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24 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3 [Next/Last]

Instruments / Technical Area	Publication Details	Published
I24-Microfocus Macromolecular Crystallography	Catabolite repression control protein antagonist, a novel player in Pseudomonas aeruginosa carbon catabolite repression control Elisabeth Sonnleitner , Flavia Bassani , Anastasia Cianciulli Sesso , Paul Brear , Branislav Lilic , Lovro Davidovski , Armin Resch , Ben F. Luisi , Isabella Moll , Udo Bläsi Frontiers In Microbiology 14 DOI: 10.3389/fmicb.2023.1195558 Diamond Proposal Number(s): [25402] Open Access Show Abstract ▼ Abstract: In the opportunistic human pathogen Pseudomonas aeruginosa (Pae), carbon catabolite repression (CCR) orchestrates the hierarchical utilization of N and C sources, and impacts virulence, antibiotic resistance and biofilm development. During CCR, the RNA chaperone Hfq and the catabolite repression control protein Crc form assemblies on target mRNAs that impede translation of proteins involved in uptake and catabolism of less preferred C sources. After exhaustion of the preferred C-source, translational repression of target genes is relieved by the regulatory RNA CrcZ, which binds to and acts as a decoy for Hfq. Here, we asked whether Crc action can be modulated to relieve CCR after exhaustion of a preferred carbon source. As Crc does not bind to RNA per se, we endeavored to identify an interacting protein. In vivo co-purification studies, co-immunoprecipitation and biophysical assays revealed that Crc binds to Pae strain O1 protein PA1677. Our structural studies support bioinformatics analyzes showing that PA1677 belongs to the isochorismatase-like superfamily. Ectopic expression of PA1677 resulted in de-repression of Hfq/Crc controlled target genes, while in the absence of the protein, an extended lag phase is observed during diauxic growth on a preferred and a non-preferred carbon source. This observations indicate that PA1677 acts as an antagonist of Crc that favors synthesis of proteins required to metabolize non-preferred carbon sources. We present a working model wherein PA1677 diminishes the formation of productive Hfq/Crc repressive complexes on target mRNAs by titrating Crc. Accordingly, we propose the name CrcA (catabolite repression control protein antagonist) for PA1677.	May 2023
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Tuning liver pyruvate kinase activity up or down with a new class of allosteric modulators Amalyn Nain-Perez , Oscar Nilsson , Aleksei Lulla , Liliana Håversen , Paul Brear , Sara Liljenberg , Marko Hyvonen , Jan Borén , Morten Grøtli European Journal Of Medicinal Chemistry 250 DOI: 10.1016/j.ejmech.2023.115177 Diamond Proposal Number(s): [18548, 25402] Open Access Show Abstract ▼ Abstract: The liver isoform of pyruvate kinase (PKL) has gained interest due to its potential capacity to regulate fatty acid synthesis involved in the progression of non-alcoholic fatty liver disease (NAFLD). Here we describe a novel series of PKL modulators that can either activate or inhibit the enzyme allosterically, from a cryptic site at the interface of two protomers in the tetrameric enzyme. Starting from urolithin D, we designed and synthesised 42 new compounds. The effect of these compounds on PKL enzymatic activity was assessed after incubation with cell lysates obtained from a liver cell line. Pronounced activation of PKL activity, up to 3.8-fold, was observed for several compounds at 10 μM, while other compounds were prominent PKL inhibitors reducing its activity to 81% at best. A structure-activity relationship identified linear-shaped sulfone-sulfonamides as activators and non-linear compounds as inhibitors. Crystal structures revealed the conformations of these modulators, which were used as a reference for designing new modulators.	Mar 2023
I04-Macromolecular Crystallography	Evolution of protease activation and specificity via alpha-2-macroglobulin-mediated covalent capture Philipp Knyphausen , Mariana Rangel Pereira , Paul Brear , Marko Hyvonen , Lutz Jermutus , Florian Hollfelder Nature Communications 14 DOI: 10.1038/s41467-023-36099-7 Diamond Proposal Number(s): [18548, 25402] Open Access Show Abstract ▼ Abstract: Tailoring of the activity and specificity of proteases is critical for their utility across industrial, medical and research purposes. However, engineering or evolving protease catalysts is challenging and often labour intensive. Here, we describe a generic method to accelerate this process based on yeast display. We introduce the protease selection system A2Mcap that covalently captures protease catalysts by repurposed alpha-2-macroglobulin (A2Ms). To demonstrate the utility of A2Mcap for protease engineering we exemplify the directed activity and specificity evolution of six serine proteases. This resulted in a variant of Staphylococcus aureus serin-protease-like (Spl) protease SplB, an enzyme used for recombinant protein processing, that no longer requires activation by N-terminal signal peptide removal. SCHEMA-based domain shuffling was used to map the specificity determining regions of Spl proteases, leading to a chimeric scaffold that supports specificity switching via subdomain exchange. The ability of A2Mcap to overcome key challenges en route to tailor-made proteases suggests easier access to such reagents in the future.	Feb 2023
I03-Macromolecular Crystallography	Systems-wide dissection of organic acid assimilation in Pseudomonas aeruginosa reveals a novel path to underground metabolism Stephen K. Dolan , Andre Wijaya , Michael Kohlstedt , Lars Gläser , Paul Brear , Rafael Silva-Rocha , Christoph Wittmann , Martin Welch Mbio DOI: 10.1128/mbio.02541-22 Diamond Proposal Number(s): [19242] Open Access Show Abstract ▼ Abstract: The human pathogen Pseudomonas aeruginosa (Pa) is one of the most frequent and severe causes of nosocomial infection. This organism is also a major cause of airway infections in people with cystic fibrosis (CF). Pa is known to have a remarkable metabolic plasticity, allowing it to thrive under diverse environmental conditions and ecological niches; yet, little is known about the central metabolic pathways that sustain its growth during infection or precisely how these pathways operate. In this work, we used a combination of ‘omics approaches (transcriptomics, proteomics, metabolomics, and 13C-fluxomics) and reverse genetics to provide systems-level insight into how the infection-relevant organic acids succinate and propionate are metabolized by Pa. Moreover, through structural and kinetic analysis of the 2-methylcitrate synthase (2-MCS; PrpC) and its paralogue citrate (CIT) synthase (GltA), we show how these two crucial enzymatic steps are interconnected in Pa organic acid assimilation. We found that Pa can rapidly adapt to the loss of GltA function by acquiring mutations in a transcriptional repressor, which then derepresses prpC expression. Our findings provide a clear example of how “underground metabolism,” facilitated by enzyme substrate promiscuity, “rewires” Pa metabolism, allowing it to overcome the loss of a crucial enzyme. This pathogen-specific knowledge is critical for the advancement of a model-driven framework to target bacterial central metabolism.	Nov 2022
I04-Macromolecular Crystallography	Crystal structure of the Rho-associated coiled-coil kinase 2 inhibitor belumosudil bound to CK2α Paul Brear , Marko Hyvonen Acta Crystallographica Section F Structural Biology Communications 78 DOI: 10.1107/S2053230X22008767 Diamond Proposal Number(s): [25402] Open Access Show Abstract ▼ Abstract: The small molecule belumosudil was initially identified as a selective inhibitor of Rho-associated coiled-coil kinase 2 (ROCK2) and has recently been approved for the treatment of graft-versus-host disease. However, recent studies have shown that many of the phenotypes displayed upon treatment with belumosudil were due to CK2α inhibition. CK2α is in itself a very promising therapeutic target for a range of conditions and has recently been put forward as a potential treatment for COVID-19. Belumosudil presents a promising starting point for the development of future CK2α inhibitors as it provides a safe, potent and orally bioavailable scaffold. Therefore, several of the major hurdles in drug development have already been overcome. Here, the crystal structure of belumosudil bound to the ATP site of CK2α is presented. This crystal structure combined with modelling studies further elucidates how belumosudil could be developed into a selective and potent CK2α or ROCK2 inhibitor.	Oct 2022
I02-Macromolecular Crystallography I04-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	A fragment-based approach leading to the discovery of inhibitors of CK2α with a novel mechanism of action Paul Brear , Claudia De Fusco , Eleanor L. Atkinson , Jessica Iegre , Nicola J. Francis-Newton , Ashok R. Venkitaraman , Marko Hyvonen , David R. Spring Rsc Medicinal Chemistry 17 DOI: 10.1039/D2MD00161F Diamond Proposal Number(s): [14043, 18548] Open Access Show Abstract ▼ Abstract: CK2 is a ubiquitous protein kinase with an anti-apoptotic role and is found to be overexpressed in multiple cancer types. To this end, the inhibition of CK2 is of great interest with regard to the development of novel anti-cancer therapeutics. ATP-site inhibition of CK2 is possible; however, this typically results in poor selectivity due to the highly conserved nature of the catalytic site amongst kinases. An alternative methodology for the modulation of CK2 activity is through allosteric inhibition. The recently identified αD site represents a promising binding site for allosteric inhibition of CK2α. The work presented herein describes the development of a series of CK2α allosteric inhibitors through iterative cycles of X-ray crystallography and enzymatic assays, in addition to both fragment growing and fragment merging design strategies. The lead fragment developed, fragment 8, exhibits a high ligand efficiency, displays no drop off in activity between enzymatic and cellular assays, and successfully engages CK2α in cells. Furthermore, X-ray crystallographic analysis provided indications towards a novel mechanism of allosteric inhibition through αD site binding. Fragments described in this paper therefore represent promising starting points for the development of highly selective allosteric CK2 inhibitors.	Sep 2022
I03-Macromolecular Crystallography I04-Macromolecular Crystallography I23-Long wavelength MX	Functional metagenomic screening identifies an unexpected β-glucuronidase Stefanie Neun , Paul Brear , Eleanor Campbell , Theodora Tryfona , Kamel El Omari , Armin Wagner , Paul Dupree , Marko Hyvonen , Florian Hollfelder Nature Chemical Biology 48 DOI: 10.1038/s41589-022-01071-x Diamond Proposal Number(s): [18548, 25402] Show Abstract ▼ Abstract: The abundance of recorded protein sequence data stands in contrast to the small number of experimentally verified functional annotation. Here we screened a million-membered metagenomic library at ultrahigh throughput in microfluidic droplets for β-glucuronidase activity. We identified SN243, a genuine β-glucuronidase with little homology to previously studied enzymes of this type, as a glycoside hydrolase 3 family member. This glycoside hydrolase family contains only one recently added β-glucuronidase, showing that a functional metagenomic approach can shed light on assignments that are currently ‘unpredictable’ by bioinformatics. Kinetic analyses of SN243 characterized it as a promiscuous catalyst and structural analysis suggests regions of divergence from homologous glycoside hydrolase 3 members creating a wide-open active site. With a screening throughput of >107 library members per day, picolitre-volume microfluidic droplets enable functional assignments that complement current enzyme database dictionaries and provide bridgeheads for the annotation of unexplored sequence space.	Jul 2022
I04-Macromolecular Crystallography	Development of small cyclic peptides targeting the CK2α/β interface Eleanor L. Atkinson , Jessica Iegre , Claudio D’amore , Paul Brear , Mauro Salvi , Marko Hyvonen , David R. Spring Chemical Communications 58, 4791 - 4794 DOI: 10.1039/D2CC00707J Diamond Proposal Number(s): [18548] Open Access Show Abstract ▼ Abstract: In this work, an iterative cycle of enzymatic assays, X-ray crystallography, molecular modelling and cellular assays were used to develop a functionalisable chemical probe for the CK2α/β PPI. The lead peptide, P8C9, successfully binds to CK2α at the PPI site, is easily synthesisable and functionalisable, highly stable in serum and small enough to accommodate further optimisation.	Apr 2022
I03-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	Anthraquinone derivatives as ADP-competitive inhibitors of liver pyruvate kinase Amalyn Nain-Perez , Anders Foller Füchtbauer , Liliana Håversen , Aleksei Lulla , Chunxia Gao , Josipa Matic , Leticia Monjas , Alexandra Rodríguez , Paul Brear , Woonghee Kim , Marko Hyvonen , Jan Borén , Adil Mardinoglu , Mathias Uhlen , Morten Grøtli European Journal Of Medicinal Chemistry 41 DOI: 10.1016/j.ejmech.2022.114270 Diamond Proposal Number(s): [25402] Show Abstract ▼ Abstract: Liver pyruvate kinase (PKL) is a major regulator of metabolic flux and ATP production during liver cell glycolysis and is considered a potential drug target for the treatment of non-alcoholic fatty liver disease (NAFLD). In this study, we report the first ADP-competitive PKL inhibitors and identify several starting points for the further optimization of these inhibitors. Modeling and structural biology guided the optimization of a PKL-specific anthraquinone-based compound. A structure–activity relationship study of 47 novel synthetic derivatives revealed PKL inhibitors with half-maximal inhibitory concentration (IC50) values in the 200 nM range. Despite the difficulty involved in studying a binding site as exposed as the ADP site, these derivatives feature expanded structural diversity and chemical spaces that may be used to improve their inhibitory activities against PKL. The obtained results expand the knowledge of the structural requirements for interactions with the ADP-binding site of PKL.	Mar 2022
I04-1-Macromolecular Crystallography (fixed wavelength)	Structure, function and regulation of a second pyruvate kinase isozyme in Pseudomonas aeruginosa Yassmin Abdelhamid , Meng Wang , Susannah L. Parkhill , Paul Brear , Xavier Chee , Taufiq Rahman , Martin Welch Frontiers In Microbiology 12 DOI: 10.3389/fmicb.2021.790742 Diamond Proposal Number(s): [14043] Open Access Show Abstract ▼ Abstract: Pseudomonas aeruginosa (PA) depends on the Entner-Doudoroff pathway (EDP) for glycolysis. The main enzymatic regulator in the lower half of the EDP is pyruvate kinase. PA contains genes that encode two isoforms of pyruvate kinase, denoted PykAPA and PykFPA. In other well-characterized organisms containing two pyruvate kinase isoforms (such as Escherichia coli) each isozyme is differentially regulated. The structure, function and regulation of PykAPA has been previously characterized in detail, so in this work, we set out to assess the biochemical and structural properties of the PykFPA isozyme. We show that pykFPA expression is induced in the presence of the diureide, allantoin. In spite of their relatively low amino acid sequence identity, PykAPA and PykFPA display broadly comparable kinetic parameters, and are allosterically regulated by a very similar set of metabolites. However, the x-ray crystal structure of PykFPA revealed significant differences compared with PykAPA. Notably, although the main allosteric regulator binding-site of PykFPA was empty, the “ring loop” covering the site adopted a partially closed conformation. Site-directed mutation of the proline residues flanking the ring loop yielded apparent “locked on” and “locked off” allosteric activation phenotypes, depending on the residue mutated. Analysis of PykFPA inter-protomer interactions supports a model in which the conformational transition(s) accompanying allosteric activation involve re-orientation of the A and B domains of the enzyme and subsequent closure of the active site.	Nov 2021

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