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Characterization of ERK docking domain inhibitors that induce apoptosis by targeting Rsk-1 and caspase-9.

Boston SR, Deshmukh R, Strome S, Priyakumar UD, MacKerell AD, Shapiro P - BMC Cancer (2011)

Bottom Line: Induction of HeLa cell apoptosis appeared to be through intrinsic mechanisms involving caspase-9 activation and decreased phosphorylation of the pro-apoptotic Bad protein.Further examination of the test compound's mechanism of action showed little effects on related MAP kinases or other cell survival proteins.These findings support the identification of a class of ERK-targeted molecules that can induce apoptosis in transformed cells by inhibiting ERK-mediated phosphorylation and inactivation of pro-apoptotic proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland 20 N. Pine St, Baltimore, MD 21201 USA.

ABSTRACT

Background: The extracellular signal-regulated kinase-1 and 2 (ERK1/2) proteins play an important role in cancer cell proliferation and survival. ERK1/2 proteins also are important for normal cell functions. Thus, anti-cancer therapies that block all ERK1/2 signaling may result in undesirable toxicity to normal cells. As an alternative, we have used computational and biological approaches to identify low-molecular weight compounds that have the potential to interact with unique ERK1/2 docking sites and selectively inhibit interactions with substrates involved in promoting cell proliferation.

Methods: Colony formation and water soluble tetrazolium salt (WST) assays were used to determine the effects of test compounds on cell proliferation. Changes in phosphorylation and protein expression in response to test compound treatment were examined by immunoblotting and in vitro kinase assays. Apoptosis was determined with immunoblotting and caspase activity assays.

Results: In silico modeling was used to identify compounds that were structurally similar to a previously identified parent compound, called 76. From this screen, several compounds, termed 76.2, 76.3, and 76.4 sharing a common thiazolidinedione core with an aminoethyl side group, inhibited proliferation and induced apoptosis of HeLa cells. However, the active compounds were less effective in inhibiting proliferation or inducing apoptosis in non-transformed epithelial cells. Induction of HeLa cell apoptosis appeared to be through intrinsic mechanisms involving caspase-9 activation and decreased phosphorylation of the pro-apoptotic Bad protein. Cell-based and in vitro kinase assays indicated that compounds 76.3 and 76.4 directly inhibited ERK-mediated phosphorylation of caspase-9 and the p90Rsk-1 kinase, which phosphorylates and inhibits Bad, more effectively than the parent compound 76. Further examination of the test compound's mechanism of action showed little effects on related MAP kinases or other cell survival proteins.

Conclusion: These findings support the identification of a class of ERK-targeted molecules that can induce apoptosis in transformed cells by inhibiting ERK-mediated phosphorylation and inactivation of pro-apoptotic proteins.

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Test compounds inhibit ERK-mediated phosphorylation of p90Rsk-1 and Bad. (A) HeLa cells were pre-treated for one hour in the presence or absence of 50 uM of the indicated test compounds and then stimulated with EGF (25 ng/ml) for 10 minutes. Immunoblots of phosphorylated p90Rsk-1 (pRsk), total Rsk (Rsk), phosphorylated ERK1/2 (ppERK), and total ERK2 (ERK2). α-tubulin was used as a loading control. Graph shows densitometry quantification of pRsk-1 to total Rsk or ppERK2 to ERK2 ratios. Data represents the mean ± SEM from three independent experiments. (B) In vitro kinase assays examining 32P incorporation into p90Rsk-1 following incubation with active ERK2 and γ-32P-ATP for 60 min. in the absence or presence of 1-25 μM of test compounds. Relative phosphate incorporation was quantified by phosphoimager analysis. (C) HeLa cells were serum starved overnight and pre-treated for 1 hr with 50 uM indicated test compounds, 10 mM U0126 (U), or 25 mM LY294002 (L) prior to stimulation with or without EGF (25 ng/ml). Immunoblot analysis of Bad phosphorylated on Ser112 (pS112) or Ser136 (pS136) and total Bad. α-tubulin was used as a loading control. Data represents the mean ± SEM from three independent experiments. * and # indicates statistical significance compared to EGF-only treatment (A and C) or untreated controls (B), p ≤ 0.05 and p ≤ 0.01, respectively. C, untreated control; (-) EGF treated control.
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Figure 4: Test compounds inhibit ERK-mediated phosphorylation of p90Rsk-1 and Bad. (A) HeLa cells were pre-treated for one hour in the presence or absence of 50 uM of the indicated test compounds and then stimulated with EGF (25 ng/ml) for 10 minutes. Immunoblots of phosphorylated p90Rsk-1 (pRsk), total Rsk (Rsk), phosphorylated ERK1/2 (ppERK), and total ERK2 (ERK2). α-tubulin was used as a loading control. Graph shows densitometry quantification of pRsk-1 to total Rsk or ppERK2 to ERK2 ratios. Data represents the mean ± SEM from three independent experiments. (B) In vitro kinase assays examining 32P incorporation into p90Rsk-1 following incubation with active ERK2 and γ-32P-ATP for 60 min. in the absence or presence of 1-25 μM of test compounds. Relative phosphate incorporation was quantified by phosphoimager analysis. (C) HeLa cells were serum starved overnight and pre-treated for 1 hr with 50 uM indicated test compounds, 10 mM U0126 (U), or 25 mM LY294002 (L) prior to stimulation with or without EGF (25 ng/ml). Immunoblot analysis of Bad phosphorylated on Ser112 (pS112) or Ser136 (pS136) and total Bad. α-tubulin was used as a loading control. Data represents the mean ± SEM from three independent experiments. * and # indicates statistical significance compared to EGF-only treatment (A and C) or untreated controls (B), p ≤ 0.05 and p ≤ 0.01, respectively. C, untreated control; (-) EGF treated control.

Mentions: ERK proteins provide a survival advantage by directly or indirectly phosphorylating and inactivating the Bcl-2 family members of pro-apoptotic proteins [3,4]. Bad is a pro-apoptotic Bcl-2 family member that is inactivated by phosphorylation at S112 by p90RSK-1 and S136 by Akt [4,35]. The intrinsic mechanisms of apoptosis induction by the test compounds were first examined by evaluating ERK-mediated phosphorylation of p90Rsk-1. Phosphorylation of p90Rsk-1 was inhibited by 40, 54, and 67% in the presence of 50 μM of test compound 76.2, 76.3, and 76.4, respectively (Figure 4A). As expected, 76.1, which was less effective in inhibiting cell proliferation, did not inhibit p90Rsk-1 phosphorylation (Figure 4A). It was also somewhat surprising that 76 did not affect p90Rsk-1 phosphorylation at this dose although 2 fold higher doses did inhibit p90Rsk-1 phosphorylation by 50% in our previous studies [17]. None of the compounds caused a statistically significant inhibition of ERK1/2 activation. These compounds are predicted to target ERK2 near the CD domain and may affect ERK activation by upstream MEK1/2 proteins, which reportedly interact with ERK through the CD domain [36]. Nonetheless, compounds 76.3, and 76.4 appeared to show selectivity for inhibiting ERK-mediated phosphorylation of p90Rsk-1 without inhibiting ERK activation. Similarly, 76.3 was the most potent inhibitor of phosphate incorporation into p90Rsk-1 as measured using in vitro kinase assays and statistically more potent than the parent compound 76 at 25 μM (Figure 4B).


Characterization of ERK docking domain inhibitors that induce apoptosis by targeting Rsk-1 and caspase-9.

Boston SR, Deshmukh R, Strome S, Priyakumar UD, MacKerell AD, Shapiro P - BMC Cancer (2011)

Test compounds inhibit ERK-mediated phosphorylation of p90Rsk-1 and Bad. (A) HeLa cells were pre-treated for one hour in the presence or absence of 50 uM of the indicated test compounds and then stimulated with EGF (25 ng/ml) for 10 minutes. Immunoblots of phosphorylated p90Rsk-1 (pRsk), total Rsk (Rsk), phosphorylated ERK1/2 (ppERK), and total ERK2 (ERK2). α-tubulin was used as a loading control. Graph shows densitometry quantification of pRsk-1 to total Rsk or ppERK2 to ERK2 ratios. Data represents the mean ± SEM from three independent experiments. (B) In vitro kinase assays examining 32P incorporation into p90Rsk-1 following incubation with active ERK2 and γ-32P-ATP for 60 min. in the absence or presence of 1-25 μM of test compounds. Relative phosphate incorporation was quantified by phosphoimager analysis. (C) HeLa cells were serum starved overnight and pre-treated for 1 hr with 50 uM indicated test compounds, 10 mM U0126 (U), or 25 mM LY294002 (L) prior to stimulation with or without EGF (25 ng/ml). Immunoblot analysis of Bad phosphorylated on Ser112 (pS112) or Ser136 (pS136) and total Bad. α-tubulin was used as a loading control. Data represents the mean ± SEM from three independent experiments. * and # indicates statistical significance compared to EGF-only treatment (A and C) or untreated controls (B), p ≤ 0.05 and p ≤ 0.01, respectively. C, untreated control; (-) EGF treated control.
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Figure 4: Test compounds inhibit ERK-mediated phosphorylation of p90Rsk-1 and Bad. (A) HeLa cells were pre-treated for one hour in the presence or absence of 50 uM of the indicated test compounds and then stimulated with EGF (25 ng/ml) for 10 minutes. Immunoblots of phosphorylated p90Rsk-1 (pRsk), total Rsk (Rsk), phosphorylated ERK1/2 (ppERK), and total ERK2 (ERK2). α-tubulin was used as a loading control. Graph shows densitometry quantification of pRsk-1 to total Rsk or ppERK2 to ERK2 ratios. Data represents the mean ± SEM from three independent experiments. (B) In vitro kinase assays examining 32P incorporation into p90Rsk-1 following incubation with active ERK2 and γ-32P-ATP for 60 min. in the absence or presence of 1-25 μM of test compounds. Relative phosphate incorporation was quantified by phosphoimager analysis. (C) HeLa cells were serum starved overnight and pre-treated for 1 hr with 50 uM indicated test compounds, 10 mM U0126 (U), or 25 mM LY294002 (L) prior to stimulation with or without EGF (25 ng/ml). Immunoblot analysis of Bad phosphorylated on Ser112 (pS112) or Ser136 (pS136) and total Bad. α-tubulin was used as a loading control. Data represents the mean ± SEM from three independent experiments. * and # indicates statistical significance compared to EGF-only treatment (A and C) or untreated controls (B), p ≤ 0.05 and p ≤ 0.01, respectively. C, untreated control; (-) EGF treated control.
Mentions: ERK proteins provide a survival advantage by directly or indirectly phosphorylating and inactivating the Bcl-2 family members of pro-apoptotic proteins [3,4]. Bad is a pro-apoptotic Bcl-2 family member that is inactivated by phosphorylation at S112 by p90RSK-1 and S136 by Akt [4,35]. The intrinsic mechanisms of apoptosis induction by the test compounds were first examined by evaluating ERK-mediated phosphorylation of p90Rsk-1. Phosphorylation of p90Rsk-1 was inhibited by 40, 54, and 67% in the presence of 50 μM of test compound 76.2, 76.3, and 76.4, respectively (Figure 4A). As expected, 76.1, which was less effective in inhibiting cell proliferation, did not inhibit p90Rsk-1 phosphorylation (Figure 4A). It was also somewhat surprising that 76 did not affect p90Rsk-1 phosphorylation at this dose although 2 fold higher doses did inhibit p90Rsk-1 phosphorylation by 50% in our previous studies [17]. None of the compounds caused a statistically significant inhibition of ERK1/2 activation. These compounds are predicted to target ERK2 near the CD domain and may affect ERK activation by upstream MEK1/2 proteins, which reportedly interact with ERK through the CD domain [36]. Nonetheless, compounds 76.3, and 76.4 appeared to show selectivity for inhibiting ERK-mediated phosphorylation of p90Rsk-1 without inhibiting ERK activation. Similarly, 76.3 was the most potent inhibitor of phosphate incorporation into p90Rsk-1 as measured using in vitro kinase assays and statistically more potent than the parent compound 76 at 25 μM (Figure 4B).

Bottom Line: Induction of HeLa cell apoptosis appeared to be through intrinsic mechanisms involving caspase-9 activation and decreased phosphorylation of the pro-apoptotic Bad protein.Further examination of the test compound's mechanism of action showed little effects on related MAP kinases or other cell survival proteins.These findings support the identification of a class of ERK-targeted molecules that can induce apoptosis in transformed cells by inhibiting ERK-mediated phosphorylation and inactivation of pro-apoptotic proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland 20 N. Pine St, Baltimore, MD 21201 USA.

ABSTRACT

Background: The extracellular signal-regulated kinase-1 and 2 (ERK1/2) proteins play an important role in cancer cell proliferation and survival. ERK1/2 proteins also are important for normal cell functions. Thus, anti-cancer therapies that block all ERK1/2 signaling may result in undesirable toxicity to normal cells. As an alternative, we have used computational and biological approaches to identify low-molecular weight compounds that have the potential to interact with unique ERK1/2 docking sites and selectively inhibit interactions with substrates involved in promoting cell proliferation.

Methods: Colony formation and water soluble tetrazolium salt (WST) assays were used to determine the effects of test compounds on cell proliferation. Changes in phosphorylation and protein expression in response to test compound treatment were examined by immunoblotting and in vitro kinase assays. Apoptosis was determined with immunoblotting and caspase activity assays.

Results: In silico modeling was used to identify compounds that were structurally similar to a previously identified parent compound, called 76. From this screen, several compounds, termed 76.2, 76.3, and 76.4 sharing a common thiazolidinedione core with an aminoethyl side group, inhibited proliferation and induced apoptosis of HeLa cells. However, the active compounds were less effective in inhibiting proliferation or inducing apoptosis in non-transformed epithelial cells. Induction of HeLa cell apoptosis appeared to be through intrinsic mechanisms involving caspase-9 activation and decreased phosphorylation of the pro-apoptotic Bad protein. Cell-based and in vitro kinase assays indicated that compounds 76.3 and 76.4 directly inhibited ERK-mediated phosphorylation of caspase-9 and the p90Rsk-1 kinase, which phosphorylates and inhibits Bad, more effectively than the parent compound 76. Further examination of the test compound's mechanism of action showed little effects on related MAP kinases or other cell survival proteins.

Conclusion: These findings support the identification of a class of ERK-targeted molecules that can induce apoptosis in transformed cells by inhibiting ERK-mediated phosphorylation and inactivation of pro-apoptotic proteins.

Show MeSH
Related in: MedlinePlus