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The unusual mechanism of inhibition of the p90 ribosomal S6 kinase (RSK) by flavonol rhamnosides.

ABSTRACT: All known protein kinases share a bilobal kinase domain with well conserved structural elements. Because of significant structural similarities of nucleotide binding pocket, the development of highly selective kinase inhibitors is a very challenging task. Flavonols, naturally occurring plant metabolites, have long been known to inhibit kinases by mimicking the adenine moiety. Interestingly, recent data show that some flavonol glycosides are more selective, although underlying mechanisms were unknown. Crystallographic data from our laboratory revealed that the N-terminal kinase domain of p90 ribosomal S6 kinase, isoform 2, binds three different flavonol rhamnosides in a highly unusual manner, distinct from other kinase inhibitor interactions. The kinase domain undergoes a reorganization of several structural elements in response to the binding of the inhibitors. Specifically, the main ?-sheet of the N-lobe undergoes a twisting rotation by ~56° around an axis passing through the N- and C-lobes, leading to the restructuring of the canonical ATP-binding pocket into pockets sterically adapted to the inhibitor shape. The flavonol rhamnosides appear to adopt compact, but strained conformations with the rhamnose moiety swept under the B-ring of flavonol, unlike the structure of the free counterparts in solution. These data suggest that the flavonol glycoside scaffold could be used as a template for new inhibitors selective for the RSK family. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

SUBMITTER: Utepbergenov D

PROVIDER: S-EPMC3683371 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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The unusual mechanism of inhibition of the p90 ribosomal S6 kinase (RSK) by flavonol rhamnosides.

Utepbergenov Darkhan D Derewenda Zygmunt S ZS

Biochimica et biophysica acta 20130327 7

All known protein kinases share a bilobal kinase domain with well conserved structural elements. Because of significant structural similarities of nucleotide binding pocket, the development of highly selective kinase inhibitors is a very challenging task. Flavonols, naturally occurring plant metabolites, have long been known to inhibit kinases by mimicking the adenine moiety. Interestingly, recent data show that some flavonol glycosides are more selective, although underlying mechanisms were unk ...[more]

PMID: 23541530

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Project description:Triple-negative breast cancers (TNBC) are notoriously difficult to treat because they lack hormone receptors and have limited targeted therapies. Recently, we demonstrated that p90 ribosomal S6 kinase (RSK) is essential for TNBC growth and survival indicating it as a target for therapeutic development. RSK phosphorylates Y-box binding protein-1 (YB-1), an oncogenic transcription/translation factor, highly expressed in TNBC (~70% of cases) and associated with poor prognosis, drug resistance and tumor initiation. YB-1 regulates the tumor-initiating cell markers, CD44 and CD49f however its role in Notch signaling has not been explored. We sought to identify novel chemical entities with RSK inhibitory activity. The Prestwick Chemical Library of 1120 off-patent drugs was screened for RSK inhibitors using both in vitro kinase assays and molecular docking. The lead candidate, luteolin, inhibited RSK1 and RSK2 kinase activity and suppressed growth in TNBC, including TIC-enriched populations. Combining luteolin with paclitaxel increased cell death and unlike chemotherapy alone, did not enrich for CD44(+) cells. Luteolin's efficacy against drug-resistant cells was further indicated in the primary x43 cell line, where it suppressed monolayer growth and mammosphere formation. We next endeavored to understand how the inhibition of RSK/YB-1 signaling by luteolin elicited an effect on TIC-enriched populations. ChIP-on-ChIP experiments in SUM149 cells revealed a 12-fold enrichment of YB-1 binding to the Notch4 promoter. We chose to pursue this because there are several reports indicating that Notch4 maintains cells in an undifferentiated, TIC state. Herein we report that silencing YB-1 with siRNA decreased Notch4 mRNA. Conversely, transient expression of Flag:YB-1(WT) or the constitutively active mutant Flag:YB-1(D102) increased Notch4 mRNA. The levels of Notch4 transcript and the abundance of the Notch4 intracellular domain (N4ICD) correlated with activation of P-RSK(S221/7) and P-YB-1(S102) in a panel of TNBC cell lines. Silencing YB-1 or RSK reduced Notch4 mRNA and this corresponded with loss of N4ICD. Likewise, the RSK inhibitors, luteolin and BI-D1870, suppressed P-YB-1(S102) and thereby reduced Notch4. In conclusion, inhibiting the RSK/YB-1 pathway with luteolin is a novel approach to blocking Notch4 signaling and as such provides a means of inhibiting TICs.

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The unusual mechanism of inhibition of the p90 ribosomal S6 kinase (RSK) by flavonol rhamnosides.

Publications

The unusual mechanism of inhibition of the p90 ribosomal S6 kinase (RSK) by flavonol rhamnosides.

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