The Structure of CDK8/CycC Implicates Specificity in the CDK/Cyclin Family and Reveals Interaction with a Deep Pocket Binder

Schneider, Edgar W.; Böttcher, Jark; Blaesse, M.; Neumann, Lars; Huber, Robert; Maskos, K.

doi:10.1016/j.jmb.2011.07.020

Cited by 118 publications

(158 citation statements)

References 62 publications

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“…To find further compounds with an elongated residence time similar to the CDK8/CycC/sorafenib complex (22,23), we performed a primary screen (hit rates, Table S1) of a kinase-focused subset of the Proteros library using the Proteros reporter displacement assay with a reporter based on a type I inhibitor for kinases (21,22). The library screen was performed according to the previously described method for active p38 alpha (21,22) and in total 4,921 compounds with different molecular weights (MW) (fragment with MW < 350 g/mol and lead-like compounds with MW > 350 g/mol) were tested.…”

Section: Resultsmentioning

confidence: 99%

“…The effect of the change of Phe to Met within the DFG motif in the activation loop can be evaluated by superimposing p38α (DFG) and CDK8/CycC (DMG) both complexed with sorafenib. No significant difference in the conformation of the DFG/DMG motifs is seen (23). However, the CDK8-specific Phe176 CDK8 , two residues beyond DMG (Leu in other CDKs), might allow a higher flexibility to the activation loop compared with other members of the CDK family.…”

Section: Discussionmentioning

confidence: 95%

“…Protein expression and purification of the corresponding CDK8(Hs1-403)/CycC(Hs1-283) constructs was performed according to the previously published protocols (23). Protein crystals were obtained within the hanging drop setup at 20°C in 20% (wt/vol) polyethylene glycol 3350 and 0.2 M sodium formate.…”

Section: Methodsmentioning

confidence: 99%

“…Using this concept, we set out to investigate the features of SKRs in the CDK8/CycC complex. We have already presented structural and kinetic data of a deep pocket binder that binds CDK8/CycC (22,23). Because the morphology of the ATP binding site is very well conserved in the CDK family, ATPcompetitive type I compound binding is rather indiscriminative (24).…”

mentioning

confidence: 99%

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Structure–kinetic relationship study of CDK8/CycC specific compounds

Schneider

Böttcher²,

Huber

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

120

136

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In contrast with the very well explored concept of structure-activity relationship, similar studies are missing for the dependency between binding kinetics and compound structure of a protein ligand complex, the structure-kinetic relationship. Here, we present a structure-kinetic relationship study of the cyclin-dependent kinase 8 (CDK8)/cyclin C (CycC) complex. The scaffold moiety of the compounds is anchored in the kinase deep pocket and extended with diverse functional groups toward the hinge region and the front pocket. These variations can cause the compounds to change from fast to slow binding kinetics, resulting in an improved residence time. The flip of the DFG motif ("DMG" in CDK8) to the inactive DFG-out conformation appears to have relatively little influence on the velocity of binding. Hydrogen bonding with the kinase hinge region contributes to the residence time but has less impact than hydrophobic complementarities within the kinase front pocket.kinetic profiling | structure-based drug design T he cyclin-dependent kinase 8/cyclin C (CDK8/CycC) complex is a potent oncogene (1-4), involved in transcription and regulation of transcriptional activity (5-10), linked to epigenetic processes (11), and regarded as an attractive drug target. The recent clinical success of the small molecule inhibitors sorafenib (BAY-43006, Bayer Pharma) and imatinib (STI-571, Novartis Pharma AG) has been attributed to their deep pocket binding mode (12). The "deep pocket" (13) is adjacent to the kinase ATP binding site and accessible in protein kinases by the rearrangement of the DFG motif (a short motif composed of the residues AspPhe-Gly near the N-terminal region of the activation loop) from the active (DFG-in) to the inactive state (DFG-out) (13,14). Binding of a type II compound to such a DFG-out conformation often includes slow binding kinetics (15) with a prolonged "residence time," which is defined as the period for which a target is occupied by a compound (16). Residence time is currently considered to be a key success factor for compound optimization during drug discovery and perhaps as important as the apparent affinity such as half-inhibitory concentration (IC 50 ) or dissociation constant (K d ) (16,17). Accordingly, residence time could be a key to providing enhanced potency in vivo (18) and a means to improve the correlation of in vitro and in vivo efficacy of drugs (19). Despite the increased acceptance of the need to understand binding kinetics, the interplay between compound-target interactions and binding kinetics is in general too complex and poorly understood to enable prediction of the essential dynamic properties. The impact of the combination of kinetic data of proteininhibitor interactions with crystallographic studies has been recognized with pioneer studies such as bovine trypsin in complex with the bovine pancreatic trypsin inhibitor (20). The concept of structure-activity relationships describing the interdependency between binding affinity and compound structure has been well explored in the l...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 95%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Structure–kinetic relationship study of CDK8/CycC specific compounds

Schneider

Böttcher²,

Huber

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

120

136

View full text Add to dashboard Cite

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“…Two phosphorylation sites have been confirmed by analyses of the phosphoproteome of HeLa cells, T410 and S413 (36), but such studies were not comprehensive in regard to the identification of possible CDK8 phosphoepitopes and do not identify the responsible kinase. CDK8 lacks a residue for phosphorylation in its T loop, making it unique among CDKs for activation at this motif (23,50). It is proposed that a glutamate in cyclin C mimics a phosphoresidue that interacts with three conserved arginines within the loop (23,50).…”

mentioning

confidence: 99%

Retroviral Cyclin Enhances Cyclin-Dependent Kinase-8 Activity

Rovnak

Brewster

Quackenbush

2012

J Virol

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Alterations in the functional levels of cyclin-dependent kinase-8 (CDK8) or its partner, cyclin C, have been clearly associated with cancers, including colon cancer, melanoma, and osteosarcoma. Walleye dermal sarcoma virus encodes a retroviral cyclin (RV-cyclin) that localizes to interchromatin granule clusters and binds CDK8. It also binds to the Aα subunit (PR65) of protein phosphatase 2A (PP2A). Binding to the Aα subunit excludes the regulatory B subunit, but not the catalytic C subunit, in a manner similar to that of T antigens of the small DNA tumor viruses. The expression of the RV-cyclin enhances the activity of immune affinity-purified CDK8 in vitro for RNA polymerase II carboxy-terminal domain (CTD) and histone H3 substrates. PP2A also enhances CDK8 kinase activity in vitro for the CTD but not for histone H3. The PP2A enhancement of CDK8 is independent of RV-cyclin expression and likely plays a role in the normal regulation of CDK8. The manipulation of endogenous PP2A activity by inhibition, amendment, or depletion confirmed its role in CDK8 activation by triggering CDK8 autophosphorylation. Although RV-cyclin and PP2A both enhance CDK8 activity, their actions are uncoupled and additive in kinase reactions. PP2A may be recruited to CDK8 in the Mediator complex by a specific PP2A B subunit or additionally by the RV-cyclin in infected cells, but the RV-cyclin appears to activate CDK8 directly and in a manner independent of its physical association with PP2A.

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From Type I to Type II: Design, Synthesis, and Characterization of Potent Pyrazin‐2‐ones as DFG‐Out Inhibitors of PDGFRβ

et al. 2016

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Reversible protein kinase inhibitors that bind in the ATP cleft can be classified as type I or type II binders. Of these, type I inhibitors address the active form, whereas type II inhibitors typically lock the kinase in an inactive form. At the molecular level, the conformation of the flexible activation loop holding the key DFG motif controls access to the ATP site, thereby determining an active or inactive kinase state. Accordingly, type I and type II kinase inhibitors bind to so-called DFG-in or DFG-out conformations, respectively. Based on our former study on highly selective platelet-derived growth factor receptor β (PDGFRβ) pyrazin-2-one type I inhibitors, we expanded this scaffold toward the deep pocket, yielding the highly potent and effective type II inhibitor 5 (4-[(4-methylpiperazin-1-yl)methyl]-N-[3-[[6-oxo-5-(3,4,5-trimethoxyphenyl)-1H-pyrazin-3-yl]methyl]phenyl]benzamide). In vitro characterization, including selectivity panel data from activity-based assays (300 kinases) and affinity-based assays (97 kinases) of these PDGFRβ type I (1; 5-(4-hydroxy-3-methoxy-phenyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazin-2-one) and II (5) inhibitors showing the same pyrazin-2-one chemotype are compared. Implications are discussed regarding the data for selectivity and efficacy of type I and type II ligands.

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The Structure of CDK8/CycC Implicates Specificity in the CDK/Cyclin Family and Reveals Interaction with a Deep Pocket Binder

Cited by 118 publications

References 62 publications

Structure–kinetic relationship study of CDK8/CycC specific compounds

Structure–kinetic relationship study of CDK8/CycC specific compounds

Retroviral Cyclin Enhances Cyclin-Dependent Kinase-8 Activity

From Type I to Type II: Design, Synthesis, and Characterization of Potent Pyrazin‐2‐ones as DFG‐Out Inhibitors of PDGFRβ

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