Second-generation CK2α inhibitors targeting the αD pocket

Jessica Iegre, Paul Brear, Claudia De Fusco, Masao Yoshida, Sophie L. Mitchell, Maxim Rossmann, Laura Carro Santos, Hannah F. Sore, Marko Hyvӧnen and David R. Spring

Chemical Science (2018) (9:3041-)
DOI: 10.1039/C7SC05122K
Pubmed: 29732088

PDB coordinates:

6EHU (3D view ), 5OTQ (3D view ), 5OTY (3D view ), 6EHK (3D view ), 5OTI (3D view ), 5OTL (3D view ), 5OTO (3D view ), 5OYF (3D view ), 5OSZ (3D view ), 5OT5 (3D view ), 5OTD (3D view ), 5OTH (3D view ), 5OT6 (3D view ), 5OUE (3D view ), 5OUM (3D view ), 5OUU (3D view ), 5OS8 (3D view ), 5OTR (3D view ), 6EII (3D view ), 5OQU (3D view ), 5ORK (3D view ), 5OSL (3D view ), 5OUL (3D view ), 5ORH (3D view ), 5ORJ (3D view ), 5OS7 (3D view )


CK2 is a critical cell cycle regulator that also promotes various anti-apoptotic mechanisms. Development of ATP-non-competitive inhibitors of CK2 is a very attractive strategy considering that the ATP binding site is highly conserved among other kinases. We have previously utilised a pocket outside the active site to develop a novel CK2 inhibitor, CAM4066. Whilst CAM4066 bound to this new pocket it was also interacting with the ATP site: herein, we describe an example of a CK2α inhibitor that binds completely outside the active site. Continue reading →

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Structural and Mechanistic Analysis of the Choline Sulfatase from Sinorhizobium melliloti: A Class I Sulfatase Specific for an Alkyl Sulfate Ester

Bert van Loo, Markus Schober, Eugene Valkov, Magdalena Heberlein, Erich Bornberg-Bauer, Kurt Faber, Marko Hyvönen, Florian Hollfelder

Journal of Molecular Biology, 430:1004-1023, 2018 
DOI: 10.1016/j.jmb.2018.02.010
Pubmed: 29458126
PDB coordinates:
6FNY (3D view )



Hydrolysis of organic sulfate esters proceeds by two distinct mechanisms, water attacking at either sulfur (S-O bond cleavage) or carbon (C-O bond cleavage). In primary and secondary alkyl sulfates attack at carbon is favored, whereas in aromatic sulfates and sulfated sugars attack at sulfur is preferred. This mechanistic distinction is mirrored in the classification of enzymes that catalyze sulfate ester hydrolysis: arylsulfatases catalyze S-O cleavage in sulfate sugars and arylsulfates and alkyl sulfatases break the C-O bond of alkyl sulfates. Continue reading →

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Molecular characterization of latent GDF8 reveals mechanisms of activation

Ryan G. Walker, Jason C. McCoy, Magdalena Czepnik, Melanie J. Mills, Adam Haggd, Kelly L. Walton, Thomas R. Cotton, Marko Hyvönen, Richard T. Lee, Paul Gregorevic, Craig A. Harrison, and Thomas B. Thompson



Growth/differentiation factor 8 (GDF8), or myostatin, negatively regulates muscle mass. GDF8 is held in a latent state through interactions with its N-terminal prodomain, much like TGF-β. Using a combination of small-angle X-ray scattering and mutagenesis, we characterized the interactions of GDF8 with its prodomain. Our results show that the prodomain:GDF8 complex can exist in a fully latent state and an activated or “triggered” state where the prodomain remains in complex with the mature domain. Continue reading →

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Structure of the human pro-myostatin precursor and determinants of growth factor latency

Thomas Cotton, Gerhard Fischer, Xuelu Wang, Jason McCoy, Magdalena Czepnik, Thomas B. Thompson, Marko Hyvönen

The EMBO Journal (2018) 37: 367-383
DOI: 10.15252/embj.201797883
Pre-publication in BiorXiv, DOI:
PDB coordinates: 5NTU (3D view ), 5NXS (3D view )


Myostatin, a key regulator of muscle mass in vertebrates, is biosynthesised as a latent precursor in muscle and is activated by sequential proteolysis of the pro-domain. To investigate the molecular mechanism by which pro-myostatin remains latent, we have solved the structure of unprocessed pro-myostatin and analysed the properties of the protein in its different forms. Continue reading →

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Computationally-guided optimization of small-molecule inhibitors of the Aurora A kinase–TPX2 protein–protein interaction

Daniel J. Cole, Matej Janecek, Jamie E. Stokes, Maxim Rossmann, John C. Faver, Grahame J. McKenzie, Ashok R. Venkitaraman, Marko Hyvönen, David R. Spring, David J. Huggins and William L. Jorgensen

Chemical Communications 53, 9372-9375 (2017)
DOI 10.1039/C7CC05379G
Pubmed: 28787041

PDB coordinates: 5OBR (3D view)


Free energy perturbation theory, in combination with enhanced sampling of protein–ligand binding modes, is evaluated in the context of fragment-based drug design, and used to design two new small-molecule inhibitors of the Aurora A kinase–TPX2 protein–protein interaction.

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A fragment-based approach leading to the discovery of a novel binding site and the selective CK2 inhibitor CAM4066.

Claudia De Fusco, Paul Brear, Jessie Iegre, Kathy H. Georgiou, Hannah F. Sore, Marko Hyvönen, David R. Spring

Bioorganic and Medicinal Chemistry, 25:3471-3482 (2017)
DOI: 10.1016/j.bmc.2017.04.037
Pubmed: 28495381

PDB coordinates: 5MMF (3D view ), 5MMR (3D view ), 5MO5 (3D view ), 5MO6 (3D view ), 5MO7 (3D view ), 5MO8 (3D view ), 5MOD (3D view ), 5MOE (3D view ), 5MOH (3D view ), 5MOT (3D view ), 5MOV (3D view ), 5MOW (3D view ), 5CU0 (3D view ), 5CU2 (3D view ), 5CT0 (3D view ), 5CTP (3D view ), 5CX9 (3D view )


Recently we reported the discovery of a potent and selective CK2α inhibitor CAM4066. This compound inhibits CK2 activity by exploiting a pocket located outside the ATP binding site (αD pocket). Here we describe in detail the journey that led to the discovery of CAM4066 using the challenging fragment linking strategy. Specifically, we aimed to develop inhibitors by linking a high-affinity fragment anchored in the αD site to a weakly binding warhead fragment occupying the ATP site. Moreover, we describe the remarkable impact that molecular modelling had on the development of this novel chemical tool. The work described herein shows potential for the development of a novel class of CK2 inhibitors.

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Structural analyses of von Willebrand factor C domains of collagen 2A and CCN3 reveal an alternative mode of binding to bone morphogenetic protein-2

Emma-Ruoqi Xu, Emily E. Blythe, Gerhard Fischer and  Marko Hyvönen

Journal of Biological Chemistry, 292:12516-12527, 2017
Pubmed ID: 28584056
PDB coordinates: 5NIR (3D view), 5NB8 (3D view)


Bone morphogenetic proteins (BMPs) are secreted growth factors that promote differentiation processes in embryogenesis and tissue development. Regulation of BMP signalling involves binding to a variety of extracellular proteins, among which are many von Willebrand factor C (vWC) domain-containing proteins. While the crystal structure of the complex of crossveinless-2 (CV-2) vWC1 and BMP-2 previously revealed one mode of the vWC:BMP binding mechanism, other vWC domains may bind to BMP differently.  Continue reading →

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Development of a multipurpose scaffold for the display of peptide loops

Maxim Rossmann, Sandra J. Greive, Tommaso Moschetti, Michael Dinan, Marko Hyvönen

Protein Engineering, Design and Selection 30: 419–430 (2017)
DOI: 10.1093/protein/gzx017
Pubmed: 228444399


Protein-protein interactions (PPIs) determine a wide range of biological processes and analysis of these dynamic networks is increasingly becoming a mandatory tool for studying protein function. Using the globular ATPase domain of recombinase RadA as a scaffold, we have developed a peptide display system (RAD display), which allows for the presentation of target peptides, protein domains or full-length proteins and their rapid recombinant production in bacteria. Continue reading →

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Structure and activation of pro-activin A

Xuelu Wang, Gerhard Fischer and Marko Hyvönen

Nature Communications 7:1205, 2016
DOI: 10.1038/ncomms12052
Pubmed: 27373274

PDB coordinates: 5HLY (3D view), 5HLZ (3D view)


Activins are growth factors with multiple roles in the development and homeostasis. Like all TGF-β family of growth factors, activins are synthesized as large precursors from which mature dimeric growth factors are released proteolytically. Here we have studied the activation of activin A and determined crystal structures of the unprocessed precursor and of the cleaved pro-mature complex. Replacing the natural furin cleavage site with a HRV 3C protease site, we show how the protein gains its bioactivity after proteolysis and is as active as the isolated mature domain. The complex remains associated in conditions used for biochemical analysis with a dissociation constant of 5 nM, but the pro-domain can be actively displaced from the complex by follistatin. Our high-resolution structures of pro-activin A share features seen in the pro-TGF-β1 and pro-BMP-9 structures, but reveal a new oligomeric arrangement, with a domain-swapped, cross-armed conformation for the protomers in the dimeric protein.

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Specific inhibition of CK2a from an anchor outside the active site

Paul Brear, Claudia De Fusco, Kathy Hadje Georgiou, Nicola J. Francis-Newton, Christopher J. Stubbs,  Hannah F. Sore, Ashok R. Venkitaraman, Chris Abell, David R. Spring and Marko Hyvönen

Chemical Science, 7(11):6839-6845, 2016
Pubmed: 28451126
PDB coordinates:
5CVH (3D view), 5CVG (3D view), 5CVF (3D view), 5CU3 (3D view), 5CU4 (3D view), 5CU6 (3D view), 5CSH (3D view), 5CSP (3D view), 5CSV (3D view), 5CSH (3D view), 5CS6 (3D view), 5CLP (3D view)


The development of selective inhibitors of protein kinases is challenging because of the significant conservation of the ATP binding site. Here, we describe a new mechanism by which the protein kinase CK2α can be selectively inhibited using features outside the ATP site. We have identified a new binding site for small molecules on CK2α adjacent to the ATP site and behind the αD loop, termed the αD pocket. An elaborated fragment anchored in this site has been linked with a low affinity fragment binding in the ATP site, creating a novel and selective inhibitor (CAM4066) that binds CK2α with a Kd of 320 nM and shows significantly improved selectivity compared to other CK2α inhibitors. CAM4066 shows target engagement in several cell lines and similar potency to clinical trial candidate CX4945. Our data demonstrate that targeting a poorly conserved, cryptic pocket allows inhibition of CK2α via a novel mechanism, enabling the development of a new generation of selective CK2α inhibitors.

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