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The 90 kDa ribosomal S6 kinases (RSK) are effectors of the Ras-ERK1/2 signaling pathway. RSK signaling controls proliferation and protein synthesis, and is altered in several types of tumors. BI-D1870 and SL0101 are two widely used inhibitors of RSK. After revision of the literature, discrepancies in the effects of the inhibitors were identified. Herein we report that while SL0101 inhibited mTORC1-p70S6K signaling, BI-D1870 increased p70S6K activation. Both effects were independent of ERK1/2 and RSK, and thus nonspecific. We also demonstrated how these opposite nonspecific effects mislead the identification of the RSK-dependent phosphorylation of rpS6 (S235/236), a known RSK and p70S6K substrate. Phosphorylation of tuberin at S1798 by RSK was proposed to mediate ERK1/2-dependent activation of mTORC1-p70S6K signaling. In glioblastoma-derived cells, phosphorylation of tuberin was abolished after RSK depletion or ERK1/2 inhibition, suggesting that RSK is its main kinase. However, RSK depletion did not reduce PMA-dependent p70S6K phosphorylation, which suggests that tuberin phosphorylation at S1798 is not the main mediator of ERK1/2-dependent activation of mTORC1. Remarkably, tuberin phosphorylation (S1798) followed the activation status of RSK in different cells and experimental conditions, suggesting that phosphorylation of that residue could be used as readout for RSK activation in cells. We confirmed the difference in the effects of SL0101 and BI-D1870 in cellular proliferation assays. Rapamycin potentiated the inhibition of proliferation induced by BI-D1870, but not by SL0101. We thus conclude that SL0101 and BI-D1870 induce distinct off-target effects in mTORC1-p70S6K signaling, and thus, the functions previously ascribed to RSK based on these inhibitors should be reassessed. [ABSTRACT FROM AUTHOR]

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 β-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). [Copyright &y& Elsevier]

Abstract: The Ser/Thr protein kinase, RSK, is important in the etiology of tumor progression including invasion and motility. The natural product kaempferol-3-O-(3″,4″-di-O-acetyl-α-l-rhamnopyranoside), called SL0101, is a highly specific RSK inhibitor. Acylation of the rhamnose moiety is necessary for high affinity binding and selectivity. However, the acetyl groups can be cleaved by esterases, which accounts for the poor in vitro biological stability of SL0101. To address this problem a series of analogs containing acetyl group replacements were synthesized and their in vitro stability evaluated. Monosubstituted carbamate analogs of SL0101 showed improved in vitro biological stability while maintaining specificity for RSK. These results should facilitate the development of RSK inhibitors derived from SL0101 as anticancer agents. [Copyright &y& Elsevier]

Abstract: Insulin stimulates trafficking of GLUT4 to the cell surface for glucose uptake into target cells, and phosphorylation of Ser703 of the Na+/H+ exchanger NHE1, which activates proton efflux. The latter has been proposed to facilitate optimal glucose uptake into cardiomyocytes. We found that the insulin-stimulated phosphorylation of Ser703 of NHE1 is mediated by p90RSK but not directly coupled to glucose uptake in 3T3-L1 adipocytes in the short-term. Inhibiting Erk1/2 activation prevented NHE1 phosphorylation but not glucose uptake in 3T3-L1 adipocytes. In contrast, both NHE1 phosphorylation and insulin-stimulated uptake of glucose into 3T3-L1 adipocytes were blocked by inhibitors of the N-terminal kinase domain of p90RSK, namely BI-D1870 and SL0101, but not the FMK inhibitor of the C-terminal kinase domain of p90RSK, though in our hands FMK did not inhibit p90RSK in 3T3-L1 adipocytes. Further experiments were consistent with phosphorylation of AS160 by PKB/Akt mediating insulin-stimulated trafficking of GLUT4 to the plasma membrane. BI-D1870 and SL0101 however, inhibited glucose uptake without blocking GLUT4 translocation. While BI-D1870 partially inhibited insulin-stimulated PKB activation in these cells, this only partially inhibited AS160 phosphorylation and did not block GLUT4 trafficking, suggesting that p90RSK might regulate glucose transport after GLUT4 translocation. Moreover, BI-D1870 also prevented PMA-induced glucose transport in 3T3-L1 adipocytes further suggesting a role for p90RSK in regulating uptake of glucose into the cells. Kinetic experiments are consistent with SL0101 being a direct competitor of 2-deoxyglucose entry into cells, and this compound might also inhibit uptake of glucose into cells via inhibiting p90RSK, as revealed by comparison with the inactive form of the inhibitor. Taken together, we propose that BI-D1870 and SL0101 might exert their inhibitory effects on glucose uptake in 3T3-L1 adipocytes at least partially through a p90RSK dependent step after GLUT4 becomes associated with the plasma membrane. [Copyright &y& Elsevier]

Abstract: Inappropriate activity of p90 ribosomal S6 kinase (RSK) has been implicated in various human cancers as well as other pathologies. We previously reported the isolation, characterization, and synthesis of the natural product kaempferol 3-O-(3″,4″-di-O-acetyl-α-l-rhamnopyranoside), termed SL0101 [Smith, J. A.; Poteet-Smith, C. E.; Xu, Y.; Errington, T. M.; Hecht, S. M.; Lannigan, D. A. Cancer Res., 2005, 65, 1027–1034: Xu, Y.-M; Smith, J. A.; Lannigan, D. A.; Hecht, S. M. Bioorg. Med. Chem., 2006, 14, 3974–3977: Maloney, D. J.; Hecht, S. M. Org. Lett., 2005, 7, 1097–1099]. SL0101 is a potent and specific inhibitor of RSK; therefore, we performed an analysis of the structural basis for the inhibitory activity of this lead compound. In in vitro kinase assays we found that acylation of the rhamnose moiety and the 4′, 5, and 7-hydroxyl groups are responsible for maintaining a high affinity interaction of RSK with SL0101. It is likely that the hydroxyl groups facilitate RSK binding through their ability to form hydrogen bonds. To determine whether the SL0101 derivatives were specific for inhibition of RSK we analyzed their ability to preferentially inhibit the growth of the human breast cancer line, MCF-7, compared to the normal human breast line, MCF-10A. We have previously validated this differential growth assay as a convenient readout for analyzing the specificity of RSK inhibitors [Smith, J. A.; Maloney, D. J.; Clark, D. E.; Xu, Y.-M.; Hecht, S. M.; Lannigan, D. A. Bioorg. Med. Chem., 2006, 14, 6034–6042]. We found that acylation of the rhamnose moiety was essential for maintaining the selectivity for RSK inhibition in intact cells. Further, the efficacy of SL0101 in intact cells is limited by cellular uptake as well as possible hydrolysis of the acetyl groups on the rhamnose moiety by ubiquitous intracellular esterases. These studies should facilitate the development of a RSK inhibitor, based on the SL0101 pharmacophore, as an anti-cancer chemotherapeutic agent. [Copyright &y& Elsevier]