ABSTRACT
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.
Subject(s)
Enzyme Activators/pharmacology , G1 Phase Cell Cycle Checkpoints/drug effects , S Phase Cell Cycle Checkpoints/drug effects , Signal Transduction/drug effects , eIF-2 Kinase/metabolism , Animals , Cluster Analysis , Cyclin D1/metabolism , DNA, Complementary/genetics , Drug Evaluation, Preclinical , Drug Interactions , Endoplasmic Reticulum Stress/drug effects , Enzyme Activation/drug effects , Enzyme Activators/chemistry , Eukaryotic Initiation Factor-2/metabolism , Humans , Mice , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Paclitaxel/pharmacology , Phosphorylation/drug effects , Retinoblastoma Protein/metabolism , Transcriptome/drug effects , Transcriptome/geneticsABSTRACT
The protein kinase Chk2 (checkpoint kinase 2) is a major effector of the replication checkpoint. Chk2 activation is initiated by phosphorylation of Thr68, in the serine-glutamine/threonine-glutamine cluster domain (SCD), by ATM. The phosphorylated SCD-segment binds to the FHA domain of a second Chk2 molecule, promoting dimerisation of the protein and triggering phosphorylation of the activation segment/T-loop in the kinase domain. We have now determined the structure of the kinase domain of human Chk2 in complexes with ADP and a small-molecule inhibitor debromohymenialdisine. The structure reveals a remarkable dimeric arrangement in which T-loops are exchanged between protomers, to form an active kinase conformation in trans. Biochemical data suggest that this dimer is the biologically active state promoted by ATM-phosphorylation, and also suggests a mechanism for dimerisation-driven activation of Chk2 by trans-phosphorylation.
Subject(s)
DNA Damage , Models, Molecular , Protein Serine-Threonine Kinases/chemistry , Trans-Activators , Adenosine Diphosphate/chemistry , Ataxia Telangiectasia Mutated Proteins , Azepines/chemistry , Catalytic Domain , Cell Cycle Proteins/chemistry , Checkpoint Kinase 2 , DNA-Binding Proteins/chemistry , Dimerization , Enzyme Activation , Humans , Phosphorylation , Protein Conformation , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary , Pyrroles/chemistry , Signal Transduction , Tumor Suppressor Proteins/chemistryABSTRACT
PURPOSE: To investigate pharmacokinetic-pharmacodynamic relationships for the trisubstituted aminopurine cyclin-dependent kinase inhibitors olomoucine, bohemine, and CYC202 (R-roscovitine; seliciclib) in the HCT116 human colon carcinoma model. EXPERIMENTAL DESIGN: The in vitro activity of the agents was determined in a human tumor panel using the sulforhodamine B assay. The concentration and time dependence was established in HCT116 cells. Molecular biomarkers, including RB phosphorylation and cyclin expression, were assessed by Western blotting. Pharmacokinetic properties were characterized in mice following analysis by liquid chromatography-tandem mass spectrometry. Based on these studies, a dosing regimen was developed for CYC202 that allowed therapeutic exposures in the HCT116 tumor xenograft. RESULTS: The antitumor potency of the agents in vitro was in the order olomoucine (IC50, 56 micromol/L) < bohemine (IC50, 27 micromol/L) < CYC202 (IC50, 15 micromol/L), corresponding to their activities as cyclin-dependent kinase inhibitors. Antitumor activity increased with exposure time up to 16 hours. The agents caused inhibition of RB and RNA polymerase II phosphorylation and depletion of cyclins. They exhibited relatively rapid clearance following administration to mice. CYC202 displayed the slowest clearance from plasma and the highest tumor uptake, with oral bioavailability of 86%. Oral dosing of CYC202 gave active concentrations in the tumor, modulation of pharmacodynamic markers, and inhibition of tumor growth. CONCLUSIONS: CYC202 showed therapeutic activity on human cancer cell lines in vitro and on xenografts. Pharmacodynamic markers are altered in vitro and in vivo, consistent with the inhibition of cyclin-dependent kinases. Such markers may be potentially useful in the clinical development of CYC202 and other cyclin-dependent kinase inhibitors.
Subject(s)
Cyclin-Dependent Kinases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/pharmacokinetics , Neoplasms, Experimental/metabolism , Animals , Area Under Curve , Blotting, Western , CDC2 Protein Kinase/antagonists & inhibitors , CDC2 Protein Kinase/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinases/metabolism , Female , HCT116 Cells , Humans , Inhibitory Concentration 50 , Kinetin/pharmacokinetics , Kinetin/pharmacology , Maximum Tolerated Dose , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasms, Experimental/pathology , Phosphorylation/drug effects , Purines/pharmacokinetics , Purines/pharmacology , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Retinoblastoma Protein/metabolism , Roscovitine , Time Factors , Tissue Distribution , Xenograft Model Antitumor AssaysABSTRACT
The tumor suppressor protein, pRb, regulates progression through the G1 phase of the cell cycle by its ability to bind to and regulate the activity of a variety of transcription factors. This function of pRb is disabled through its phosphorylation by the cyclin-dependent kinase (CDK) family of serine/threonine kinases. In many human cancers, genetic alteration such as loss of CDK inhibitor function and deregulated G1 cyclin expression leads to inappropriate phosphorylation and hence inactivation of this tumor suppressor. Identification of cell-permeable small molecules that block pRb phosphorylation in these tumors could therefore lead to development of an effective anticancer treatment. As a result, we have developed a high-throughput assay to detect changes in the level of pRb phosphorylation in cells. Signal detection is by a time-resolved fluorescence-based cellular immunosorbant assay on a fixed monolayer of cells. This comprises a mouse monoclonal antibody that recognizes the phosphorylated form of serine 608 on pRb, a known site of CDK phosphorylation, and a Europium-labeled secondary antibody for signal detection. The assay is reproducible and amenable to automation and has been used to screen 2000 compounds in a search for cell-permeable small molecules that will block pRb phosphorylation.
