Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site

Chen, Hongming; Tucker, J.A.; Wang, Xiaotao; Gavine, Paul R.; Phillips, Chris; Augustin, Martin; Schreiner, Patrick; Steinbacher, Stefan; Preston, Marian; Ogg, D.

doi:10.1107/s2059798316004502

Cited by 16 publications

(18 citation statements)

References 49 publications

(41 reference statements)

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“…The trifluoro-ethyl group orients towards the hydrophobic back pocket, and a putative fluorine-sulfur contact is observed with the sulfur of the gatekeeper residue (M465). 23 Second, we compared our DCLK1 docking structures to the structure of ERK5 in complex with XMD8–92 (PDB:5BYY) 24 to uncover the structural basis of DCLK1-IN-1 selectivity. The residues lining the pocket of the ATP-binding site of the two proteins are highly conserved, with the exception of the gatekeeper.…”

Section: Resultsmentioning

confidence: 99%

Discovery of a selective inhibitor of doublecortin like kinase 1

et al. 2020

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Doublecortin like kinase 1 (DCLK1) is an understudied kinase that is upregulated in a wide range of cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about its potential as a therapeutic target. We leveraged chemoproteomic profiling and structure-based design to develop the first selective, in vivo -compatible chemical probe of the DCLK1 kinase domain, DCLK1-IN-1. We demonstrate activity of DCLK1-IN-1 against clinically relevant patient-derived PDAC organoid models and use a combination of RNA sequencing, proteomics and phosphoproteomics analysis to reveal that DCLK1 inhibition modulates proteins and pathways associated with cell motility in this context. DCLK1-IN-1 will serve as a versatile tool to investigate DCLK1 biology and establish its role in cancer.

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Section: Resultsmentioning

confidence: 99%

Discovery of a selective inhibitor of doublecortin like kinase 1

et al. 2020

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“…These observations have prompted commercial and academic MEK5 or ERK5 drug discovery programmes in the hope of developing novel anti-inflammatory or anti-cancer therapeutics. Commercial MEK5 or ERK5 inhibitor programmes include ActivX, Kyorin Pharmaceutical Co. 28 , Bayer AG 29 , Boehringer Ingelheim 30 and AstraZeneca 31 . The first ERK5 inhibitor (ERK5i) to be described was XMD8-92 from the Dana-Farber Cancer Institute and the Scripps Research Institute, USA 32,33 .…”

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confidence: 99%

Paradoxical activation of the protein kinase-transcription factor ERK5 by ERK5 kinase inhibitors

et al. 2020

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The dual protein kinase-transcription factor, ERK5, is an emerging drug target in cancer and inflammation, and small-molecule ERK5 kinase inhibitors have been developed. However, selective ERK5 kinase inhibitors fail to recapitulate ERK5 genetic ablation phenotypes, suggesting kinase-independent functions for ERK5. Here we show that ERK5 kinase inhibitors cause paradoxical activation of ERK5 transcriptional activity mediated through its unique C-terminal transcriptional activation domain (TAD). Using the ERK5 kinase inhibitor, Compound 26 (ERK5-IN-1), as a paradigm, we have developed kinase-active, drug-resistant mutants of ERK5. With these mutants, we show that induction of ERK5 transcriptional activity requires direct binding of the inhibitor to the kinase domain. This in turn promotes conformational changes in the kinase domain that result in nuclear translocation of ERK5 and stimulation of gene transcription. This shows that both the ERK5 kinase and TAD must be considered when assessing the role of ERK5 and the effectiveness of anti-ERK5 therapeutics. 1 1234567890():,;E xtracellular signal-regulated kinase 5 (ERK5, also known as Big MAP Kinase or BMK1) 1,2 is a member of the mitogenactivated protein kinase (MAPK) family, which also includes ERK1/2 3 , the JNKs 4 and the p38 kinases 5 . Like ERK1/2, ERK5 is the effector kinase of a three-tiered MAPK pathway, comprising MEKK2 and MEKK3 (the MKKKs), MEK5 (MKK) and finally ERK5 (MAPK). ERK5 is encoded by the MAPK7 gene and includes an N-terminal kinase domain that shares 50% identity with ERK2 1,2 . However, it also contains a large, unique C-terminal extension that includes a nuclear localisation signal (NLS) and a transcriptional activation domain (TAD) (Fig. 1a) 6 . The ERK5 pathway is activated by mitogens 7 , agonists of the Tolllike receptor-2 8 and cellular stresses 9 . Upon cellular stimulation, activated MEK5 phosphorylates the TEY motif in the ERK5 activation-loop, leading to activation of its kinase domain 10 . The ERK5 C-terminus also becomes auto-phosphorylated and promotes ERK5 translocation from the cytosol to the nucleus 11,12 , where ERK5 has been shown to interact with MEF2 transcription factors such as MEF2D 7,13,14 . The C-terminus can also be regulated by other protein kinases, including ERK1/2 15 and CDK1 16,17 , which phosphorylate C-terminal residues independently of ERK5 kinase activity. Thus, the C-terminus mediates NATURE COMMUNICATIONS | https://doi.

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“…For a review on the classification of kinase inhibitors see [67]. A type IV ERK5 inhibitor, cpd 5, has been identified by Chen et al [68]. Type IV inhibitors do not bind the ATP or peptide substrate binding sites and are considered to act as allosteric inhibitors.…”

Section: Development Of Mek5 and Erk5 Kinase Inhibitorsmentioning

confidence: 99%

“…Although cpd 5 does not bind directly to the ATP-binding site, it displaces the kinase P-loop into the ATP-binding site and thus is ATP competitive. This inhibitor has off-target effects against five other kinases at concentrations required to inhibit ERK5 in cells [68] but it has not been tested against BRD4.…”

Section: Development Of Mek5 and Erk5 Kinase Inhibitorsmentioning

confidence: 99%

Small molecule ERK5 kinase inhibitors paradoxically activate ERK5 signalling: be careful what you wish for…

Cook

Tucker

Lochhead

2020

Biochemical Society Transactions

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ERK5 is a protein kinase that also contains a nuclear localisation signal and a transcriptional transactivation domain. Inhibition of ERK5 has therapeutic potential in cancer and inflammation and this has prompted the development of ERK5 kinase inhibitors (ERK5i). However, few ERK5i programmes have taken account of the ERK5 transactivation domain. We have recently shown that the binding of small molecule ERK5i to the ERK5 kinase domain stimulates nuclear localisation and paradoxical activation of its transactivation domain. Other kinase inhibitors paradoxically activate their intended kinase target, in some cases leading to severe physiological consequences highlighting the importance of mitigating these effects. Here, we review the assays used to monitor ERK5 activities (kinase and transcriptional) in cells, the challenges faced in development of small molecule inhibitors to the ERK5 pathway, and classify the molecular mechanisms of paradoxical activation of protein kinases by kinase inhibitors.

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Discovery of a novel allosteric inhibitor-binding site in ERK5: comparison with the canonical kinase hinge ATP-binding site

Abstract: Crystal structures of the MAP kinase ERK5 in complex with XMD8-92 and four novel inhibitors reveal an allosteric binding site between the kinase P-loop and αC helix. Binding at this site displaces the P-loop into the ATP-binding site and was shown to be ATP-competitive.

Cited by 16 publications

References 49 publications

Discovery of a selective inhibitor of doublecortin like kinase 1

Discovery of a selective inhibitor of doublecortin like kinase 1

Paradoxical activation of the protein kinase-transcription factor ERK5 by ERK5 kinase inhibitors

Small molecule ERK5 kinase inhibitors paradoxically activate ERK5 signalling: be careful what you wish for…

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