ABSTRACT
BACKGROUND: Eight human catalytic phosphoinositide 3-kinase (PI3K) isoforms exist which are subdivided into three classes. While class I isoforms have been well-studied in cancer, little is known about the functions of class II PI3Ks. MATERIALS AND METHODS: The expression pattern and functions of the class II PI3KC2ß isoform were investigated in a panel of tumour samples and cell lines. RESULTS: Overexpression of PI3KC2ß was found in subsets of tumours and cell lines from acute myeloid leukemia (AML), glioblastoma multiforme (GBM), medulloblastoma (MB), neuroblastoma (NB), and small cell lung cancer (SCLC). Specific pharmacological inhibitors of PI3KC2ß or RNA interference impaired proliferation of a panel of human cancer cell lines and primary cultures. Inhibition of PI3KC2ß also induced apoptosis and sensitised the cancer cells to chemotherapeutic agents. CONCLUSION: Together, these data show that PI3KC2ß contributes to proliferation and survival in AML, brain tumours and neuroendocrine tumours, and may represent a novel target in these malignancies.
Subject(s)
Brain Neoplasms , Cerebellar Neoplasms , Leukemia, Myeloid, Acute , Neuroendocrine Tumors , Acute Disease , Brain Neoplasms/drug therapy , Brain Neoplasms/genetics , Cell Line, Tumor , Cell Proliferation , Cerebellar Neoplasms/drug therapy , Cerebellar Neoplasms/genetics , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Lung Neoplasms , Neuroendocrine Tumors/drug therapy , Neuroendocrine Tumors/genetics , Phosphatidylinositol 3-Kinases/metabolismABSTRACT
The recently discovered enzyme tyrosyl-DNA phosphodiesterase 2 (TDP2) has been implicated in the topoisomerase-mediated repair of DNA damage. In the clinical setting, it has been hypothesized that TDP2 may mediate drug resistance to topoisomerase II (topo II) inhibition by etoposide. Therefore, selective pharmacological inhibition of TDP2 is proposed as a novel approach to overcome intrinsic or acquired resistance to topo II-targeted drug therapy. Following a high-throughput screening (HTS) campaign, toxoflavins and deazaflavins were identified as the first reported sub-micromolar and selective inhibitors of this enzyme. Toxoflavin derivatives appeared to exhibit a clear structure-activity relationship (SAR) for TDP2 enzymatic inhibition. However, we observed a key redox liability of this series, and this, alongside early in vitro drug metabolism and pharmacokinetics (DMPK) issues, precluded further exploration. The deazaflavins were developed from a singleton HTS hit. This series showed distinct SAR and did not display redox activity; however low cell permeability proved to be a challenge.
Subject(s)
Phosphoric Diester Hydrolases/drug effects , Pyrimidinones/pharmacology , Topoisomerase II Inhibitors/pharmacology , Triazines/pharmacology , Structure-Activity Relationship , Topoisomerase II Inhibitors/chemistryABSTRACT
Topoisomerases regulate DNA topology by the transient cleavage and religation of DNA during transcription and replication. Topoisomerase II (Topo II) poisons such as etoposide can induce abortive DNA strand breaks in which Topo II remains covalently bound to a 5' DNA strand terminus via a phosphotyrosyl linker. Tyrosyl DNA phosphodiesterase 2 (Tdp2) is a recently discovered human 5'-tyrosyl DNA phosphodiesterase that repairs this topoisomerase-mediated DNA damage, thereby playing a central role in maintaining normal DNA topology in cells. Cellular depletion of Tdp2 has been shown to result in increased susceptibility and sensitivity to Topo II-induced DNA double-strand breaks, thereby revealing Tdp2 as a potentially attractive anticancer target. No drug-like inhibitors of Tdp2 have been identified to date, and assays suitable for high-throughput screening (HTS) have not been widely reported. Here we have identified a new and effective chromogenic substrate for Tdp2 and developed a homogeneous and robust HTS assay. A second novel Tdp2 assay was also developed to cross-validate hit matter identified from an HTS. In addition, a new and specific Tdp2 antibody is described. Together, these new tools will aid in the identification of novel Tdp2 inhibitors and the investigation of the role of Tdp2 in cancer.
Subject(s)
Antibodies/immunology , High-Throughput Screening Assays/methods , Phosphodiesterase Inhibitors/pharmacology , Phosphoric Diester Hydrolases/analysis , Phosphoric Diester Hydrolases/immunology , Base Sequence , Cell Line, Tumor , Drug Evaluation, Preclinical/methods , Gene Knockdown Techniques , Humans , Molecular Sequence Data , Nitrophenols/metabolism , Phosphoric Diester Hydrolases/genetics , Reproducibility of ResultsABSTRACT
BACKGROUND: Eight human catalytic phosphoinositide 3-kinase (PI3K) isoforms exist which are subdivided into three classes. While class I isoforms have been well-studied in cancer, little is known about the functions of class II PI3Ks. MATERIALS AND METHODS: The expression pattern and functions of the class II PI3KC2ß isoform were investigated in a panel of tumour samples and cell lines. RESULTS: Overexpression of PI3KC2ß was found in subsets of tumours and cell lines from acute myeloid leukemia (AML), glioblastoma multiforme (GBM), medulloblastoma (MB), neuroblastoma (NB), and small cell lung cancer (SCLC). Specific pharmacological inhibitors of PI3KC2ß or RNA interference impaired proliferation of a panel of human cancer cell lines and primary cultures. Inhibition of PI3KC2ß also induced apoptosis and sensitised the cancer cells to chemotherapeutic agents. CONCLUSION: Together, these data show that PI3KC2ß contributes to proliferation and survival in AML, brain tumours and neuroendocrine tumours, and may represent a novel target in these malignancies.
Subject(s)
Brain Neoplasms/pathology , Cell Proliferation , Isoenzymes/metabolism , Leukemia, Myeloid, Acute/pathology , Neuroendocrine Tumors/pathology , Phosphatidylinositol 3-Kinases/metabolism , Survival Rate , Animals , Cell Line, Tumor , Electrophoresis, Polyacrylamide Gel , Enzyme Inhibitors/pharmacology , Humans , Inhibitory Concentration 50 , Mice , Oligonucleotide Array Sequence Analysis , Phosphoinositide-3 Kinase InhibitorsABSTRACT
A potent inhibitor of PI3Kδ that is ≥ 200 fold selective for the remaining three Class I PI3K isoforms and additional kinases is described. The hypothesis for selectivity is illustrated through structure activity relationships and crystal structures of compounds bound to a K802T mutant of PI3Kγ. Pharmacokinetic data in rats and mice support the use of 3 as a useful tool compound to use for in vivo studies.
Subject(s)
Phosphoinositide-3 Kinase Inhibitors , Protein Kinase Inhibitors/chemistry , Tryptophan/chemistry , Animals , Binding Sites , Computer Simulation , Female , Injections, Intravenous , Liver/metabolism , Male , Mice , Mice, Inbred BALB C , Mutation , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Protein Structure, Tertiary , Rats , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis, biological activity, and further profiling of these compounds are described. This work resulted in the discovery of 17, GDC-0941, which is a potent, selective, orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Indazoles/pharmacology , Neoplasms/drug therapy , Phosphoinositide-3 Kinase Inhibitors , Sulfonamides/pharmacology , Administration, Oral , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Biological Availability , Cell Line, Tumor , Drug Screening Assays, Antitumor , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/therapeutic use , Humans , Indazoles/administration & dosage , Indazoles/pharmacokinetics , Indazoles/therapeutic use , Magnetic Resonance Spectroscopy , Molecular Structure , Sulfonamides/administration & dosage , Sulfonamides/pharmacokinetics , Sulfonamides/therapeutic useABSTRACT
Multidrug resistance mediated by P-glycoprotein (Pgp) or multidrug-resistance-associated protein (MRP) remains a major obstacle for successful treatment of cancer. Inhibition of Pgp and MRP transport is important for high efficacy of anticancer drugs. While several Pgp inhibitors have entered clinical trials, the development of specific MRP1 inhibitors is still in its infancy. In our screening program, we have identified a pyrrolopyrimidine (4) as a novel and selective MRP1 inhibitor. Subsequent SAR work on the 4-position of the template revealed the phenethylpiperazine side chain as a potent replacement of the benzylthio group of the lead molecule. Introduction of groups at the 2-position seems to have no detrimental effect on activity. Modifications to the nitrile group at the 7-position resulted in the identification of analogues with groups, such as amides, with superior pharmacokinetic profiles. In vivo efficacy has been demonstrated by xenograft studies on selected compounds.
Subject(s)
Drug Resistance, Multiple , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Pyrimidines/chemical synthesis , Pyrroles/chemical synthesis , ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , Animals , Area Under Curve , Biological Availability , Biological Transport , Cell Line, Tumor , Combinatorial Chemistry Techniques , Daunorubicin/metabolism , Daunorubicin/pharmacology , Drug Synergism , Female , Half-Life , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Multidrug Resistance-Associated Proteins/biosynthesis , Pyrimidines/chemistry , Pyrimidines/pharmacology , Pyrroles/chemistry , Pyrroles/pharmacology , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
In our continued effort to identify selective MRP1 modulators, we have developed two novel templates, 3 and 4, through rational drug design by identifying the key pharmacophore interaction at the 7-position of the pyrrolopyrimidine template 1. Further synthesis and SAR work on these novel templates gave a number of potent MRP1 modulators with great selectivity against Pgp. Additional studies to reduce the CYP3A4 inhibition are also reported. Several compounds of these classes were subjected to in vivo xenograft studies and in vivo efficacies were demonstrated.
Subject(s)
Drug Resistance, Multiple , Multidrug Resistance-Associated Proteins/antagonists & inhibitors , Pyrimidines/chemical synthesis , Pyrroles/chemical synthesis , ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , Animals , Area Under Curve , Biological Availability , Cell Line, Tumor , Combinatorial Chemistry Techniques , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme Inhibitors , Doxorubicin/pharmacology , Drug Synergism , Female , Half-Life , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Models, Molecular , Multidrug Resistance-Associated Proteins/biosynthesis , Pyrimidines/chemistry , Pyrimidines/pharmacology , Pyrroles/chemistry , Pyrroles/pharmacology , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
The syntheses and SAR studies of various quinazolinone compounds are described for the dual inhibition of Pgp and MRP1 in multidrug resistance.
