Mechanism-Based Screen for G1/S Checkpoint Activators Identifies a Selective Activator of EIF2AK3/PERK Signalling

Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes

Mechanism-based screen for G1/S checkpoint activators identifies a selective activator of EIF2AK3/PERK signalling.

Human cancers often contain genetic alterations that disable G1/S checkpoint control and loss of this checkpoint is thought to critically contribute to cancer generation by permitting inappropriate proliferation and distorting fate-driven cell cycle exit. The identification of cell permeable small molecules that activate the G1/S checkpoint may therefore represent a broadly applicable and clinically effective strategy for the treatment of cancer. Here we describe the identification of several novel small molecules that trigger G1/S checkpoint activation and characterise the mechanism of action for one, CCT020312, in detail. Transcriptional profiling by cDNA microarray combined with reverse genetics revealed phosphorylation of the eukaryotic initiation factor 2-alpha (EIF2A) through the eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3/PERK) as the mechanism of action of this compound. While EIF2AK3/PERK activation classically follows endoplasmic reticulum (ER) stress signalling that sets off a range of different cellular responses, CCT020312 does not trigger these other cellular responses but instead selectively elicits EIF2AK3/PERK signalling. Phosphorylation of EIF2A by EIF2A kinases is a known means to block protein translation and hence restriction point transit in G1, but further supports apoptosis in specific contexts. Significantly, EIF2AK3/PERK signalling has previously been linked to the resistance of cancer cells to multiple anticancer chemotherapeutic agents, including drugs that target the ubiquitin/proteasome pathway and taxanes. Consistent with such findings CCT020312 sensitizes cancer cells with defective taxane-induced EIF2A phosphorylation to paclitaxel treatment. Our work therefore identifies CCT020312 as a novel small molecule chemical tool for the selective activation of EIF2A-mediated translation control with utility for proof-of-concept applications in EIF2A-centered therapeutic approaches, and as a chemical starting point for pathway selective agent development. We demonstrate that consistent with its mode of action CCT020312 is capable of delivering potent, and EIF2AK3 selective, proliferation control and can act as a sensitizer to chemotherapy-associated stresses as elicited by taxanes

AHR- and DNA-damage-mediated gene expression responses induced by benzo(a)pyrene in human cell lines

Carcinogens induce complex transcriptional responses in cells that may hold key mechanistic information. Benzo(a)pyrene (BaP) modulation of transcription may occur through the activation of the aryl hydrocarbon receptor (AHR) or through responses to DNA damage. To characterize further the expression profiles induced by BaP in HepG2 and MCF-7 cells obtained in our previous study, they were compared to those induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which activates AHR but does not bind to DNA, and anti-benzo(a)pyrene- trans-7,8-dihydrodiol-9,10-epoxide (BPDE), which binds directly to DNA but does not activate AHR. A total of 22 genes had altered expression in MCF-7 cells after both BaP and TCDD exposure, and a total of 29 genes had altered expression in HepG2 cells. In both cell lines, xenobiotic metabolism was upregulated through induction of NQO1, MGST1, and CYP1B1. A total of 78 expression changes were induced by both BaP and BPDE in MCF-7 cells, and a total of 29 expression changes were induced by both BaP and BPDE in HepG2 cells. These genes were predominantly involved in cell cycle regulation, apoptosis, and DNA repair. BaP and BPDE caused the repression of histone genes in both cell lines, suggesting that regulation of these genes is an important component of the DNA damage response. Interestingly, overlap of the BPDE and TCDD gene expression profiles was also observed. Furthermore, some genes were modulated by BaP but not by TCDD or BPDE, including induction of CRY1 and MAK, which may represent novel signaling pathways that are independent of both AHR activation and DNA damage. Promoter analysis identified candidate genes for direct transcriptional regulation by either AHR or p53. These analyses have further dissected and characterized the complex cellular response to BaP

3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 Contains a Phosphatidylinositol Cofactor

3-Hydroxybenzoate 6-hydroxylase (3HB6H, EC 1.13.14.26) is a FAD-dependent monooxygenase involved in the catabolism of aromatic compounds in soil microorganisms. 3HB6H is unique among flavoprotein hydroxylases in that it harbors a phospholipid ligand. The purified protein obtained from expressing the gene encoding 3HB6H from Rhodococcus jostii RHA1 in the host Escherichia coli contains a mixture of phosphatidylglycerol and phosphatidylethanolamine, which are the major constituents of E. coli's cytoplasmic membrane. Here, we purified 3HB6H (RjHB6H) produced in the host R. jostii RHA#2 by employing a newly developed actinomycete expression system. Biochemical and biophysical analysis revealed that Rj3HB6H possesses similar catalytic and structural features as 3HB6H, but now contains phosphatidylinositol, which is a specific constituent of actinomycete membranes. Native mass spectrometry suggests that the lipid cofactor stabilizes monomer-monomer contact. Lipid analysis of 3HB6H from Pseudomonas alcaligenes NCIMB 9867 (Pa3HB6H) produced in E. coli supports the conclusion that 3HB6H enzymes have an intrinsic ability to bind phospholipids with different specificity, reflecting the membrane composition of their bacterial host

3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1 contains a phosphatidylinositol cofactor

3-Hydroxybenzoate 6-hydroxylase (3HB6H, EC 1.13.14.26) is a FAD-dependent monooxygenase involved in the catabolism of aromatic compounds in soil microorganisms. 3HB6H is unique among flavoprotein hydroxylases in that it harbors a phospholipid ligand. The purified protein obtained from expressing the gene encoding 3HB6H from Rhodococcus jostii RHA1 in the host Escherichia coli contains a mixture of phosphatidylglycerol and phosphatidylethanolamine, which are the major constituents of E. coli's cytoplasmic membrane. Here, we purified 3HB6H (RjHB6H) produced in the host R. jostii RHA#2 by employing a newly developed actinomycete expression system. Biochemical and biophysical analysis revealed that Rj3HB6H possesses similar catalytic and structural features as 3HB6H, but now contains phosphatidylinositol, which is a specific constituent of actinomycete membranes. Native mass spectrometry suggests that the lipid cofactor stabilizes monomer-monomer contact. Lipid analysis of 3HB6H from Pseudomonas alcaligenes NCIMB 9867 (Pa3HB6H) produced in E. coli supports the conclusion that 3HB6H enzymes have an intrinsic ability to bind phospholipids with different specificity, reflecting the membrane composition of their bacterial host.</p

Interaction of CCT020312 with paclitaxel.

<p><b>A) Paclitaxel-associated EIF2A phosphorylation in U-2OS and HCT116 cells.</b> U-2OS and HCT116 were exposed to 10 µM CCT020312 (CCT), DMSO or paclitaxel (taxol) for 4 hours. Cell lysates were analyzed by immunoblotting for P-S51-EIF2A and tubulin. <b>B) Proliferation inhibition by paclitaxel in the presence of CCT020312.</b> Cells were treated with increasing amounts (1–25 nM) of paclitaxel in the absence and presence of a fixed dose (2.5 µM) of CCT020312. Arrows denote proliferation inhibition in cells treated with 2.5 µM CCT020312 only. Cell lines were as indicated. <b>C) Multiple drugs effect analysis:</b> U-2OS and HCT116 cells were treated with escalating doses of paclitaxel, CCT020312 or their combination. Combination indices (CIs) for each dose are shown. The calculated mean CI and standard error is indicated. “CI excl” assumes agents act by a competing, mutually exclusive mechanism of action, “CI no-exl. assumes agents act through distinct, exclusive mechanisms of action <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028568#pone.0028568-Chou2" target="_blank">[57]</a>.</p

Cellular responses to CCT020312 treatment.

<p><b>A) Concentration-dependence of P-S608-pRB loss in CCT020312-exposed cells.</b> HT29 cells seeded in 96-well plates were exposed to CCT020312 for 24 hours. Ser608 pRB phosphorylation was quantified using the cell-based immunoassay for the detection of pRB-P-Ser608 as employed for the primary screen (Barrie et al. 2003). Signals normalized to protein content (BCA assay) are shown. Error bars represent the standard error of the mean (n−3). The range for linear response is indicated. <b>B) C) Effects of CCT020312 on cell cycle progression and DNA synthesis.</b> HT29 cells were treated for 16 and 24 hours with 10 µM CCT020312. Cells were stained with propidium iodide and analysed by flow-cytometry (B). Cells were treated with CCT020312 for 16 or 24 hours. BrdU was added to the medium for the final two hours. Cells were stained with anti-BrdU antibody and analysed by flow-cytometry (C). <b>D) Accumulation of Ser608 unphosphorylated pRB in CCT020312 exposed cells.</b> HT29 cells were incubated in the presence of the vehicle (MOCK) or CCT020312 for 24 hours and analysed by immunoblotting. NP-pRB denotes use of the antibody for detection of the non-phosphorylated Ser608 pRB site. <b>E) Marker expression 24 h post CCT020312 exposure.</b> HT29 cells were exposed to 10 µM CCT020312 or vehicle (MOCK) for 24 hours. Lysates were analysed by immunoblot for marker proteins as indicated. Membrane staining with amido black documents loading. <b>F) CCT020312 induces a rapid loss of D cyclin expression.</b> HT29 cells were treated with 10 µM CCT020312 for the times indicated and lysates analyzed as in E. Tubulin probing documents loading.</p

EIF2A phosphorylation in CCT020312-treated cells.

<p><b>A) Detection of EIF2A phosphorylation following CCT020312 treatment.</b> HT29 human colon cancer and MCF-7 human breast cancer cells were treated with 10 µM CCT020312 (+) or vehicle () for the times indicated. Lysates were prepared and immunoblot-analysis performed using antibodies to detect EIF2A and Ser51-phosphorylated EIF2A (P-S51EIF2A) as indicated. <b>B) Structure-activity relationships.</b> Representatives from different chemical series were tested using HT29 cells for their respective ability to activate EIF2A phosphorylation. <b>C) Analogue structures and potency of analogues for reducing P-S608-pRB and growth.</b></p