Identification of a novel orally bioavailable ERK5 inhibitor with selectivity over p38α and BRD4

Myers, Stephanie M.; Miller, Duncan C.; Molyneux, Lauren; Arasta, Mercedes; Bawn, Ruth H.; Blackburn, Timothy J.; Cook, Simon J.; Edwards, Noel; Endicott, Jane; Golding, Bernard T.; Griffin, Roger J.; Hammonds, Tim; Hardcastle, Ian R.; Harnor, Suzannah J.; Heptinstall, Amy B; Lochhead, Pamela A.; Martin, Mathew P.; Martin, Nick C; Newell, David R.; Owen, Paul; Pang, Leon; Reuillon, Tristan; Rigoreau, Laurent; Thomas, Huw D.; Tucker, J.A.; Wang, Lan-Zhen; Wong, Ai-Ching; Noble, M.E.M.; Wedge, Stephen R.; Cano, Céline

doi:10.1016/j.ejmech.2019.05.057

Cited by 16 publications

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“…Thus, dual ERK5-BRD inhibitors, such as XMD8-92 and compound 26, are no longer suitable for dissecting the contribution that ERK5 plays within the cell. Selective ERK5 inhibition, without activity against BRD proteins, can be achieved with AX15836 or the new ERK5i compound 46 38 making them useful small molecules for interrogating the cellular roles of ERK5. Critically, AX15836 failed to replicate the effects of genetic ablation of ERK5 on cytokine release from immune cells or cell proliferation 28 .…”

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

et al. 2020

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“…This study also suggested that the ERK5 C-terminal domain is important for the biological function of ERK5 [64]. Heedful researchers have since used BRD4 inhibitors, such as JQ1, to delineate the role of BRD4 in their systems [25,45,55,56] and testing of ERK5 inhibitors against BRD4 is now an essential step in ERK5 drug discovery. Adding to the complexity, we have found that cpd 26 (XMD17-109) and AX15836, cause a conformational change in the kinase domain which leads to exposure of the C-terminal NLS and paradoxical activation of the ERK5 TAD [55] (see below and Figure 2).…”

Section: Development Of Mek5 and Erk5 Kinase Inhibitorsmentioning

confidence: 81%

“…The best validated ERK5 interacting proteins and substrates are the MEF2A, C and D transcription factors [10,17,54]. Thus, the MEF2 proteins remain the most appropriate direct reporters of ERK5 activity and have been used in cellular reporter systems for monitoring MEK5 and/or ERK5 inhibition [55][56][57]58]. The simplest version consists of multimerised MEF2 binding sites and basic promoter elements to drive the expression of a reporter (typically firefly luciferase) in response to EGF ( Figure 1B top) [57], making it a significant advance in ERK5 selectivity over AP-1.…”

Section: The Challenge Of Monitoring Erk5 Inhibition In Cellsmentioning

confidence: 99%

“…This system has been shown to work for MEF2A, C and D [17] and although it has more components, it is a far more direct readout of ERK5 activation since ERK5 interacts directly with the MEF2-GAL4 reporter construct to drive luciferase expression. We have used this assay to measure the pharmacological inhibition of MEK5 and ERK5 [38,55,56].…”

Section: The Challenge Of Monitoring Erk5 Inhibition In Cellsmentioning

confidence: 99%

“…In cell-based assays, an inhibitor that blocks LRRK2 activity (such as JWG-048 [45]), and not ERK5 activity, could be used to confirm ERK5 kinase involvement. A multi-site collaboration reported cpd 46; this inhibitor is distinct from the XMD8-92 series, has no activity towards BRD4 or LRRK2, is highly selective for ERK5 and is also suitable for use in animal studies [56]. Bayer have reported BAY-885, which like AX15836 is very selective for ERK5 and did not significantly inhibit other kinases or BRD4 [57].…”

Section: Development Of Mek5 and Erk5 Kinase Inhibitorsmentioning

confidence: 99%

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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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Recent advances in the therapeutic development of ERK inhibitors

Kumar

Hassan²

2022

Protein Kinase Inhibitors

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Identification of a novel orally bioavailable ERK5 inhibitor with selectivity over p38α and BRD4

Abstract: Id e n tific a tio n of a N ov el O r ally Bio av ail a bl e ERK 5 In hi bit o r wi t h S el e c tivity ov e r p 3 8 a n d α BRD 4. E u r o p e a n Jou r n al of M e di ci n al C h e mi s t ry, 1 7 8 . p p. 5 3 0-5 4 3. IS S N 0 2 2 3-5 2 3 4 Do w nlo a d e d fro m: h t t

Cited by 16 publications

References 37 publications

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

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…

Recent advances in the therapeutic development of ERK inhibitors

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