ABSTRACT
To identify new chemical series with enhanced binding affinity to the BTB domain of B-cell lymphoma 6 protein, we targeted a subpocket adjacent to Val18. With no opportunities for strong polar interactions, we focused on attaining close shape complementarity by ring fusion onto our quinolinone lead series. Following exploration of different sized rings, we identified a conformationally restricted core which optimally filled the available space, leading to potent BCL6 inhibitors. Through X-ray structure-guided design, combined with efficient synthetic chemistry to make the resulting novel core structures, a >300-fold improvement in activity was obtained by the addition of seven heavy atoms.
Subject(s)
BTB-POZ Domain , Protein Binding , Proto-Oncogene Proteins c-bcl-6ABSTRACT
Histone demethylases are epigenetic modulators that play key roles in regulating gene expression related to many critical cellular functions and are emerging as promising therapeutic targets in a number of tumor types. We previously identified histone demethylase family members as overexpressed in the pediatric sarcoma, rhabdomyosarcoma. Here we show high sensitivity of rhabdomyosarcoma cells to a pan-histone demethylase inhibitor, JIB-04 and identify a key role for the histone demethylase KDM4B in rhabdomyosarcoma cell growth through an RNAi-screening approach. Decreasing KDM4B levels affected cell cycle progression and transcription of G1/S and G2/M checkpoint genes including CDK6 and CCNA2, which are bound by KDM4B in their promoter regions. However, after sustained knockdown of KDM4B, rhabdomyosarcoma cell growth recovered. We show that this can be attributed to acquired molecular compensation via recruitment of KDM4A to the promoter regions of CDK6 and CCNA2 that are otherwise bound by KDM4B. Furthermore, upfront silencing of both KDM4B and KDM4A led to RMS cell apoptosis, not seen by reducing either alone. To circumvent compensation and elicit stronger therapeutic responses, our study supports targeting histone demethylase sub-family proteins through selective poly-pharmacology as a therapeutic approach.
ABSTRACT
Residues in the histone substrate binding sites that differ between the KDM4 and KDM5 subfamilies were identified. Subsequently, a C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one series was designed to rationally exploit these residue differences between the histone substrate binding sites in order to improve affinity for the KDM4-subfamily over KDM5-subfamily enzymes. In particular, residues E169 and V313 (KDM4A numbering) were targeted. Additionally, conformational restriction of the flexible pyridopyrimidinone C8-substituent was investigated. These approaches yielded potent and cell-penetrant dual KDM4/5-subfamily inhibitors including 19a (KDM4A and KDM5B Kiâ¯=â¯0.004 and 0.007⯵M, respectively). Compound cellular profiling in two orthogonal target engagement assays revealed a significant reduction from biochemical to cell-based activity across multiple analogues; this decrease was shown to be consistent with 2OG competition, and suggests that sub-nanomolar biochemical potency will be required with C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one compounds to achieve sub-micromolar target inhibition in cells.
Subject(s)
Enzyme Inhibitors/pharmacology , Jumonji Domain-Containing Histone Demethylases/antagonists & inhibitors , Pyridines/pharmacology , Pyrimidinones/pharmacology , Cell Line, Tumor , Crystallography, X-Ray , Drug Screening Assays, Antitumor/methods , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Humans , Hydrophobic and Hydrophilic Interactions , Jumonji Domain-Containing Histone Demethylases/chemistry , Jumonji Domain-Containing Histone Demethylases/metabolism , Molecular Structure , Protein Binding , Pyridines/chemical synthesis , Pyridines/chemistry , Pyridines/metabolism , Pyrimidinones/chemical synthesis , Pyrimidinones/chemistry , Pyrimidinones/metabolism , Structure-Activity RelationshipABSTRACT
Multiparameter optimization of a series of 5-((4-aminopyridin-2-yl)amino)pyrazine-2-carbonitriles resulted in the identification of a potent and selective oral CHK1 preclinical development candidate with in vivo efficacy as a potentiator of deoxyribonucleic acid (DNA) damaging chemotherapy and as a single agent. Cellular mechanism of action assays were used to give an integrated assessment of compound selectivity during optimization resulting in a highly CHK1 selective adenosine triphosphate (ATP) competitive inhibitor. A single substituent vector directed away from the CHK1 kinase active site was unexpectedly found to drive the selective cellular efficacy of the compounds. Both CHK1 potency and off-target human ether-a-go-go-related gene (hERG) ion channel inhibition were dependent on lipophilicity and basicity in this series. Optimization of CHK1 cellular potency and in vivo pharmacokinetic-pharmacodynamic (PK-PD) properties gave a compound with low predicted doses and exposures in humans which mitigated the residual weak in vitro hERG inhibition.
Subject(s)
4-Aminopyridine/analogs & derivatives , Checkpoint Kinase 1/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pyrazines/pharmacology , 4-Aminopyridine/chemical synthesis , 4-Aminopyridine/chemistry , 4-Aminopyridine/pharmacology , Checkpoint Kinase 1/metabolism , Dose-Response Relationship, Drug , Humans , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrazines/chemical synthesis , Pyrazines/chemistry , Structure-Activity RelationshipABSTRACT
CCT245737 is the first orally active, clinical development candidate CHK1 inhibitor to be described. The IC50 was 1.4 nM against CHK1 enzyme and it exhibited>1,000-fold selectivity against CHK2 and CDK1. CCT245737 potently inhibited cellular CHK1 activity (IC50 30-220 nM) and enhanced gemcitabine and SN38 cytotoxicity in multiple human tumor cell lines and human tumor xenograft models. Mouse oral bioavailability was complete (100%) with extensive tumor exposure. Genotoxic-induced CHK1 activity (pS296 CHK1) and cell cycle arrest (pY15 CDK1) were inhibited both in vitro and in human tumor xenografts by CCT245737, causing increased DNA damage and apoptosis. Uniquely, we show CCT245737 enhanced gemcitabine antitumor activity to a greater degree than for higher doses of either agent alone, without increasing toxicity, indicating a true therapeutic advantage for this combination. Furthermore, development of a novel ELISA assay for pS296 CHK1 autophosphorylation, allowed the quantitative measurement of target inhibition in a RAS mutant human tumor xenograft of NSCLC at efficacious doses of CCT245737. Finally, CCT245737 also showed significant single-agent activity against a MYC-driven mouse model of B-cell lymphoma. In conclusion, CCT245737 is a new CHK1 inhibitor clinical development candidate scheduled for a first in man Phase I clinical trial, that will use the novel pS296 CHK1 ELISA to monitor target inhibition.
Subject(s)
4-Aminopyridine/analogs & derivatives , Carcinoma, Non-Small-Cell Lung/drug therapy , Checkpoint Kinase 1/drug effects , Lung Neoplasms/drug therapy , Lymphoma, B-Cell/drug therapy , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Pyrazines/pharmacology , Xenograft Model Antitumor Assays , 4-Aminopyridine/pharmacokinetics , 4-Aminopyridine/pharmacology , Animals , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Apoptosis/drug effects , CDC2 Protein Kinase , Camptothecin/analogs & derivatives , Camptothecin/pharmacology , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Checkpoint Kinase 1/metabolism , Checkpoint Kinase 2/antagonists & inhibitors , Cyclin-Dependent Kinases/antagonists & inhibitors , DNA Damage/drug effects , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Drug Synergism , HT29 Cells , Humans , Irinotecan , Mice , Mice, Inbred BALB C , Mice, Nude , Mice, Transgenic , Pyrazines/pharmacokinetics , GemcitabineABSTRACT
Inhibitors of checkpoint kinase 1 (CHK1) are of current interest as potential antitumor agents, but the most advanced inhibitor series reported to date are not orally bioavailable. A novel series of potent and orally bioavailable 3-alkoxyamino-5-(pyridin-2-ylamino)pyrazine-2-carbonitrile CHK1 inhibitors was generated by hybridization of two lead scaffolds derived from fragment-based drug design and optimized for CHK1 potency and high selectivity using a cell-based assay cascade. Efficient in vivo pharmacokinetic assessment was used to identify compounds with prolonged exposure following oral dosing. The optimized compound (CCT244747) was a potent and highly selective CHK1 inhibitor, which modulated the DNA damage response pathway in human tumor xenografts and showed antitumor activity in combination with genotoxic chemotherapies and as a single agent.
Subject(s)
Aminopyridines/pharmacology , Antineoplastic Agents/pharmacology , Colonic Neoplasms/drug therapy , Neuroblastoma/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Kinases/chemistry , Pyrimidines/pharmacology , Administration, Oral , Aminopyridines/chemical synthesis , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemical synthesis , Checkpoint Kinase 1 , Child , Colonic Neoplasms/enzymology , DNA Damage/drug effects , Drug Design , Humans , Mice , Mice, Nude , Mice, Transgenic , N-Myc Proto-Oncogene Protein , Neuroblastoma/enzymology , Nuclear Proteins/genetics , Oncogene Proteins/genetics , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemical synthesis , Protein Kinases/metabolism , Pyrimidines/chemical synthesis , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: Many tumors exhibit defective cell-cycle checkpoint control and increased replicative stress. CHK1 is critically involved in the DNA damage response and maintenance of replication fork stability. We have therefore discovered a novel potent, highly selective, orally active ATP-competitive CHK1 inhibitor, CCT244747, and present its preclinical pharmacology and therapeutic activity. EXPERIMENTAL DESIGN: Cellular CHK1 activity was assessed using an ELISA assay, and cytotoxicity a SRB assay. Biomarker modulation was measured using immunoblotting, and cell-cycle effects by flow cytometry analysis. Single-agent oral CCT244747 antitumor activity was evaluated in a MYCN-driven transgenic mouse model of neuroblastoma by MRI and in genotoxic combinations in human tumor xenografts by growth delay. RESULTS: CCT244747 inhibited cellular CHK1 activity (IC(50) 29-170 nmol/L), significantly enhanced the cytotoxicity of several anticancer drugs, and abrogated drug-induced S and G(2) arrest in multiple tumor cell lines. Biomarkers of CHK1 (pS296 CHK1) activity and cell-cycle inactivity (pY15 CDK1) were induced by genotoxics and inhibited by CCT244747 both in vitro and in vivo, producing enhanced DNA damage and apoptosis. Active tumor concentrations of CCT244747 were obtained following oral administration. The antitumor activity of both gemcitabine and irinotecan were significantly enhanced by CCT244747 in several human tumor xenografts, giving concomitant biomarker modulation indicative of CHK1 inhibition. CCT244747 also showed marked antitumor activity as a single agent in a MYCN-driven neuroblastoma. CONCLUSION: CCT244747 represents the first structural disclosure of a highly selective, orally active CHK1 inhibitor and warrants further evaluation alone or combined with genotoxic anticancer therapies.
Subject(s)
Aminopyridines/administration & dosage , Neoplasms, Experimental , Neuroblastoma , Protein Kinases , Pyrimidines/administration & dosage , Administration, Oral , Animals , Antineoplastic Agents/administration & dosage , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Cell Cycle Checkpoints/drug effects , Checkpoint Kinase 1 , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mice , Mice, Transgenic , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Neuroblastoma/drug therapy , Neuroblastoma/metabolism , Protein Kinase Inhibitors/administration & dosage , Protein Kinases/genetics , Protein Kinases/metabolismABSTRACT
PURPOSE: Deregulated phosphatidylinositol 3-kinase pathway signaling through AGC kinases including AKT, p70S6 kinase, PKA, SGK and Rho kinase is a key driver of multiple cancers. The simultaneous inhibition of multiple AGC kinases may increase antitumor activity and minimize clinical resistance compared with a single pathway component. EXPERIMENTAL DESIGN: We investigated the detailed pharmacology and antitumor activity of the novel clinical drug candidate AT13148, an oral ATP-competitive multi-AGC kinase inhibitor. Gene expression microarray studies were undertaken to characterize the molecular mechanisms of action of AT13148. RESULTS: AT13148 caused substantial blockade of AKT, p70S6K, PKA, ROCK, and SGK substrate phosphorylation and induced apoptosis in a concentration and time-dependent manner in cancer cells with clinically relevant genetic defects in vitro and in vivo. Antitumor efficacy in HER2-positive, PIK3CA-mutant BT474 breast, PTEN-deficient PC3 human prostate cancer, and PTEN-deficient MES-SA uterine tumor xenografts was shown. We show for the first time that induction of AKT phosphorylation at serine 473 by AT13148, as reported for other ATP-competitive inhibitors of AKT, is not a therapeutically relevant reactivation step. Gene expression studies showed that AT13148 has a predominant effect on apoptosis genes, whereas the selective AKT inhibitor CCT128930 modulates cell-cycle genes. Induction of upstream regulators including IRS2 and PIK3IP1 as a result of compensatory feedback loops was observed. CONCLUSIONS: The clinical candidate AT13148 is a novel oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity, which shows a distinct mechanism of action from other AKT inhibitors. AT13148 will now be assessed in a first-in-human phase I trial.
Subject(s)
Antineoplastic Agents/administration & dosage , Neoplasms , Phosphatidylinositol 3-Kinase/metabolism , Protein Kinase Inhibitors/administration & dosage , Apoptosis/drug effects , Apoptosis/genetics , Cell Line, Tumor , Gene Expression Regulation, Neoplastic/drug effects , Humans , Neoplasms/drug therapy , Neoplasms/metabolism , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation/drug effects , Pyrimidines/administration & dosage , Pyrroles/administration & dosage , Signal Transduction/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Pyrazolopyridine inhibitors with low micromolar potency for CHK1 and good selectivity against CHK2 were previously identified by fragment-based screening. The optimization of the pyrazolopyridines to a series of potent and CHK1-selective isoquinolines demonstrates how fragment-growing and scaffold morphing strategies arising from a structure-based understanding of CHK1 inhibitor binding can be combined to successfully progress fragment-derived hit matter to compounds with activity in vivo. The challenges of improving CHK1 potency and selectivity, addressing synthetic tractability, and achieving novelty in the crowded kinase inhibitor chemical space were tackled by multiple scaffold morphing steps, which progressed through tricyclic pyrimido[2,3-b]azaindoles to N-(pyrazin-2-yl)pyrimidin-4-amines and ultimately to imidazo[4,5-c]pyridines and isoquinolines. A potent and highly selective isoquinoline CHK1 inhibitor (SAR-020106) was identified, which potentiated the efficacies of irinotecan and gemcitabine in SW620 human colon carcinoma xenografts in nude mice.
Subject(s)
Antineoplastic Agents/chemical synthesis , Isoquinolines/chemical synthesis , Protein Kinase Inhibitors/chemical synthesis , Protein Kinases/metabolism , Pyrazines/chemical synthesis , Pyridines/chemical synthesis , Adenosine Triphosphate/chemistry , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Binding Sites , Biological Availability , Cell Line, Tumor , Checkpoint Kinase 1 , Crystallography, X-Ray , Drug Screening Assays, Antitumor , Humans , Isoquinolines/chemistry , Isoquinolines/pharmacology , Mice , Mice, Nude , Molecular Conformation , Neoplasm Transplantation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Pyrazines/chemistry , Pyrazines/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Stereoisomerism , Structure-Activity Relationship , Transplantation, HeterologousABSTRACT
CHK2 is a checkpoint kinase involved in the ATM-mediated response to double-strand DNA breaks. Its potential as a drug target is still unclear, but inhibitors of CHK2 may increase the efficacy of genotoxic cancer therapies in a p53 mutant background by eliminating one of the checkpoints or DNA repair pathways contributing to cellular resistance. We report here the identification and characterization of a novel CHK2 kinase inhibitor, CCT241533. X-ray crystallography confirmed that CCT241533 bound to CHK2 in the ATP pocket. This compound inhibits CHK2 with an IC(50) of 3 nmol/L and shows minimal cross-reactivity against a panel of kinases at 1 µmol/L. CCT241533 blocked CHK2 activity in human tumor cell lines in response to DNA damage, as shown by inhibition of CHK2 autophosphorylation at S516, band shift mobility changes, and HDMX degradation. CCT241533 did not potentiate the cytotoxicity of a selection of genotoxic agents in several cell lines. However, this compound significantly potentiates the cytotoxicity of two structurally distinct PARP inhibitors. Clear induction of the pS516 CHK2 signal was seen with a PARP inhibitor alone, and this activation was abolished by CCT241533, implying that the potentiation of PARP inhibitor cell killing by CCT241533 was due to inhibition of CHK2. Consequently, our findings imply that CHK2 inhibitors may exert therapeutic activity in combination with PARP inhibitors.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Quinazolines/pharmacology , Bleomycin/administration & dosage , Cell Cycle/drug effects , Cell Line, Tumor , Checkpoint Kinase 2 , Crystallography, X-Ray , Drug Synergism , Enzyme Activation/drug effects , HCT116 Cells , HT29 Cells , HeLa Cells , Heterocyclic Compounds, 3-Ring/administration & dosage , Heterocyclic Compounds, 3-Ring/pharmacology , Humans , Models, Molecular , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Quinazolines/administration & dosage , Quinazolines/chemistryABSTRACT
AKT is frequently deregulated in cancer, making it an attractive anticancer drug target. CCT128930 is a novel ATP-competitive AKT inhibitor discovered using fragment- and structure-based approaches. It is a potent, advanced lead pyrrolopyrimidine compound exhibiting selectivity for AKT over PKA, achieved by targeting a single amino acid difference. CCT128930 exhibited marked antiproliferative activity and inhibited the phosphorylation of a range of AKT substrates in multiple tumor cell lines in vitro, consistent with AKT inhibition. CCT128930 caused a G(1) arrest in PTEN-null U87MG human glioblastoma cells, consistent with AKT pathway blockade. Pharmacokinetic studies established that potentially active concentrations of CCT128930 could be achieved in human tumor xenografts. Furthermore, CCT128930 also blocked the phosphorylation of several downstream AKT biomarkers in U87MG tumor xenografts, indicating AKT inhibition in vivo. Antitumor activity was observed with CCT128930 in U87MG and HER2-positive, PIK3CA-mutant BT474 human breast cancer xenografts, consistent with its pharmacokinetic and pharmacodynamic properties. A quantitative immunofluorescence assay to measure the phosphorylation and total protein expression of the AKT substrate PRAS40 in hair follicles is presented. Significant decreases in pThr246 PRAS40 occurred in CCT128930-treated mouse whisker follicles in vivo and human hair follicles treated ex vivo, with minimal changes in total PRAS40. In conclusion, CCT128930 is a novel, selective, and potent AKT inhibitor that blocks AKT activity in vitro and in vivo and induces marked antitumor responses. We have also developed a novel biomarker assay for the inhibition of AKT in human hair follicles, which is currently being used in clinical trials.
Subject(s)
Antineoplastic Agents , Biomarkers, Pharmacological/analysis , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/pharmacokinetics , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Pyrimidines/pharmacology , Pyrimidines/pharmacokinetics , Pyrroles/pharmacology , Pyrroles/pharmacokinetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , HCT116 Cells , Hair Follicle/metabolism , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasms/enzymology , Protein Kinase Inhibitors/chemistry , Pyrimidines/chemistry , Pyrroles/chemistry , Xenograft Model Antitumor AssaysABSTRACT
Genotoxic antitumor agents continue to be the mainstay of current cancer chemotherapy. These drugs cause DNA damage and activate numerous cell cycle checkpoints facilitating DNA repair and the maintenance of genomic integrity. Most human tumors lack functional p53 and consequently have compromised G(1)-S checkpoint control. This has led to the hypothesis that S and G(2)-M checkpoint abrogation may selectively enhance genotoxic cell killing in a p53-deficient background, as normal cells would be rescued at the G(1)-S checkpoint. CHK1 is a serine/threonine kinase associated with DNA damage-linked S and G(2)-M checkpoint control. SAR-020106 is an ATP-competitive, potent, and selective CHK1 inhibitor with an IC(50) of 13.3 nmol/L on the isolated human enzyme. This compound abrogates an etoposide-induced G(2) arrest with an IC(50) of 55 nmol/L in HT29 cells, and significantly enhances the cell killing of gemcitabine and SN38 by 3.0- to 29-fold in several colon tumor lines in vitro and in a p53-dependent fashion. Biomarker studies have shown that SAR-020106 inhibits cytotoxic drug-induced autophosphorylation of CHK1 at S296 and blocks the phosphorylation of CDK1 at Y15 in a dose-dependent fashion both in vitro and in vivo. Cytotoxic drug combinations were associated with increased gammaH2AX and poly ADP ribose polymerase cleavage consistent with the SAR-020106-enhanced DNA damage and tumor cell death. Irinotecan and gemcitabine antitumor activity was enhanced by SAR-020106 in vivo with minimal toxicity. SAR-020106 represents a novel class of CHK1 inhibitors that can enhance antitumor activity with selected anticancer drugs in vivo and may therefore have clinical utility.
Subject(s)
Isoquinolines/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Pyrazines/pharmacology , Xenograft Model Antitumor Assays , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Biomarkers, Tumor/metabolism , Camptothecin/administration & dosage , Camptothecin/analogs & derivatives , Camptothecin/pharmacology , Cell Death/drug effects , Cell Line, Tumor , Checkpoint Kinase 1 , DNA Damage , Deoxycytidine/administration & dosage , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Drug Synergism , G2 Phase/drug effects , Humans , Irinotecan , Isoquinolines/administration & dosage , Isoquinolines/chemistry , Isoquinolines/pharmacokinetics , Mice , Mice, Nude , Mutagens/toxicity , Phosphorylation/drug effects , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Pyrazines/administration & dosage , Pyrazines/chemistry , Pyrazines/pharmacokinetics , GemcitabineABSTRACT
Cyclin dependent kinase 4 is a key regulator of the cell cycle and its activity is frequently deregulated in cancer. The activity of cyclin dependent kinase 4 is controlled by multiple mechanisms, including phosphorylation of tyrosine 17. This site is equivalent to tyrosine 15 of cyclin dependent kinase 1, which undergoes inhibitory phosphorylation by WEE1 and MYT1; however, the kinases that phosphorylate cyclin dependent kinase 4 on tyrosine 17 are still unknown. In the present study, we generated a phosphospecific antibody to the tyrosine 17-phosphorylated form of cyclin dependent kinase 4, and showed that this site is phosphorylated to a low level in asynchronously proliferating HCT116 cells. We purified tyrosine 17 kinases from HeLa cells and found that the Src family non-receptor tyrosine kinase C-YES contributes a large fraction of the tyrosine 17 kinase activity in HeLa lysates. C-YES also phosphorylated cyclin dependent kinase 4 when transfected into HCT116 cells, and treatment of cells with Src family kinase inhibitors blocked the tyrosine 17 phosphorylation of cyclin dependent kinase 4. Taken together, the results obtained in the present study provide the first evidence that Src family kinases, but not WEE1 or MYT1, phosphorylate cyclin dependent kinase 4 on tyrosine 17, and help to resolve how the phosphorylation of this site is regulated.
Subject(s)
Cyclin-Dependent Kinase 4/metabolism , Tyrosine/metabolism , src-Family Kinases/metabolism , Amino Acid Sequence , Cell Cycle Proteins/metabolism , Cyclin-Dependent Kinase 4/chemistry , HeLa Cells , Humans , Membrane Proteins , Molecular Sequence Data , Nuclear Proteins/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins c-yes/isolation & purification , Proto-Oncogene Proteins c-yes/metabolism , Sequence Homology, Amino AcidABSTRACT
The P2X7 receptor, a plasma membrane ATP-gated ion channel that plays a role in lymphocyte apoptosis, has been suggested to be involved in the development of chronic lymphocytic leukemia (CLL). P2X7 is polymorphic with 1513A and 1513C alleles encoding fully active and nonfunctional proteins, respectively. We evaluated the significance of the P2X7-A1513C polymorphism on CLL risk by genotyping 424 patients and 428 healthy controls. To empower detection of an association, we included in our analysis 106 familial cases. Allele frequencies were identical in cases and controls irrespective of whether cases were familial or sporadic (frequency of the C allele was 0.17 and 0.17, respectively). The odds ratio of CLL associated with the C allele was 1.03 (95% confidence interval: 0.80-1.31). A meta-analysis of this study and five other smaller published studies provides no evidence of relationship between this P2X7 polymorphism and risk of CLL (odds ratio = 0.99, 95% confidence interval: 0.74-1.32).