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Characterization of the cellular action of the MSK inhibitor SB-747651A
Shaista Naqvi1, Andrew Macdonald, Claire E McCoy
1MRC Protein Phosphorylation Unit, Sir James Black Complex, University of Dundee, Dundee DD1 5EH, Scotland, U.K.
Abstract:
MSK1 (mitogen- and stress-activated kinase 1) and MSK2 are nuclear protein kinases that regulate transcription downstream of the ERK1/2 (extracellular-signal-regulated kinase 1/2) and p38α MAPKs (mitogen-activated protein kinases) via the phosphorylation of CREB (cAMP-response-element-binding protein) and histone H3. Previous studies on the function of MSKs have used two inhibitors, H89 and Ro 31-8220, both of which have multiple off-target effects. In the present study, we report the characterization of the in vitro and cellular properties of an improved MSK1 inhibitor, SB-747651A. In vitro, SB-747651A inhibits MSK1 with an IC50 value of 11 nM. Screening of an in vitro panel of 117 protein kinases revealed that, at 1 microM, SB-747651A inhibited four other kinases, PRK2 (double-stranded-RNA-dependent protein kinase 2), RSK1 (ribosomal S6 kinase 1), p70S6K (S6K is S6 kinase) (p70RSK) and ROCK-II (Rho-associated protein kinase 2), with a similar potency to MSK1. In cells, SB-747651A fully inhibited MSK activity at 5-10 microM. SB-747651A was found to inhibit the production of the anti-inflammatory cytokine IL-10 (interleukin-10) in wild-type, but not MSK1/2-knockout, macrophages following LPS (lipopolysaccharide) stimulation. Both SB-747651A and MSK1/2 knockout resulted in elevated pro-inflammatory cytokine production by macrophages in response to LPS. Comparison of the effects of SB-747651A, both in vitro and in cells, demonstrated that SB-747651A exhibited improved selectivity over H89 and Ro 31-8220 and therefore represents a useful tool to study MSK function in cells.
Insights
A new MSK1 inhibitor, SB-747651A, offers improved selectivity over older compounds. This tool aids research into mitogen- and stress-activated kinase functions and their role in cytokine regulation.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Mitogen- and stress-activated kinases (MSKs) regulate transcription via CREB and histone H3 phosphorylation.
- Existing MSK inhibitors (H89, Ro 31-8220) possess significant off-target effects.
- Understanding MSK function requires selective chemical probes.
Purpose of the Study:
- To characterize the in vitro and cellular properties of a novel MSK1 inhibitor, SB-747651A.
- To assess the selectivity of SB-747651A compared to existing inhibitors.
- To evaluate SB-747651A's utility in studying MSK-mediated cellular responses.
Main Methods:
- In vitro kinase inhibition assays to determine IC50 values.
- Kinase panel screening to assess off-target effects.
- Cellular assays using macrophages to measure cytokine production (IL-10, pro-inflammatory cytokines) following LPS stimulation.
- Comparison with MSK1/2-knockout models.
Main Results:
- SB-747651A inhibits MSK1 with an IC50 of 11 nM.
- SB-747651A shows improved selectivity compared to H89 and Ro 31-8220.
- SB-747651A inhibits IL-10 production in macrophages and, along with MSK1/2 knockout, elevates pro-inflammatory cytokine release.
Conclusions:
- SB-747651A is a potent and selective MSK1 inhibitor.
- SB-747651A effectively probes MSK1/2 function in cellular contexts.
- This inhibitor provides a valuable tool for investigating MSK roles in inflammatory responses.
