogenic and neurotoxic risks. A survey was carried for this purpose whereby 78% of the Lebanese population was found to consume at least one type of caffeinated beverages. Gas Chromatography Mass Spectrometry analysis revealed that the average acrylamide level in caffeinated beverages is 29,176 μg/kg sample. The daily consumption of acrylamide from Lebanese coffee (10.9 μg/kg-bw/day), hot chocolate (1.2 μg/kg-bw/day) and Espresso (7.4 μg/kg-bw/day) was found to be higher than the risk intake for carcinogenicity and neurotoxicity as set by World Health Organization (WHO; 0.3-2 μg/kg-bw/day) at both the mean (average consumers) and high (high consumers) dietary exposures. On the other hand, American coffee (0.37 μg/kg-bw/day) was shown to pose no carcinogenic or neurotoxic risks among the Lebanese community for consumers with a mean dietary exposure. The study shows alarming results that call for regulating the caffeinated product industry by setting legislations and standard protocols for product preparation in order to limit the acrylamide content and protect consumers. In order to avoid carcinogenic and neurotoxic risks, we propose that WHO/FAO set acrylamide levels in caffeinated beverages to 7000 μg acrylamide/kg sample, a value which is 4-folds lower than the average acrylamide levels of 29,176 μg/kg sample found in caffeinated beverages sold in the Lebanese market. Alternatively, consumers of caffeinated products, especially Lebanese coffee and espresso, would have to lower their daily consumption to 0.3-0.4 cups/day.
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http://dx.doi.org/10.1016/j.chemosphere.2018.05.185DOI Listing
October 2018

2-Oxoglutarate regulates binding of hydroxylated hypoxia-inducible factor to prolyl hydroxylase domain 2.

Chem Commun (Camb) 2018 Mar 9;54(25):3130-3133. Epub 2018 Mar 9.

Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.

Prolyl hydroxylation of hypoxia inducible factor (HIF)-α, as catalysed by the Fe(ii)/2-oxoglutarate (2OG)-dependent prolyl hydroxylase domain (PHD) enzymes, has a hypoxia sensing role in animals. We report that binding of prolyl-hydroxylated HIF-α to PHD2 is ∼50 fold hindered by prior 2OG binding; thus, when 2OG is limiting, HIF-α degradation might be inhibited by PHD binding.
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http://dx.doi.org/10.1039/c8cc00387dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5885369PMC
March 2018

Lysine-241 Has a Role in Coupling 2OG Turnover with Substrate Oxidation During KDM4-Catalysed Histone Demethylation.

Chembiochem 2018 05 6;19(9):917-921. Epub 2018 Apr 6.

Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA, UK.

The JmjC histone lysyl demethylases (KDMs) play important roles in modulating histone methylation states and have the potential to be regulated by oxygen availability. Lys241 of the KDM4 subfamily is proposed to be important in oxygen binding by KDM4A. We report evidence that, although Lys241 is unlikely to be directly involved in oxygen binding, it has an important role in coupling 2-oxoglutarate cosubstrate oxidation with lysine demethylase activity. The results suggest that compounds promoting the uncoupling of substrate oxidation are of interest as JmjC-KDM inhibitors.
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http://dx.doi.org/10.1002/cbic.201800002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5969224PMC
May 2018

Molecular and cellular mechanisms of HIF prolyl hydroxylase inhibitors in clinical trials.

Chem Sci 2017 Nov 11;8(11):7651-7668. Epub 2017 Sep 11.

Chemistry Research Laboratory , Department of Chemistry , University of Oxford , Oxford OX1 3TA , UK . Email:

Inhibition of the human 2-oxoglutarate (2OG) dependent hypoxia inducible factor (HIF) prolyl hydroxylases (human PHD1-3) causes upregulation of HIF, thus promoting erythropoiesis and is therefore of therapeutic interest. We describe cellular, biophysical, and biochemical studies comparing four PHD inhibitors currently in clinical trials for anaemia treatment, that describe their mechanisms of action, potency against isolated enzymes and in cells, and selectivities representatives of other human 2OG oxygenase subfamilies. The 'clinical' PHD inhibitors are potent inhibitors of PHD catalyzed hydroxylation of the HIF-α oxygen dependent degradation domains (ODDs), and selective against most, but not all, representatives of other human 2OG dependent dioxygenase subfamilies. Crystallographic and NMR studies provide insights into the different active site binding modes of the inhibitors. Cell-based results reveal the inhibitors have similar effects on the upregulation of HIF target genes, but differ in the kinetics of their effects and in extent of inhibition of hydroxylation of the N- and C-terminal ODDs; the latter differences correlate with the biophysical observations.
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http://dx.doi.org/10.1039/c7sc02103hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802278PMC
November 2017

In Silico Fragment-Based Design Identifies Subfamily B1 Metallo-β-lactamase Inhibitors.

J Med Chem 2018 02 10;61(3):1255-1260. Epub 2018 Jan 10.

School of Chemistry, University of Leeds , Leeds LS2 9JT, United Kingdom.

Zinc ion-dependent β-lactamases (MBLs) catalyze the hydrolysis of almost all β-lactam antibiotics and resist the action of clinically available β-lactamase inhibitors. We report how application of in silico fragment-based molecular design employing thiol-mediated metal anchorage leads to potent MBL inhibitors. The new inhibitors manifest potent inhibition of clinically important B1 subfamily MBLs, including the widespread NDM-1, IMP-1, and VIM-2 enzymes; with lower potency, some of them also inhibit clinically relevant Class A and D serine-β-lactamases. The inhibitors show selectivity for bacterial MBL enzymes compared to that for human MBL fold nucleases. Cocrystallization of one inhibitor, which shows potentiation of Meropenem activity against MBL-expressing Enterobacteriaceae, with VIM-2 reveals an unexpected binding mode, involving interactions with residues from conserved active site bordering loops.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01728DOI Listing
February 2018

Cyclobutanone Mimics of Intermediates in Metallo-β-Lactamase Catalysis.

Chemistry 2018 Apr 17;24(22):5734-5737. Epub 2018 Jan 17.

Department of Chemistry, University of Waterloo, 200 University Ave. W., Waterloo, Ontario, N2L 3G1, Canada.

The most important resistance mechanism to β-lactam antibiotics involves hydrolysis by two β-lactamase categories: the nucleophilic serine and the metallo-β-lactamases (SBLs and MBLs, respectively). Cyclobutanones are hydrolytically stable β-lactam analogues with potential to inhibit both SBLs and MBLs. We describe solution and crystallographic studies on the interaction of a cyclobutanone penem analogue with the clinically important MBL SPM-1. NMR experiments using F-labeled SPM-1 imply the cyclobutanone binds to SPM-1 with micromolar affinity. A crystal structure of the SPM-1:cyclobutanone complex reveals binding of the hydrated cyclobutanone through interactions with one of the zinc ions, stabilisation of the hydrate by hydrogen bonding to zinc-bound water, and hydrophobic contacts with aromatic residues. NMR analyses using a C-labeled cyclobutanone support assignment of the bound species as the hydrated ketone. The results inform on how MBLs bind substrates and stabilize tetrahedral intermediates. They support further investigations on the use of transition-state and/or intermediate analogues as inhibitors of all β-lactamase classes.
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http://dx.doi.org/10.1002/chem.201705886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947706PMC
April 2018

Crystallographic analyses of isoquinoline complexes reveal a new mode of metallo-β-lactamase inhibition.

Chem Commun (Camb) 2017 May;53(43):5806-5809

Department of Chemistry, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.

Crystallographic analyses of the VIM-5 metallo-β-lactamase (MBL) with isoquinoline inhibitors reveal non zinc ion binding modes. Comparison with other MBL-inhibitor structures directed addition of a zinc-binding thiol enabling identification of potent B1 MBL inhibitors. The inhibitors potentiate meropenem activity against clinical isolates harboring MBLs.
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http://dx.doi.org/10.1039/c7cc02394dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5516270PMC
May 2017

NMR-filtered virtual screening leads to non-metal chelating metallo-β-lactamase inhibitors.

Chem Sci 2017 Feb 14;8(2):928-937. Epub 2016 Dec 14.

Department of Chemistry , University of Oxford , 12 Mansfield Road , Oxford , OX1 3TA , UK . Email: ; Email:

There are no clinically useful inhibitors of metallo-β-lactamases (MBLs), which are a growing problem because they hydrolyse almost all β-lactam antibacterials. Inhibition by most reported MBL inhibitors involves zinc ion chelation. A structure-based virtual screening approach combined with NMR filtering led to the identification of inhibitors of the clinically relevant Verona Integron-encoded MBL (VIM)-2. Crystallographic analyses reveal a new mode of MBL inhibition involving binding adjacent to the active site zinc ions, but which does not involve metal chelation. The results will aid efforts to develop new types of clinically useful inhibitors targeting MBLs/MBL-fold metallo-enzymes involved in antibacterial and anticancer drug resistance.
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http://dx.doi.org/10.1039/c6sc04524cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5369532PMC
February 2017

Structural and stereoelectronic insights into oxygenase-catalyzed formation of ethylene from 2-oxoglutarate.

Proc Natl Acad Sci U S A 2017 05 18;114(18):4667-4672. Epub 2017 Apr 18.

Department of Chemistry, University of Oxford, Oxford, OX1 3TA, United Kingdom

Ethylene is important in industry and biological signaling. In plants, ethylene is produced by oxidation of 1-aminocyclopropane-1-carboxylic acid, as catalyzed by 1-aminocyclopropane-1-carboxylic acid oxidase. Bacteria catalyze ethylene production, but via the four-electron oxidation of 2-oxoglutarate to give ethylene in an arginine-dependent reaction. Crystallographic and biochemical studies on the ethylene-forming enzyme reveal a branched mechanism. In one branch, an apparently typical 2-oxoglutarate oxygenase reaction to give succinate, carbon dioxide, and sometimes pyrroline-5-carboxylate occurs. Alternatively, Grob-type oxidative fragmentation of a 2-oxoglutarate-derived intermediate occurs to give ethylene and carbon dioxide. Crystallographic and quantum chemical studies reveal that fragmentation to give ethylene is promoted by binding of l-arginine in a nonoxidized conformation and of 2-oxoglutarate in an unprecedented high-energy conformation that favors ethylene, relative to succinate formation.
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http://dx.doi.org/10.1073/pnas.1617760114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5422762PMC
May 2017

F-NMR Reveals the Role of Mobile Loops in Product and Inhibitor Binding by the São Paulo Metallo-β-Lactamase.

Angew Chem Int Ed Engl 2017 03 2;56(14):3862-3866. Epub 2017 Mar 2.

Department of Chemistry, University of Oxford, 12 Mansfield Road, OX1 3TA, Oxford, UK.

Resistance to β-lactam antibiotics mediated by metallo-β-lactamases (MBLs) is a growing problem. We describe the use of protein-observe F-NMR (PrOF NMR) to study the dynamics of the São Paulo MBL (SPM-1) from β-lactam-resistant Pseudomonas aeruginosa. Cysteinyl variants on the α3 and L3 regions, which flank the di-Zn active site, were selectively F-labeled using 3-bromo-1,1,1-trifluoroacetone. The PrOF NMR results reveal roles for the mobile α3 and L3 regions in the binding of both inhibitors and hydrolyzed β-lactam products to SPM-1. These results have implications for the mechanisms and inhibition of MBLs by β-lactams and non-β-lactams and illustrate the utility of PrOF NMR for efficiently analyzing metal chelation, identifying new binding modes, and studying protein binding from a mixture of equilibrating isomers.
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http://dx.doi.org/10.1002/anie.201612185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5396265PMC
March 2017

Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases.

Nat Commun 2016 08 26;7:12673. Epub 2016 Aug 26.

Chemistry Research Laboratory, Department of Chemistry, Oxford Centre for Integrative Systems Biology, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK.

The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1-3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel-Lindau protein (VHL)-elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors.
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http://dx.doi.org/10.1038/ncomms12673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007464PMC
August 2016

Normal tissue radioprotection by amifostine via Warburg-type effects.

Sci Rep 2016 08 10;6:30986. Epub 2016 Aug 10.

Cancer Research UK, Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK.

The mechanism of Amifostine (WR-2721) mediated radioprotection is poorly understood. The effects of amifostine on human basal metabolism, mouse liver metabolism and on normal and tumor hepatic cells were studied. Indirect calorimetric canopy tests showed significant reductions in oxygen consumption and of carbon dioxide emission in cancer patients receiving amifostine. Glucose levels significantly decreased and lactate levels increased in patient venous blood. Although amifostine in vitro did not inhibit the activity of the prolyl-hydroxylase PHD2, experiments with mouse liver showed that on a short timescale WR-1065 induced expression of the Hypoxia Inducible Factor HIF1α, lactate dehydrogenase LDH5, glucose transporter GLUT2, phosphorylated pyruvate dehydrogenase pPDH and PDH-kinase. This effect was confirmed on normal mouse NCTC hepatocytes, but not on hepatoma cells. A sharp reduction of acetyl-CoA and ATP levels in NCTC cells indicated reduced mitochondrial usage of pyruvate. Transient changes of mitochondrial membrane potential and reactive oxygen species ROS production were evident. Amifostine selectively protects NCTC cells against radiation, whilst HepG2 neoplastic cells are sensitized. The radiation protection was correlates with HIF levels. These findings shed new light on the mechanism of amifostine cytoprotection and encourage clinical research with this agent for the treatment of primary and metastatic liver cancer.
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http://dx.doi.org/10.1038/srep30986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4978965PMC
August 2016

Interaction of Avibactam with Class B Metallo-β-Lactamases.

Antimicrob Agents Chemother 2016 10 23;60(10):5655-62. Epub 2016 Sep 23.

Centre d'Ingénierie des Protéines, Université de Liège, Liège, Belgium

β-Lactamases are the most important mechanisms of resistance to the β-lactam antibacterials. There are two mechanistic classes of β-lactamases: the serine β-lactamases (SBLs) and the zinc-dependent metallo-β-lactamases (MBLs). Avibactam, the first clinically useful non-β-lactam β-lactamase inhibitor, is a broad-spectrum SBL inhibitor, which is used in combination with a cephalosporin antibiotic (ceftazidime). There are multiple reports on the interaction of avibactam with SBLs but few such studies with MBLs. We report biochemical and biophysical studies on the binding and reactivity of avibactam with representatives from all 3 MBL subfamilies (B1, B2, and B3). Avibactam has only limited or no activity versus MBL-mediated resistance in pathogens. Avibactam does not inhibit MBLs and binds only weakly to most of the MBLs tested; in some cases, avibactam undergoes slow hydrolysis of one of its urea N-CO bonds followed by loss of CO2, in a process different from that observed with the SBLs studied. The results suggest that while the evolution of MBLs that more efficiently catalyze avibactam hydrolysis should be anticipated, pursuing the development of dual-action SBL and MBL inhibitors based on the diazabicyclooctane core of avibactam may be productive.
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http://dx.doi.org/10.1128/AAC.00897-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5038302PMC
October 2016

The road to avibactam: the first clinically useful non-β-lactam working somewhat like a β-lactam.

Future Med Chem 2016 Jun 21;8(10):1063-84. Epub 2016 Jun 21.

Department of Chemistry, University of Oxford, UK.

Avibactam, which is the first non-β-lactam β-lactamase inhibitor to be introduced for clinical use, is a broad-spectrum serine β-lactamase inhibitor with activity against class A, class C, and, some, class D β-lactamases. We provide an overview of efforts, which extend to the period soon after the discovery of the penicillins, to develop clinically useful non-β-lactam compounds as antibacterials, and, subsequently, penicillin-binding protein and β-lactamase inhibitors. Like the β-lactam inhibitors, avibactam works via a mechanism involving covalent modification of a catalytically important nucleophilic serine residue. However, unlike the β-lactam inhibitors, avibactam reacts reversibly with its β-lactamase targets. We discuss chemical factors that may account for the apparently special nature of β-lactams and related compounds as antibacterials and β-lactamase inhibitors, including with respect to resistance. Avenues for future research including non-β-lactam antibacterials acting similarly to β-lactams are discussed.
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http://dx.doi.org/10.4155/fmc-2016-0078DOI Listing
June 2016