ABSTRACT
Analogues of (dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one (NU7441), a potent inhibitor of DNA-dependent protein kinase (DNA-PK; IC50 = 42 ± 2 nM), have been synthesized in which water-solubilizing groups [NHCO(CH2)nNR¹R², where n = 1 or 2 and the moiety R¹R²N was derived from a library of primary and secondary amines, e.g., morpholine] were placed at the 1-position. Several of the newly synthesized compounds exhibited high potency against DNA-PK and potentiated the cytotoxicity of ionizing radiation (IR) in vitro 10-fold or more (e.g., 2-(4-ethylpiperazin-1-yl)-N-(4-(2-morpholino-4-oxo-4H-chromen-8-yl)dibenzo[b,d]thio-phen-1-yl)acetamide, 39; DNA-PK IC50 = 5.0 ± 1 nM, IR dose modification ratio = 13). Furthermore, 39 was shown to potentiate not only IR in vitro but also DNA-inducing cytotoxic anticancer agents, both in vitro and in vivo. Counter-screening against other members of the phosphatidylinositol 3-kinase (PI-3K) related kinase (PIKK) family unexpectedly revealed that some of the compounds were potent mixed DNA-PK and PI-3K inhibitors.
Subject(s)
DNA-Activated Protein Kinase/antagonists & inhibitors , Morpholines/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/pharmacology , HeLa Cells , Humans , Morpholines/chemistryABSTRACT
Substitution at the 7-position of the chromen-4-one pharmacophore of 8-(dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one NU7441, a potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor, with allyl, n-propyl or methyl enabled the resolution by chiral HPLC of atropisomers. Biological evaluation against DNA-PK of each pair of atropisomers showed a marked difference in potency, with biological activity residing exclusively in the laevorotatory enantiomer.
Subject(s)
Chromones/chemistry , Chromones/pharmacology , DNA-Activated Protein Kinase/antagonists & inhibitors , Morpholines/chemistry , Morpholines/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Animals , Binding Sites , Chromones/chemical synthesis , DNA-Activated Protein Kinase/chemistry , DNA-Activated Protein Kinase/metabolism , Humans , Models, Molecular , Morpholines/chemical synthesis , Protein Kinase Inhibitors/chemical synthesis , Stereoisomerism , Structure-Activity Relationship , SwineABSTRACT
DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by topoisomerase II poisons. Nonhomologous end joining (NHEJ) is a major pathway for DSB repair and requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK catalytic subunit (DNA-PKcs) is structurally similar to PI-3K, which promotes cell survival and proliferation and is upregulated in many cancers. KU-0060648 is a dual inhibitor of DNA-PK and PI-3K in vitro. KU-0060648 was investigated in a panel of human breast and colon cancer cells. The compound inhibited cellular DNA-PK autophosphorylation with IC(50) values of 0.019 µmol/L (MCF7 cells) and 0.17 µmol/L (SW620 cells), and PI-3K-mediated AKT phosphorylation with IC(50) values of 0.039 µmol/L (MCF7 cells) and more than 10 µmol/L (SW620 cells). Five-day exposure to 1 µmol/L KU-0060648 inhibited cell proliferation by more than 95% in MCF7 cells but only by 55% in SW620 cells. In clonogenic survival assays, KU-0060648 increased the cytotoxicity of etoposide and doxorubicin across the panel of DNA-PKcs-proficient cells, but not in DNA-PKcs-deficient cells, thus confirming that enhanced cytotoxicity was due to DNA-PK inhibition. In mice bearing SW620 and MCF7 xenografts, concentrations of KU-0060648 that were sufficient for in vitro growth inhibition and chemosensitization were maintained within the tumor for at least 4 hours at nontoxic doses. KU-0060648 alone delayed the growth of MCF7 xenografts and increased etoposide-induced tumor growth delay in both in SW620 and MCF7 xenografts by up to 4.5-fold, without exacerbating etoposide toxicity to unacceptable levels. The proof-of-principle in vitro and in vivo chemosensitization with KU-0060648 justifies further evaluation of dual DNA-PK and PI-3K inhibitors.
Subject(s)
Antineoplastic Agents/pharmacology , Chromones/pharmacology , DNA-Activated Protein Kinase/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Thiophenes/pharmacology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Chromones/administration & dosage , Drug Resistance, Neoplasm , Enzyme Activation , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/pharmacokinetics , Female , Humans , MCF-7 Cells , Mice , Neoplasms/drug therapy , Neoplasms/metabolism , Thiophenes/administration & dosage , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Introduction of an O-alkoxyphenyl substituent at the 8-position of the 2-morpholino-4H-chromen-4-one pharmacophore enabled regions of the ATP-binding site of DNA-dependent protein kinase (DNA-PK) to be probed further. Structure-activity relationships have been elucidated for inhibition of DNA-PK and PI3K (p110α), with N-(2-(cyclopropylmethoxy)-4-(2-morpholino-4-oxo-4H-chromen-8-yl)phenyl)-2-morpholinoacetamide 11a being identified as a potent and selective DNA-PK inhibitor (IC(50)=8 nM).
Subject(s)
Chromones/chemistry , DNA-Activated Protein Kinase/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Adenosine Triphosphate/chemistry , Chromones/chemical synthesis , Chromones/pharmacology , DNA-Activated Protein Kinase/metabolism , Humans , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Binding , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Structure, Tertiary , Structure-Activity RelationshipABSTRACT
Replacement of the core heterocycle of a defined series of chromen-4-one DNA-PK inhibitors by the isomeric chromen-2-one (coumarin) and isochromen-1-one (isocoumarin) scaffolds was investigated. Structure-activity relationships for DNA-PK inhibition were broadly consistent, albeit with a reduction of potency compared with the parent chromenone.
Subject(s)
Adenosine Triphosphate/metabolism , Coumarins/antagonists & inhibitors , DNA-Activated Protein Kinase/chemistry , Isocoumarins/antagonists & inhibitors , Antineoplastic Agents , Binding Sites , Chromones , DNA-Activated Protein Kinase/metabolism , Humans , Inhibitory Concentration 50 , Structure-Activity RelationshipABSTRACT
Hypoxia inducible factor-1 (HIF-1) is as a key transcriptional mediator of the hypoxic response in eukaryotic cells, regulating the expression of a myriad of genes involved in oxygen transport, glucose uptake and glycolysis and angiogenesis. Deregulation of HIF-1 activity occurs in many human cancers, usually at the level of the HIF-1alpha subunit. HIF-1 is regulated by a variety of mechanisms including transcription, translation post-translational modification, protein-protein interaction and degradation. Our understanding of the key signalling pathways that regulate HIF-1 has significantly progressed in recent years and has highlighted the potential for targeting the HIF-1 pathway as a basis for the development of new cancer therapies.
Subject(s)
DNA-Binding Proteins/genetics , Gene Expression Regulation , Nuclear Proteins/genetics , Transcription Factors/genetics , Animals , Cell Hypoxia , Helix-Loop-Helix Motifs , Humans , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Transcription, GeneticABSTRACT
The hypoxia-inducible factor 1 (HIF-1) transcriptional complex is regulated by cellular oxygen levels and growth factors. The phosphoinosotide 3-kinase (PI-3K)-Akt/protein kinase B (PKB) pathway has been shown to regulate HIF-1 activity in response to oncogenic signals and growth factors. We assessed whether the HDM2 oncoprotein, a direct target of Akt/PKB, could regulate HIF-1alpha expression and HIF-1 activity under normoxic conditions. We found that growth factor stimulation, overexpression of Akt/PKB, or loss of PTEN resulted in enhanced expression of both HIF-1alpha and HDM2. Growth factor-mediated induction of HIF-1alpha was ablated by transient expression of a dominant negative form of Akt/PKB or by treatment with LY294002. Transient expression of HDM2 led to increased expression of HIF-1alpha. Pulse-chase and cycloheximide experiments revealed that HDM2 did not significantly affect the half-life of HIF-1alpha. Growth factor-induced HIF-1alpha and HDM2 proteins were localized to the nucleus, and induction of both proteins was observed in both p53(+/+) and p53(-/-) HCT116 cells to comparable levels. Importantly, insulin-like growth factor 1-induced HIF-1alpha expression was observed in p53-null mouse embryo fibroblasts (MEFs) but was significantly impaired in p53 Mdm2 double-null MEFs, indicating a requirement for Mdm2 in this process. Finally, we showed that phosphorylation at Ser166 in HDM2 contributed in part to growth factor-mediated induction of HIF-1alpha. Our study has important implications for the role of the PI-3K-Akt/PKB-HDM2 pathway in tumor progression and angiogenesis.
Subject(s)
Gene Expression Regulation , Growth Substances/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/metabolism , Transcription Factors/metabolism , Animals , Cell Line, Tumor , Chromones/metabolism , Enzyme Inhibitors/metabolism , Genes, Reporter , Humans , Hypoxia-Inducible Factor 1, alpha Subunit , Mice , Morpholines/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-akt , Proto-Oncogene Proteins c-mdm2 , Transcription Factors/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
An understanding of underlying mechanisms involved in the activation of HIF-1 in response to both hypoxic stress and oncogenic signals has important implications for how these processes may become deregulated in human cancer. Changes in microenvironmental stimuli such as hypoxia and growth factors in combination with genetic lesions, such as loss or inactivation of p53, PTEN or pVHL or oncogenic activation, can all lead to increased HIF-1 activity. This provides cancer cells with a distinct advantage for survival and proliferation, resulting in their ability to form vascular tumours, which are aggressive and metastatic. Accordingly, upregulation of HIF-1alpha, a key component of HIF-1, correlates with a poor treatment outcome using conventional therapies. A variety of mechanisms exist that regulate expression of HIF-1alpha. In recent years, it has become clear that an extensive network of signalling cascades converge on HIF-1alpha to regulate the transcriptional response. A better understanding of this regulation may provide a basis for the development of new cancer therapies.