Identifying requirements for RSK2 specific inhibitors

Identifying isoform-specific inhibitors for closely related kinase family members remains a substantial challenge. The necessity for achieving this specificity is exemplified by the RSK family, downstream effectors of ERK1/2, which have divergent physiological effects. The natural product, SL0101, a flavonoid glycoside, binds specifically to RSK1/2 through a binding pocket generated by an extensive conformational rearrangement within the RSK N-terminal kinase domain (NTKD). In modelling experiments a single amino acid that is divergent in RSK3/4 most likely prevents the required conformational rearrangement necessary for SL0101 binding. Kinetic analysis of RSK2 association with SL0101 and its derivatives identified that regions outside of the NTKD contribute to stable inhibitor binding. An analogue with an n-propyl-carbamate at the 4” position on the rhamnose moiety was identified that forms a highly stable inhibitor complex with RSK2 but not with RSK1. These results identify a SL0101 modification that will aid the identification of RSK2 specific inhibitors.     

Influence of rhamnose substituents on the potency of SL0101, an inhibitor of the Ser/Thr kinase, RSK

We have previously reported the isolation of kaempferol 3-O-(3',4'-di-O-acetyl-alpha-l-rhamnopyranoside) from Forsteronia refracta [Xu, Y.-M.; Smith, J. A.; Lannigan, D. A.; Hecht, S. M. Biorg. Med. Chem.2006, 14, 3974-3977.]. This flavonoid glycoside, termed SL0101, is a specific inhibitor of p90 ribosomal S6 kinase (RSK) with a dissociation constant of 1 microM. In intact cells, however, the EC50 for inhibition of RSK activity is 50 microM, which suggests that the efficacy of SL0101 could be limited by cellular uptake. Therefore, we investigated the possibility of developing a more potent RSK inhibitor by synthesizing SL0101 analogs with increased hydrophobic character. The total syntheses of kaempferol 3-O-(3',4'-di-O-butyryl-alpha-L-rhamnopyranoside) (Bu-SL0101) and kaempferol 3-O-(2',3',4'-tri-O-acetyl-alpha-L-rhamnopyranoside) (3Ac-SL0101) were performed. The IC50 for inhibition of RSK activity in in vitro kinase assays for the analogs was similar to that obtained for SL0101. 3Ac-SL0101 demonstrated the same remarkable specificity for inhibiting RSK activity in intact cells as SL0101; however, Bu-SL0101 was not completely specific. 3Ac-SL0101 was approximately 2-fold more potent at inhibiting MCF-7 cell proliferation compared to SL0101 and preferentially decreased MCF-7 cell growth, as compared to the growth of the normal human breast line, MCF-10A. Thus the discovery of 3Ac-SL0101 as a more potent RSK-specific inhibitor than SL0101 should facilitate the development of RSK inhibitors as anti-cancer chemotherapeutic agents.     

Analogs of the RSK inhibitor SL0101: optimization of in vitro biological stability

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.     

Insights into the Inhibition of the p90 Ribosomal S6 Kinase (RSK) by the Flavonol Glycoside SL0101 from the 1.5 Å Crystal Structure of the N-Terminal Domain of RSK2 with Bound Inhibitor

The p90 ribosomal S6 family of kinases (RSK) are potential drug targets, due to their involvement in cancer and other pathologies. There are currently only two known selective inhibitors of RSK, but the basis for selectivity is not known. One of these inhibitors is a naturally occurring kaempferol-α-l-diacetylrhamnoside, SL0101. Here, we report the crystal structure of the complex of the N-terminal kinase domain of the RSK2 isoform with SL0101 at 1.5 ? resolution. The refined atomic model reveals unprecedented structural reorganization of the protein moiety, as compared to the nucleotide-bound form. The entire N-lobe, the hinge region, and the αD-helix undergo dramatic conformational changes resulting in a rearrangement of the nucleotide binding site with concomitant formation of a highly hydrophobic pocket spatially suited to accommodate SL0101. These unexpected results will be invaluable in further optimization of the SL0101 scaffold as a promising lead for a novel class of kinase inhibitors.     

A2B adenosine and P2Y2 receptors stimulate mitogen-activated protein kinase in human embryonic kidney-293 cells. cross-talk between cyclic AMP and protein kinase c pathways

Mitogen-activated protein kinase (MAPK) cascades underlie long-term mitogenic, morphogenic, and secretory activities of purinergic receptors. In HEK-293 cells, N-ethylcarboxamidoadenosine (NECA) activates endogenous A2BARs that signal through Gs and Gq/11. UTP activates P2Y2 receptors and signals only through Gq/11. The MAPK isoforms, extracellular-signal regulated kinase 1/2 (ERK), are activated by NECA and UTP. H-89 blocks ERK activation by forskolin, but weakly affects the response to NECA or UTP. ERK activation by NECA or UTP is unaffected by a tyrosine kinase inhibitor (genistein), attenuated by a phospholipase C inhibitor (U73122), and is abolished by a MEK inhibitor (PD098059) or dominant negative Ras. Inhibition of protein kinase C (PKC) by GF 109203X failed to block ERK activation by NECA or UTP, however, another PKC inhibitor, Ro 31-8220, which unlike GF 109203X, can block the zeta-isoform, and prevents UTP- but not NECA-induced ERK activation. In the presence of forskolin, Ro 31-8220 loses its ability to block UTP-stimulated ERK activation. PKA has opposing effects on B-Raf and c-Raf-1, both of which are found in HEK-293 cells. The data are explained by a model in which ERK activity is modulated by differential effects of PKC zeta and PKA on Raf isoforms.     

Divergent off-target effects of RSK N-terminal and C-terminal kinase inhibitors in cardiac myocytes

P90 ribosomal S6 kinases (RSK) are ubiquitously expressed and regulate responses to neurohumoral stimulation. To study the role of RSK signalling on cardiac myocyte function and protein phosphorylation, pharmacological RSK inhibitors were tested. Here, the ATP competitive N-terminal kinase domain-targeting compounds D1870 and SL0101 and the allosteric C-terminal kinase domain-targeting FMK were evaluated regarding their ability to modulate cardiac myocyte protein phosphorylation. Exposure to D1870 and SL0101 significantly enhanced phospholamban (PLN) Ser16 and cardiac troponin I (cTnI) Ser22/23 phosphorylation in response to D1870 and SL0101 upon exposure to phenylephrine (PE) that activates RSK. In contrast, FMK pretreatment significantly reduced phosphorylation of both proteins in response to PE. D1870-mediated enhancement of PLN Ser16 phosphorylation was also observed after exposure to isoprenaline or noradrenaline (NA) stimuli that do not activate RSK. Inhibition of β-adrenoceptors by atenolol or cAMP-dependent protein kinase (PKA) by H89 prevented the D1870-mediated increase in PLN phosphorylation, suggesting that PKA is the kinase responsible for the observed phosphorylation. Assessment of changes in cAMP formation by FRET measurements revealed increased cAMP formation in vicinity to PLN after exposure to D1870 and SL0101. D1870 inhibited phosphodiesterase activity similarly as established PDE inhibitors rolipram or 3-isobutyl-1-methylxanthine. Assessment of catecholamine-mediated force development in rat ventricular muscle strips revealed significantly reduced EC₅₀ for NA after D1870 pretreatment (DMSO/NA: 2.33 μmol/L vs. D1870/NA: 1.30 μmol/L). The data reveal enhanced cardiac protein phosphorylation by D1870 and SL0101 that was not detectable in response to FMK. This disparate effect might be attributed to off-target inhibition of PDEs with impact on muscle function as demonstrated for D1870.     

A clickable inhibitor reveals context-dependent autoactivation of p90 RSK

p90 ribosomal protein S6 kinases (RSKs) integrate upstream signals through two catalytic domains. Autophosphorylation of Ser386 by the regulatory C-terminal kinase domain (CTD) is thought to be essential for activation of the N-terminal kinase domain (NTD), which phosphorylates multiple downstream targets. We recently reported fmk, an irreversible inhibitor of the CTD of RSK1 and RSK2. Here we describe fmk-pa, a propargylamine variant that has improved cellular potency and a 'clickable' tag for assessing the extent and selectivity of covalent RSK modification. Copper-catalyzed conjugation of an azidoalkyl reporter (the click reaction) revealed that fmk-pa achieves selective and saturable modification of endogenous RSK1 and RSK2 in mammalian cells. Saturating concentrations of fmk-pa inhibited Ser386 phosphorylation and downstream signaling in response to phorbol ester stimulation, but had no effect on RSK activation by lipopolysaccharide. RSK autoactivation by the CTD is therefore context dependent, which suggests that NTD and CTD inhibitors should have distinct physiological effects.     

Differential regulation of NHE1 phosphorylation and glucose uptake by inhibitors of the ERK pathway and p90RSK in 3T3-L1 adipocytes

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.     

EGFR and PKC are involved in the activation of ERK1/2 and p90 RSK and the subsequent proliferation of SNU-407 colon cancer cells by muscarinic acetylcholine receptors

We have previously shown that muscarinic acetylcholine receptors (mAChRs) enhance SNU-407 colon cancer cell proliferation via the ERK1/2 pathway. Here, we examined the signaling pathways linking mAChR stimulation to ERK1/2 activation and the subsequent proliferation of SNU-407 cells. The inhibition of the epidermal growth factor receptor (EGFR) by AG1478 or protein kinase C (PKC) by GF109203X significantly reduced carbachol-stimulated ERK1/2 activation and cell proliferation. Cotreatment of the cells with AG1478 and GF109203X produced an additive effect on carbachol-stimulated ERK1/2 activation, suggesting that the EGFR and PKC pathways act in parallel. The p90 ribosomal S6 kinases (RSKs) are downstream effectors of ERK1/2 and are known to have important roles in cell proliferation. In SNU-407 cells, carbachol treatment induced RSK activation in an atropine-sensitive manner, and this RSK activation was decreased by the inhibition of either EGFR or PKC. Moreover, the RSK-specific inhibitor BRD7389 almost completely blocked carbachol-stimulated cell proliferation. Together, these data indicate that EGFR and PKC are involved in mAChR-mediated activation of ERK1/2 and RSK and the subsequent proliferation of SNU-407 colon cancer cells.     

Involvement of different protein kinases and phospholipases A2 in phorbol ester (TPA)-induced arachidonic acid liberation in bovine platelets

The effect of various phospholipase A2 and protein kinase inhibitors on the arachidonic acid liberation in bovine platelets induced by the protein kinase activator 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied. TPA stimulates arachidonic acid release mainly by activating group IV cytosolic PLA2 (cPLA2), since inhibitors of this enzyme markedly inhibited arachidonic acid formation. However, group VI Ca2+-independent PLA2 (iPLA2) seems to contribute to the arachidonic acid liberation too, since the relatively specific iPLA2 inhibitor bromoenol lactone (BEL) decreased arachidonic acid generation in part. The pronounced inhibition of the TPA-induced arachidonic acid release by the protein kinase C (PKC) inhibitors GF 109203X and Ro 31-82220, respectively, and by the p38 MAP kinase inhibitor SB 202190 suggests that the activation of the PLA2s by TPA is mediated via PKC and p38 MAP kinase.     

Computational and Biochemical Discovery of RSK2 as a Novel Target for Epigallocatechin Gallate (EGCG)

The most active anticancer component in green tea is epigallocatechin-3-gallate (EGCG). Protein interaction with EGCG is a critical step for mediating the effects of EGCG on the regulation of various key molecules involved in signal transduction. By using computational docking screening methods for protein identification, we identified a serine/threonine kinase, 90-kDa ribosomal S6 kinase (RSK2), as a novel molecular target of EGCG. RSK2 includes two kinase catalytic domains in the N-terminal (NTD) and the C-terminal (CTD) and RSK2 full activation requires phosphorylation of both terminals. The computer prediction was confirmed by an in vitro kinase assay in which EGCG inhibited RSK2 activity in a dose-dependent manner. Pull-down assay results showed that EGCG could bind with RSK2 at both kinase catalytic domains in vitro and ex vivo. Furthermore, results of an ATP competition assay and a computer-docking model showed that EGCG binds with RSK2 in an ATP-dependent manner. In RSK2^+/+ and RSK2^-/- murine embryonic fibroblasts, EGCG decreased viability only in the presence of RSK2. EGCG also suppressed epidermal growth factor-induced neoplastic cell transformation by inhibiting phosphorylation of histone H3 at Ser10. Overall, these results indicate that RSK2 is a novel molecular target of EGCG.     

Selective secretion of protein kinase C isozymes by thrombin-stimulated human platelets

The protein kinase C (PKC) was secreted from thrombin-stimulated human platelets in a time- and dose-dependent manner. The PKC specific inhibitors Ro31-8220 (0.05 microM) and GF 109203X (0.5 microM) totally inhibited the secreted kinase activity. Western blot analysis of the secretory components showed reactivity to PKCalpha, PKCbetaII, and PKCdelta antibodies, but not to PKCbetaI, and p42/44 MAPK, although they were present in lysed platelets. The fractionation of platelets secreted components showed that PKC activity increased in both soluble and microparticle fractions after thrombin treatments. This is the first report demonstrating that activated human platelets selectively secrete protein kinase C isozymes. Protein kinase C secreted by platelets in this unique manner may have an extracellular role in the plasma, and may regulate cellular functions, including remodeling of vascular endothelial cells.     

Structural basis for the activity of the RSK-specific inhibitor, SL0101

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-alpha-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.     

Synthesis,activity evaluation,and docking analysis of barbituric acid aryl hydrazone derivatives as RSK2 inhibitors

The 90 kDa ribosomal S6 kinases (RSKs), especially RSK2, have attracted attention for the development of new anticancer agents. Through structural optimization of the hit compound 1 from our previous study, a series of barbituric acid aryl hydrazone analogues were designed and synthesized as potential RSK2 inhibitors. The most potent one, compound 9, showed a higher activity against RSK2 with an IC₅₀ value of 1.95 μM. To analyze and elucidate their structure-activity relationship, the homology model of RSK2 N-terminal kinase domain was built and molecular docking simulations were performed, which provide helpful clues to design new inhibitors with desired activities.     

Interactions of opioid and chemokine receptors: oligomerization of mu,kappa, and delta with CCR5 on immune cells

In this study, we examined the signaling pathways for extracellular signal-related protein kinase (ERK) activation by three structurally different peroxisome proliferator activated receptor-gamma (PPARgamma) agonists. In murine C2C12 myoblasts, treatment with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), ciglitazone, and GW1929 leads to ERK1/2 phosphorylation in a time- and concentration-dependent manner. Consistent with ERK phosphorylation, mitogen activated protein/ERK kinase (MEK) phosphorylation as well as Raf-1 kinase activity are also accordingly stimulated, while the constitutive Ser259 phosphorylation of Raf-1 is decreased. The ERK phosphorylation induced by PPARgamma agonists is not blocked by the PKC inhibitors GF109203X and Ro31-8220, the PI3K inhibitor wortmannin, the Ras inhibitor FPTI, the negative mutant of Ras, or the PPARgamma antagonist bisphenol A diglycidil ether. Expression of PPARgamma2 without DNA binding domain or with a nonphosphorylatable mutant (S112A) fails to change ERK phosphorylation by 15d-PGJ(2). On the contrary, the ERK phosphorylation by PPARgamma agonists is inhibited by the MEK inhibitor PD98059, GSH, and permeable SOD mimetic MnTBAP. Chemiluminescence study reveals that these three PPARgamma agonists are able to induce superoxide anion production, with an efficacy similar to their action on ERK phosphorylation. Consistent with this notion, we also show that superoxide anion donor 2,3-dimethoxy-1,4-naphoquinone elicits ERK phosphorylation. In this study, we for the first time demonstrate a novel mechanism, independent of Ras activation but initiated by superoxide anion production, for PPARgamma agonists to trigger the Raf-MEK-ERK1/2 signaling pathway.     

Protein kinase Calpha mediates feedback inhibition of EGF receptor transactivation induced by Gq-coupled receptor agonists

While a great deal of attention has been focused on G-protein-coupled receptor (GPCR)-induced epidermal growth factor receptor (EGFR) transactivation, it has been known for many years that the tyrosine kinase activity of the EGFR is inhibited in cells treated with tumor-promoting phorbol esters, a process termed EGFR transmodulation. Because many GPCR agonists that elicit EGFR transactivation also stimulate the G_q/phospholipase C (PLC)/protein kinase C (PKC) pathway, we hypothesized that PKC-mediated inhibition of EGFR transactivation operates physiologically as a feedback loop that regulates the intensity and/or duration of GPCR-elicited EGFR transactivation. In support of this hypothesis, we found that treatment of intestinal epithelial IEC-18 cells with the PKC inhibitors GF 109203X or Ro 31-8220 or chronic exposure of these cells to phorbol-12,13-dibutyrate (PDB) to downregulate PKCs, markedly enhanced the increase in EGFR tyrosine phosphorylation induced by angiotensin II or vasopressin in these cells. Similarly, PKC inhibition enhanced EGFR transactivation in human colonic epithelial T84 cells stimulated with carbachol, as well as in bombesin-stimulated Rat-1 fibroblasts stably transfected with the bombesin receptor. Furthermore, cell treatment with inhibitors with greater specificity towards PKCα,  including Gö6976, Ro 31-7549 or Ro 32-0432, also increased GPCR-induced EGFR transactivation in IEC-18, T84 and Rat-1 cells. Transfection of siRNAs targeting PKCα  also enhanced bombesin-induced EGFR tyrosine phosphorylation in Rat-1 cells. Thus, multiple lines of evidence support the hypothesis that conventional PKC isoforms, especially PKCα, mediate feedback inhibition of GPCR-induced EGFR transactivation.     

Characterization of the p90 ribosomal S6 kinase 2 carboxyl-terminal domain as a protein kinase

The carboxyl-terminal domain (CTD) of the p90 ribosomal S6 kinases (RSKs) is an important regulatory domain in RSK and a model for kinase regulation of FXXFXF(Y) motifs in AGC kinases. Its properties had not been studied. We reconstituted activation of the CTD in Escherichia coli by co-expression with active ERK2 mitogen-activated protein kinase (MAPK). GST-RSK2-(aa373-740) was phosphorylated in the P-loop (Thr(577)) by MAPK, accompanied by increased phosphorylation on the hydrophobic motif site, Ser(386). Activated GST-RSK2-(aa373-740) phosphorylates synthetic peptides based on Ser(386). The peptide RRQLFRGFSFVAK, which was termed CTDtide, was phosphorylated with K(m) and V(max) values of approximately 140 microm and approximately 1 micromol/min/mg, respectively. Residues Leu at p -5 and Arg at p -3 are important for substrate recognition, but a hydrophobic residue at p +4 is not. RSK2 CTD is a much more selective peptide kinase than MAPK-activated protein kinase 2. CTDtide was used to probe regulation of hemagglutinin-tagged RSK proteins immunopurified from epidermal growth factor-stimulated BHK-21 cells. K100A but not K451A RSK2 phosphorylates CTDtide, indicating a requirement for the CTD. RSK2-(aa1-389) phosphorylates the S6 peptide, and this activity is inactivated by S386A mutation, but RSK2-(aa1-389) does not phosphorylate CTDtide. In contrast, RSK2-(aa373-740) containing only the CTD phosphorylates CTDtide robustly. Thus, CTDtide is phosphorylated by the CTD but not the NH(2)-terminal domain (NTD). Epidermal growth factor activates the CTD and NTD in parallel. Activity of the CTD for peptide phosphorylation correlates with Thr(577) phosphorylation. CTDtide activity is constrained in full-length RSK2. Interestingly, mutation of the conserved lysine in the ATP-binding site of the NTD completely eliminates S6 kinase activity, but a similar mutation of the CTD does not completely ablate kinase activity for intramolecular phosphorylation of Ser(386), even though it greatly reduces CTDtide activity. The standard lysine mutation used routinely to study kinase functions in vivo may be unsatisfactory when the substrate is intramolecular or in a tight complex.