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37 items found (0 items not approved), displaying 1 to 10.[First/Prev] 1, 2, 3, 4 [Next/Last]

Instruments / Technical Area	Publication Details	Published
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
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
I04-1-Macromolecular Crystallography (fixed wavelength)	Divergent binding mode for a protozoan BRC repeat to RAD51 Teodors Pantelejevs , Marko Hyvonen Biochemical Journal DOI: 10.1042/BCJ20220141 Diamond Proposal Number(s): [18548] Open Access Show Abstract ▼ Abstract: Interaction of BRCA2 through ca. 30 amino acid residue motifs, BRC repeats, with RAD51 is a conserved feature of the double-strand DNA break repair by homologous recombination in eukaryotes. In humans the binding of the eight BRC repeats is defined by two sequence motifs, FxxA and LFDE, interacting with distinct sites on RAD51. Little is known of the interaction of BRC repeats in other species, especially in protozoans, where variable number of BRC repeats are found in BRCA2 proteins. Here we have studied in detail the interactions of the two BRC repeats in Leishmania infantum BRCA2 with RAD51. We show LiBRC1 is a high-affinity repeat and determine the crystal structure of its complex with LiRAD51. Using truncation mutagenesis of the LiBRC1 repeat, we demonstrate that high affinity binding is maintained in the absence of an LFDE-like motif and suggest compensatory structural features. These observations point towards a divergent evolution of BRC repeats, where a common FxxA-binding ancestor evolved additional contacts for affinity maturation and fine-tuning.	May 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-Macromolecular Crystallography	Poxviruses and paramyxoviruses use a conserved mechanism of STAT1 antagonism to inhibit interferon signaling Callum Talbot-Cooper , Teodors Pantelejevs , John P. Shannon , Christian R. Cherry , Marcus T. Au , Marko Hyvonen , Heather D. Hickman , Geoffrey L. Smith Cell Host & Microbe 296 DOI: 10.1016/j.chom.2022.01.014 Diamond Proposal Number(s): [25402] Open Access Show Abstract ▼ Abstract: The induction of interferon (IFN)-stimulated genes by STATs is a critical host defense mechanism against virus infection. Here, we report that a highly expressed poxvirus protein, 018, inhibits IFN-induced signaling by binding to the SH2 domain of STAT1, thereby preventing the association of STAT1 with an activated IFN receptor. Despite encoding other inhibitors of IFN-induced signaling, a poxvirus mutant lacking 018 was attenuated in mice. The 2.0 Å crystal structure of the 018:STAT1 complex reveals a phosphotyrosine-independent mode of 018 binding to the SH2 domain of STAT1. Moreover, the STAT1-binding motif of 018 shows similarity to the STAT1-binding proteins from Nipah virus, which, similar to 018, block the association of STAT1 with an IFN receptor. Overall, these results uncover a conserved mechanism of STAT1 antagonism that is employed independently by distinct virus families.	Feb 2022
I03-Macromolecular Crystallography	Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats Laurens H. Lindenburg , Teodors Pantelejevs , Fabrice Gielen , Pedro Zuazua-Villar , Maren Butz , Eric Rees , Clemens F. Kaminski , Jessica A. Downs , Marko Hyvonen , Florian Hollfelder Proceedings Of The National Academy Of Sciences 118 DOI: 10.1073/pnas.2017708118 Diamond Proposal Number(s): [18548] Show Abstract ▼ Abstract: Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by −2.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended β-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation.	Nov 2021

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