Publications

Serendipitous Identification of a Covalent Activator of Liver Pyruvate Kinase

Umberto Maria Battisti, Chunxia Gao, Oscar Nilsson, Fady Akladios, Aleksei Lulla, Agnieszka Bogucka, Amalyn Nain-Perez, Liliana Håversen, Woonghee Kim, Jan Boren, Marko Hyvönen, Mathias Uhlen, Adil Mardinoglu, Morten Grøtli

ChemBioChem, accepted article 17th Oct (2022)
DOI: 0.1002/cbic.202200339
Pubmed: 36250581

Abstract

Enzymes are effective biological catalysts that accelerate almost all metabolic reactions in living organisms. Synthetic modulators of enzymes are useful tools for the study of enzymatic reactions and can provide starting points for the design of new drugs. Here, we report on the discovery of a class of biologically active compounds that covalently modifies lysine residues in human liver pyruvate kinase (PKL), leading to allosteric activation of the enzyme (EC50 = 0.29 µM). Surprisingly, the allosteric activation control point resides on the lysine residue K282 present in the catalytic site of PKL. These findings were confirmed by structural data, MS/MS experiments, and molecular modelling studies. Altogether, our study provides a molecular basis for the activation mechanism and establishes a framework for further development of human liver pyruvate kinase covalent activators.

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Crystal structure of the Rho-associated coiled-coil kinase 2 inhibitor belumosudil bound to CK2α

Paul Brear and Marko Hyvönen

Acta Crystallographica section F 78: 348-353 (2022)
DOI: 10.1107/S2053230X22008767

PDB coordinates:
7z39 (3D view)

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. Continue reading →

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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 Hyvönen and David R. Spring

Journal volume: RSC Med. Chem., 2022, Advance Article
DOI: 10.1039/D2MD00161F

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. Continue reading →

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Functional metagenomic screening identifies an unexpected β-glucuronidase

Stefanie Neun, Paul Brear, Eleanor Campbell, Theodora Tryfona, Kamel El Omari, Armin Wagner, Paul Dupree, Marko Hyvönen, Florian Hollfelder

Nature Chemical Biology. Online ahead of print. 07 July 2022 Jul.
DOI: 10.1038/s41589-022-01071-x
Pubmed: 35799064

PDB coordinates:
7QE1 (3D view), 7QE2 (3D view), 7QEF (3D view), 7QEA (3D view), 7QG4 (3D view), 7QEE (3D view)

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. Continue reading →

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The common H202D variant in GDF-15 does not affect its bioactivity but can significantly interfere with measurement of its circulating levels

Yanislava Karusheva, Matt Ratcliff, Audrey Melvin, Alexander Moerseburg, Naveed Sattar, Peter Barker, Keith Burling, Anna Backmark, Robert Roth, Lutz Jermutus, Esther Guiu-Jurado, Matthias Blueher, Paul Welsh, Marko Hyvönen, Stephen O’Rahilly

The Journal of Applied Laboratory Medicine, Advance Article 07 July 2022.
DOI: 10.1093/jalm/jfac055
MedRxiv preprint DOI: 022.01.03.22268655

GDF15 bioactivity

Abstract

Genetic variants in proteins can interfere with measurement of their circulating concentrations. Given the growing biomedical importance of GDF-15, we wished to establish whether a common histidine to aspartate variant present in position 6 of the mature GDF-15 protein (H202D variant) interfered with its measurement by two commonly used immunoassays. We first examined the detectability of recombinant monomers, homodimers and heterodimers of GDF-15 by assays and/or reagents used in two widely used immunoassays (Roche Elecsys GDF-15 and the R&D antibody combinations used in their Quantikine and DuoSet ELISAs). The Roche assay detected the H and D containing peptides similarly but the assays based on the R&D reagents consistently underreported concentrations of the D-containing variant peptide. Measurements of plasma concentrations of GDF-15 in genotyped human participants showed that the R&D reagents reported values in heterozygotes were ~25% lower, and in homozygotes, 50% lower than the Roche assay. Continue reading →

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Divergent binding mode for a protozoan BRC repeat to RAD51

Teodors Pantelejevs and Marko Hyvönen

Biochemical Journal, 479: 1031–1043 (2022)
DOI: 10.1042/BCJ20220141
Pubmed: 35502837
BioRxiv preprint: 2022.01.25.477309v1

PDB coordinates:
7QV8 (3D view)
L.infantum RAD51 complexed with BRC repeat

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. Continue reading →

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Development of small cyclic peptides targeting the CK2α/β interface

Eleanor L. Atkinson, Jessica Iegre, Claudio D’Amore, Paul Brear, Mauro Salvi, Marko Hyvönen and David R. Spring

Chemical Communications, 58:4791-4794 (2022)
DOI: 10.1039/D2CC00707J
Pubmed: 35343996

PDB coordinates:

6YZH (3D view)
6Q4Q (3D view)

TOC graphic

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.

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Anthraquinone derivatives as ADP-competitive inhibitors of liver pyruvate kinase

Amalyn Nain-Pereza, Anders Foller Füchtbauera, Liliana Håversen, Aleksei Lulla, Chunxia Gao, Josipa Matic, Leticia, Monjasa Alexandra Rodríguez, Paul Brear, Woonghee Kim, Marko Hyvönen, Jan Borén, Adil Mardinoglu, Mathias Uhlen, Morten Grøtli

European Journal of Medicinal Chemistry 23:114270 (2021)
DOI: 10.1016/j.ejmech.2022.114270
Pubmed: 35290845

PDB coordinates:

5SCL (3D view), 5SDT (3D view), 5SC8 (3D view), 5SCB (3D view), 5SC9 (3D view), 5SCA (3D view), 5SCK (3D view), 7QZU (3D view), 5SCE (3D view), 5SCC (3D view), 5SCD (3D view), 5SCF (3D view), 5SCH (3D view), 5SCG (3D view), 5SCI (3D view), 5SCJ (3D view), 7QDN (3D view)

Liver Pyruvate Kinase

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. Continue reading →

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Unraveling the Mechanics of a Repeat-Protein Nanospring: From Folding of Individual Repeats to Fluctuations of the Superhelix

Marie Synakewicz, Rohan S Eapen, Albert Perez-Riba, Pamela J E Rowling, Daniela Bauer, Andreas Weißl, Gerhard Fischer, Marko Hyvönen, Matthias Rief, Laura S Itzhaki, Johannes Stigler

ACS Nano, 2022 (online before print version)
DOI: doi: 10.1021/acsnano.1c09162
Pubmed: 35258937

Abstract

Tandem-repeat proteins comprise small secondary structure motifs that stack to form one-dimensional arrays with distinctive mechanical properties that are proposed to direct their cellular functions. Here, we use single-molecule optical tweezers to study the folding of consensus-designed tetratricopeptide repeats (CTPRs), superhelical arrays of short helix-turn-helix motifs. We find that CTPRs display a spring-like mechanical response in which individual repeats undergo rapid equilibrium fluctuations between partially folded and unfolded conformations. We rationalize the force response using Ising models and dissect the folding pathway of CTPRs under mechanical load, revealing how the repeat arrays form from the center toward both termini simultaneously. Continue reading →

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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 Hyvönen, Heather D. Hickman, Geoffrey L.Smith

Cell Host & Microbe, in press.
DOI: 10.1016/j.chom.2022.01.014
Pubmed: 35182467

PDB coordinates:: 7NUF (3D view)
STAT1 complexed with 018

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.

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