Publications

Teodors Pantelejevs, Pedro Zuazua-Villar, Oliwia Koczy, Stephen J Walsh, Naomi Stephanie Robertson, Andrew J. Counsell, David R Spring, Jessica A. Downs and Marko Hyvönen

Chemical Science, accepted manuscript (2023)
DOI: 10.1039/D3SC03331G

PDB coordinates: 8c3j (3D view), 8br9 (3D view), 8c3n (3D view)

Abstract

Stapling is a macrocyclisation method that connects amino acid side chains of a peptide to improve its pharmacological properties. We describe an approach for stapled peptide preparation and biochemical evaluation that combines recombinant expression of fusion constructs of target peptides and cysteine-reactive divinyl-heteroaryl chemistry as an alternative to solid-phase synthesis. We then employ this workflow to prepare and evaluate BRC-repeat-derived inhibitors of the RAD51 recombinase, showing that a diverse range of secondary structure elements in the BRC repeat can be stapled without compromising binding and function. Using X-ray crystallography, we elucidate the atomic-level features of the staple moieties. We then demonstrate that BRC-repeat-derived stapled peptides can disrupt RAD51 function in cells following ionising radiation treatment.

Maria Reinecke, Paul Brear, Larsen Vornholz, Benedict-Tilmann Berger, Florian Seefried, Stephanie Wilhelm, Patroklos Samaras, Laszlo Gyenis, David William Litchfield, Guillaume Médard, Susanne Müller, Jürgen Ruland, Marko Hyvönen, Mathias Wilhelm, Bernhard Kuster

Journal volume:pages (year)
DOI: 10.1038/s41589-023-01459-3
Pubmed: 37904048

PDB coordinates:
7zwe (3D view ), 7a4q (3D view ), 7zwg (3D view ).

Abstract

Medicinal chemistry has discovered thousands of potent protein and lipid kinase inhibitors. These may be developed into therapeutic drugs or chemical probes to study kinase biology. Because of polypharmacology, a large part of the human kinome currently lacks selective chemical probes. To discover such probes, we profiled 1,183 compounds from drug discovery projects in lysates of cancer cell lines using Kinobeads. The resulting 500,000 compound-target interactions are available in ProteomicsDB and we exemplify how this molecular resource may be used. For instance, the data revealed several hundred reasonably selective compounds for 72 kinases. Cellular assays validated GSK986310C as a candidate SYK (spleen tyrosine kinase) probe and X-ray crystallography uncovered the structural basis for the observed selectivity of the CK2 inhibitor GW869516X. Continue reading →

 Victor Bolsanelli Cioffi, Maria Fernanda de Castro‑Amarante, Aleksei Lulla, Robert Andreata‑Santos, Mario Costa Cruz, Ana Carolina Ramos Moreno, Mariângela de Oliveira Silva, Bianca de Miranda Peres, Lucio Holanda Gondim de Freitas Junior, Carolina Borsoi Moraes, Edison Luiz Durigon, Nicola Coker Gordon, Marko Hyvönen, Luís Carlos de Souza Ferreira & Andrea Balan

Scientific Reports 13: 16821 (2023)
DOI: 10.1038/s41598-023-43720-8
Pubmed: 37798298

Abstract

Amongst the potential contribution of protein or peptide-display systems to study epitopes with relevant immunological features, the RAD display system stands out as a highly stable scaffold protein that allows the presentation of constrained target peptides. Here, we employed the RAD display system to present peptides derived from the SARS-CoV-2 Spike (S) protein as a tool to detect specific serum antibodies and to generate polyclonal antibodies capable of inhibiting SARS-CoV-2 infectivity in vitro. 44 linear S-derived peptides were genetically fused with the RAD scaffold (RAD-SCoV-epitopes) and screened for antigenicity with sera collected from COVID-19-infected patients. In a second step, selected RAD-SCoV-epitopes were used to immunize mice and generate antibodies. Phenotypic screening showed that some of these antibodies were able to recognize replicating viral particles in VERO CCL-81 and most notably seven of the RAD-SCoV-epitopes were able to induce antibodies that inhibited viral infection. Continue reading →

Maximilian A J Harman, Steven J Stanway, Heather Scott, Yuliya Demydchuk, Gustavo Arruda Bezerra, Simone Pellegrino, Liuhong Chen, Paul Brear, Aleksei Lulla, Marko Hyvönen, Paul J Beswick, Michael J Skynner

Journal of Medicinal Chemistry 6(14):9881-9893(2023)
DOI: 0.1021/acs.jmedchem.3c00710
Pubmed: 37433017
PDB coordinates: 8b9p (3D view), 8bfw (3D view), 8bn1 (3D view), 8byj (3D view)

Abstract

Angiotensin-converting enzyme 2 (ACE2) is a metalloprotease that cleaves angiotensin II, a peptide substrate involved in the regulation of hypertension. Here, we identified a series of constrained bicyclic peptides, Bicycle, inhibitors of human ACE2 by panning highly diverse bacteriophage display libraries. These were used to generate X-ray crystal structures which were used to inform the design of additional Bicycles with increased affinity and inhibition of ACE2 enzymatic activity. This novel structural class of ACE2 inhibitors is among the most potent ACE2 inhibitors yet described in vitro, representing a valuable tool to further probe ACE2 function and for potential therapeutic utility.

Katherine U. Gaynor, Marina Vaysburd, Maximilian A. J. Harman, Anna Albecka, Phillip Jeffrey, Paul Beswick, Guido Papa, Liuhong Chen, Donna Mallery, Brian McGuinness, Katerine Van Rietschoten, Steven Stanway, Paul Brear, Aleksei Lulla, Katarzyna Ciazynska, Veronica T. Chang, Jo Sharp, Megan Neary, Helen Box, Jo Herriott, Edyta Kijak, Lee Tatham, Eleanor G. Bentley, Parul Sharma, Adam Kirby, Ximeng Han, James P. Stewart, Andrew Owen, John A. G. Briggs, Marko Hyvönen, Michael J. Skynner & Leo C. James

Nature Communications, 14:3583, (2023)
DOI: 10.1038/s41467-023-39158-1 
Pubmed: 37328472

PDB coordinates: 8AAA (3D view), 7Z8O (3D view)
Plasmids in Addgene: pExp-His-ZBasic-RBD,

Abstract

COVID-19 has stimulated the rapid development of new antibody and small molecule therapeutics to inhibit SARS-CoV-2 infection. Here we describe a third antiviral modality that combines the drug-like advantages of both. Bicycles are entropically constrained peptides stabilized by a central chemical scaffold into a bi-cyclic structure. Rapid screening of diverse bacteriophage libraries against SARS-CoV-2 Spike yielded unique Bicycle binders across the entire protein. Exploiting Bicycles’ inherent chemical combinability, we converted early micromolar hits into nanomolar viral inhibitors through simple multimerization. Continue reading →

Amalyn Nain-Perez , Oscar Nilsson , Aleksei Lulla , Liliana Håversen, Paul Brear, Sara Liljenberg , Marko Hyvönen, Jan Borén, Morten Grøtli

European Journal of Medicinal Chemistry, 15:250:115177 (2023)
DOI: 10.1016/j.ejmech.2023.115177
Pubmed: 36753880
PDB coordinates:
7FRW (3D view ), 7FRV (3D view ), 7FRX (3D view ), 7FRZ (3D view ), 7FS0 (3D view ), 7FS1 (3D view ), 7FS2 (3D view ), 7FS3 (3D view ), 7FS4 (3D view ), 7FS5 (3D view ), 7FS6 (3D view ), 7FS7 (3D view ), 7FS8 (3D view ), 7FS9 (3D view ), 7FSA (3D view ), 7FSB (3D view ), 7FSC (3D view ), 7FSD (3D view )

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. Continue reading →

Philipp Knyphausen, Mariana Rangel Pereira, Paul Brear, Marko Hyvönen, Lutz Jermutus, Florian Hollfelder

Nature Communications 14:768 (2023)
DOI: 10.1038/s41467-023-36099-7
Pubmed: 36765057
PDB coordinates:6YV5 (3D view ), 6YV6 (3D view )

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 A2M^cap that covalently captures protease catalysts by repurposed alpha-2-macroglobulin (A2Ms). To demonstrate the utility of A2M^cap 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. Continue reading →

Katrin Fischer, Aleksei Lulla, Tsz Y So, Pehuén Pereyra-Gerber, Matthew I. J. Raybould, Timo N. Kohler, Tomasz S. Kaminski, Juan Carlos Yam-Puc, Robert Hughes, Florian Leiß-Maier, Paul Brear, Nicholas J. Matheson,Charlotte M. Deane, Marko Hyvönen, James E. D. Thaventhiran, Florian Hollfelder

BioRxiv, posted 12 Jan 2023
DOI: 10.1101/2023.01.10.523494
PDB coordinates: 8BE1 (3D view)
Plasmids in Addgene: pExp-His-ZBasic-RBD, pExp-His-ZBasic-RBD-Avi

Abstract

Monoclonal antibodies are increasingly used to prevent and treat viral infections, playing a pivotal role in pandemic response efforts. Antibody secreting cells (ASCs, plasma cells and plasmablasts) are an excellent source of high-affinity antibodies with therapeutic potential. Current methodologies to study antigen-specific ASCs either have low throughput, require expensive and labour-intensive screening or are technically demanding and therefore not accessible to the wider research community. Here, we present a straightforward technology for the rapid discovery of monoclonal antibodies from ASCs: we combine microfluidic encapsulation of single cells into an antibody capture hydrogel with antigen bait sorting by conventional flow cytometry. Continue reading →

Jerome Jatzlau, Wiktor Burdzinski, Michael Trumpp, Leon Obendorf, Kilian Roßmann, Katharina Ravn, Marko Hyvönen, Francesca Bottanelli, Johannes Broichhagen & Petra Knaus 

Communications Biology 6: 34 (2023)
DOI: 10.1038/s42003-022-04388-4
Pubmed: 36635368

Abstract

TGFβs, BMPs and Activins regulate numerous developmental and homeostatic processes and signal through hetero-tetrameric receptor complexes composed of two types of serine/threonine kinase receptors. Each of the 33 different ligands possesses unique affinities towards specific receptor types. However, the lack of specific tools hampered simultaneous testing of ligand binding towards all BMP/TGFβ receptors. Here we present a N-terminally Halo- and SNAP-tagged TGFβ/BMP receptor library to visualize receptor complexes in dual color. In combination with fluorescently labeled ligands, we established a Ligand Surface Binding Assay (LSBA) for optical quantification of receptor-dependent ligand binding in a cellular context. We highlight that LSBA is generally applicable to test (i) binding of different ligands such as Activin A, TGFβ1 and BMP9, (ii) for mutant screens and (iii) evolutionary comparisons. This experimental set-up opens opportunities for visualizing ligand-receptor binding dynamics, essential to determine signaling specificity and is easily adaptable for other receptor signaling pathways.

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.