ABSTRACT
Bladder cancer (BlC) is the fourth most common cancer in males worldwide, but few systemic chemotherapy options for its effective treatment exist. The development of new molecularly-targeted agents against BlC is therefore an urgent issue. The Hippo signaling pathway, with its upstream LATS kinases and downstream transcriptional co-activators YAP1 and TAZ, plays a pivotal role in diverse cell functions, including cell proliferation. Recent studies have shown that overexpression of YAP1 occurs in advanced BlCs and is associated with poor patient prognosis. Accessing data from our previous screening of a chemical library of compounds targeting the Hippo pathway, we identified DMPCA (N-(3,4-dimethoxyphenethyl)-6-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-amine) as an agent able to induce the phosphorylation of LATS1 and YAP1/TAZ in BlC cells, thereby suppressing their viability both in vitro and in mouse xenografts. Our data indicate that DMPCA has a potent anti-tumor effect, and raise the possibility that this agent may represent a new and effective therapeutic option for BlC.
Subject(s)
Urinary Bladder Neoplasms , Animals , Humans , Male , Mice , Acyltransferases/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Amines , Carbazoles , Protein Serine-Threonine Kinases , Signal Transduction/physiology , Transcription Factors/metabolism , Urinary Bladder Neoplasms/drug therapy , YAP-Signaling ProteinsABSTRACT
TEAD is a transcription factor responsible for the output of the tumor suppressor Hippo pathway. The transcriptional activity of TEAD requires molecular interaction with its transcriptional coactivator, YAP. Aberrant activation of TEAD is deeply involved in tumorigenesis and is associated with poor prognosis, suggesting that inhibitors targeting the YAP-TEAD system are promising as antitumor agents. In this study, we identified NPD689, an analog of the natural product alkaloid emetine, as an inhibitor of the YAP-TEAD interaction. NPD689 suppressed the transcriptional activity of TEAD and reduced the viability of human malignant pleural mesothelioma and non-small cell lung cancer cells but not the viability of normal human mesothelial cells. Our results suggest that NPD689 is not only a new useful chemical tool for elucidating the biological role of the YAP-TEAD system but also has potential as a starting compound for developing a cancer therapeutic agent that targets the YAP-TEAD interaction.
Subject(s)
Antineoplastic Agents , Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Adaptor Proteins, Signal Transducing/metabolism , Antineoplastic Agents/pharmacology , Emetine , Lung Neoplasms/pathology , Transcription Factors/metabolism , YAP-Signaling Proteins , TEA Domain Transcription Factors/metabolismABSTRACT
The transcriptional coactivator with PDZ-binding motif (TAZ) (WWTR1) induces epithelial-mesenchymal transition and enhances drug resistance in multiple cancers. TAZ has been shown to interact with transcription factors in the nucleus, but when phosphorylated, translocates to the cytoplasm and is degraded through proteasomes. Here, we identified a compound TAZ inhibitor 4 (TI-4) that shifted TAZ localization to the cytoplasm independently of its phosphorylation. We used affinity beads to ascertain a putative target of TI-4, chromosomal segregation 1 like (CSE1L), which is known to be involved in the recycling of importin α and as a biomarker of cancer malignancy. We found that TI-4 suppressed TAZ-mediated transcription in a CSE1L-dependent manner. CSE1L overexpression increased nuclear levels of TAZ, whereas CSE1L silencing delayed its nuclear import. We also found via the in vitro coimmunoprecipitation experiments that TI-4 strengthened the interaction between CSE1L and importin α5 and blocked the binding of importin α5 to TAZ. WWTR1 silencing attenuated CSE1L-promoted colony formation, motility, and invasiveness of human lung cancer and glioblastoma cells. Conversely, CSE1L silencing blocked TAZ-promoted colony formation, motility, and invasiveness in human lung cancer and glioblastoma cells. In human cancer tissues, the expression level of CSE1L was found to correlate with nuclear levels of TAZ. These findings support that CSE1L promotes the nuclear accumulation of TAZ and enhances malignancy in cancer cells.
Subject(s)
Cell Nucleus/metabolism , Cellular Apoptosis Susceptibility Protein/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Trans-Activators/metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic , Gene Silencing , Green Fluorescent Proteins/metabolism , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Models, Biological , Neoplasm Invasiveness , Neoplasms/genetics , Phosphorylation , Photobleaching , Protein Binding , Protein Transport , Subcellular Fractions/metabolism , Transcriptional Coactivator with PDZ-Binding Motif Proteins , Tumor Stem Cell Assay , alpha Karyopherins/metabolismABSTRACT
Yes-associated protein 1 (YAP1) and its paralogue PDZ-binding motif (TAZ) play pivotal roles in cell proliferation, migration, and invasion, and abnormal activation of these TEAD transcriptional coactivators is found in diverse cancers in humans and mice. Targeting YAP1/TAZ signaling is thus a promising therapeutic avenue but, to date, few selective YAP1/TAZ inhibitors have been effective against cancer cells either in vitro or in vivo. We screened chemical libraries for potent YAP1/TAZ inhibitors using a highly sensitive luciferase reporter system to monitor YAP1/TAZ-TEAD transcriptional activity in cells. Among 29 049 low-molecular-weight compounds screened, we obtained nine hits, and the four of these that were the most effective shared a core structure with the natural product alantolactone (ALT). We also tested 16 other structural derivatives of ALT and found that natural ALT was the most efficient at increasing ROS-induced LATS kinase activities and thus YAP1/TAZ phosphorylation. Phosphorylated YAP1/TAZ proteins were subject to nuclear exclusion and proteosomic degradation such that the growth of ALT-treated tumor cells was inhibited both in vitro and in vivo. Our data show for the first time that ALT can be used to target the ROS-YAP pathway driving tumor cell growth and so could be a potent anticancer drug.
Subject(s)
Acyltransferases/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Antineoplastic Agents, Phytogenic/pharmacology , Biological Products/pharmacology , Lactones/pharmacology , Reactive Oxygen Species/metabolism , Sesquiterpenes, Eudesmane/pharmacology , Acyltransferases/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Auranofin/pharmacology , Cell Movement , Cell Nucleus/metabolism , Cell Proliferation/drug effects , Cell Self Renewal , DNA-Binding Proteins/metabolism , Drug Discovery , Female , Inula/chemistry , Luciferases , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness , Nuclear Proteins/metabolism , Phosphorylation/drug effects , Proteolysis/drug effects , Small Molecule Libraries , TEA Domain Transcription Factors , Tongue Neoplasms/chemically induced , Tongue Neoplasms/prevention & control , Transcription Factors/metabolism , Transcriptional Activation , YAP-Signaling ProteinsABSTRACT
Carcinoma-associated fibroblasts are fibroblasts activated by surrounding cancer cells. Carcinoma-associated fibroblasts exhibit enhanced cell migration, which plays an important role in cancer metastasis. Previously, we demonstrated enhanced migration of NIH3T3 fibroblasts when they were cultured in the presence of MCF7 breast cancer cells. Human fibroblasts displayed a similar phenomenon even when they were co-cultured with cancer cells other than MCF7 cells. In this study, we screened â¼16,000 compounds from the RIKEN Natural Products Depository chemical library for inhibitors of enhanced NIH3T3 cell migration in the presence of MCF7. We identified NPD8733 as an inhibitor of cancer cell-enhanced fibroblast migration. This inhibition was observed not only in a wound-healing co-culture assay but also in a Transwell migration assay. Using NPD8733 and a structurally similar but inactive derivative, NPD8126, on immobilized beads, we found that NPD8733, but not NPD8126, specifically binds to valosin-containing protein (VCP)/p97, a member of the ATPase-associated with diverse cellular activities (AAA+) protein family. Using VCP truncation variants, we found that NPD8733 binds to the D1 domain of VCP. Because VCP's D1 domain is important for its function, we concluded that NPD8733 may act on VCP by binding to this domain. siRNA-mediated silencing of VCP in NIH3T3 fibroblasts, but not in MCF7 cells, reduced the migration of the co-cultured NIH3T3 fibroblasts. These results indicate that MCF7 activates the migration of NIH3T3 cells through VCP and that NPD8733 binds VCP and thereby inhibits its activity.
Subject(s)
Cell Movement/drug effects , Fibroblasts/cytology , Fibroblasts/drug effects , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , Valosin Containing Protein/metabolism , Animals , Coculture Techniques , Drug Evaluation, Preclinical , Humans , Ligands , MCF-7 Cells , Mice , NIH 3T3 Cells , Protein Domains , Valosin Containing Protein/chemistryABSTRACT
Discovery of small-molecule inducers of unique phenotypic changes combined with subsequent target identification often provides new insights into cellular functions. Here, we applied integrated profiling based on cellular morphological and proteomic changes to compound screening. We identified an indane derivative, NPD9055, which is mechanistically distinct from reference compounds with known modes of action. Employing a chemical proteomics approach, we then showed that NPD9055 binds subunits of heterotrimeric G-protein Gi. An in vitro [35S]GTPγS-binding assay revealed that NPD9055 inhibited GDP/GTP exchange on a Gαi subunit induced by a G-protein-coupled receptor agonist, but not on another G-protein from the Gαs family. In intact HeLa cells, NPD9055 induced an increase in intracellular Ca2+ levels and ERK/MAPK phosphorylation, both of which are regulated by Gßγ, following its dissociation from Gαi. Our observations suggest that NPD9055 targets Gαi and thus regulates Gßγ-dependent cellular processes, most likely by causing the dissociation of Gßγ from Gαi.
Subject(s)
Drug Discovery , Heterotrimeric GTP-Binding Proteins/metabolism , Phenotype , Proteomics , Small Molecule Libraries/pharmacology , Cell Line, Tumor , HumansABSTRACT
The X-linked inhibitor of apoptosis protein baculovirus IAP repeat (XIAP BIR3) domain is a promising therapeutic target for cancer treatment. For the mirror-image screening campaign to identify drug candidates from an unexplored mirror-image natural product library, a facile synthetic protocol for XIAP BIR3 domain synthesis was established by a native chemical ligation strategy using conserved cysteines present among BIR domains. The native and mirror-image XIAP BIR3 domains with an appropriate functional group for labeling were prepared using the established protocol. Taking advantage of the resulting synthetic proteins, several bioassay systems were developed to characterize inhibitors of the protein-protein interaction between the XIAP BIR3 domain and the second mitochondria-derived activator of caspases.
Subject(s)
X-Linked Inhibitor of Apoptosis Protein/antagonists & inhibitors , Amino Acid Sequence , Biological Assay , Humans , Protein Binding , Protein Conformation , Protein Domains , Protein Folding , Sequence Homology, Amino Acid , X-Linked Inhibitor of Apoptosis Protein/chemistry , X-Linked Inhibitor of Apoptosis Protein/metabolismABSTRACT
Tunneling nanotubes (TNTs), the long membrane extensions connecting distant cells, have emerged as a novel form of cell-to-cell communication. However, it is not fully understood how and to what extent TNTs contribute to intercellular spread of pathogens including HIV-1. In this study, we show that HIV-1 promotes TNT formation per se via its protein Nef and a cellular protein M-Sec, which appears to mediate approximately half of viral spread among monocyte-derived macrophages (MDMs). A small compound that inhibits M-Sec-induced TNT formation reduced HIV-1 production by almost half in MDMs. Such inhibition was not observed with Nef-deficient mutant HIV-1 that fails to promote TNT formation and replicates less efficiently than the wild-type HIV-1 in MDMs. The TNT inhibitor-sensitive/Nef-promoting viral production was also observed in a T cell line ectopically expressing M-Sec, but not in another M-Sec(-) T cell line. Our results suggest the importance of TNTs in HIV-1 spread among MDMs and might answer the long-standing question how Nef promotes HIV-1 production in a cell type-specific manner.
Subject(s)
Cell Communication/physiology , HIV-1/metabolism , HIV-1/pathogenicity , Macrophages/virology , nef Gene Products, Human Immunodeficiency Virus/metabolism , Blotting, Western , Cell Line , Cytokines/metabolism , Flow Cytometry , Fluorescent Antibody Technique , Humans , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
Plants have a remarkable ability to perceive and respond to various wavelengths of light and initiate regulation of different cascades of light signaling and molecular components. While the perception of red light and the mechanisms of its signaling involving phytochromes are largely known, knowledge of the mechanisms of blue light signaling is still limited. Chemical genetics involves the use of diverse small active or synthetic molecules to evaluate biological processes. By combining chemicals and analyzing the effects they have on plant morphology, we identified a chemical, 3-bromo-7-nitroindazole (3B7N), that promotes hypocotyl elongation of wild-type Arabidopsis only under continuous blue light. Further evaluation with loss-of-function mutants confirmed that 3B7N inhibits photomorphogenesis through cryptochrome-mediated light signaling. Microarray analysis demonstrated that the effect of 3B7N treatment on gene expression in cry1cry2 is considerably smaller than that in the wild type, indicating that 3B7N specifically interrupts cryptochrome function in the control of seedling development in a light-dependent manner. We demonstrated that 3B7N directly binds to CRY1 protein using an in vitro binding assay. These results suggest that 3B7N is a novel chemical that directly inhibits plant cryptochrome function by physical binding. The application of 3B7N can be used on other plants to study further the blue light mechanism and the genetic control of cryptochromes in the growth and development of plant species.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Cryptochromes/genetics , Indazoles/pharmacology , Light , Seedlings/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Cryptochromes/metabolism , Gene Expression Profiling/methods , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/radiation effects , Gene Expression Regulation, Plant/drug effects , Gene Expression Regulation, Plant/radiation effects , Hypocotyl/genetics , Hypocotyl/metabolism , Immunoblotting , Indazoles/chemistry , Indazoles/metabolism , Light Signal Transduction/drug effects , Light Signal Transduction/genetics , Light Signal Transduction/radiation effects , Molecular Structure , Morphogenesis/drug effects , Morphogenesis/genetics , Morphogenesis/radiation effects , Mutation , Oligonucleotide Array Sequence Analysis , Protein Binding , Reverse Transcriptase Polymerase Chain Reaction , Seedlings/growth & development , Seedlings/metabolismABSTRACT
Developing antiviral therapies for influenza A virus (IAV) infection is an ongoing process because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. The ideal strategy is to develop drugs that target well-conserved, functionally restricted, and unique surface structures without affecting host cell function. We recently identified the antiviral compound, RK424, by screening a library of 50,000 compounds using cell-based infection assays. RK424 showed potent antiviral activity against many different subtypes of IAV in vitro and partially protected mice from a lethal dose of A/WSN/1933 (H1N1) virus in vivo. Here, we show that RK424 inhibits viral ribonucleoprotein complex (vRNP) activity, causing the viral nucleoprotein (NP) to accumulate in the cell nucleus. In silico docking analysis revealed that RK424 bound to a small pocket in the viral NP. This pocket was surrounded by three functionally important domains: the RNA binding groove, the NP dimer interface, and nuclear export signal (NES) 3, indicating that it may be involved in the RNA binding, oligomerization, and nuclear export functions of NP. The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424. Surface plasmon resonance (SPR) and pull-down assays showed that RK424 inhibited both the NP-RNA and NP-NP interactions, whereas size exclusion chromatography showed that RK424 disrupted viral RNA-induced NP oligomerization. In addition, in vitro nuclear export assays confirmed that RK424 inhibited nuclear export of NP. The amino acid residues comprising the NP pocket play a crucial role in viral replication and are highly conserved in more than 7,000 NP sequences from avian, human, and swine influenza viruses. Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules. Taken together, these results describe a promising new approach to developing influenza virus drugs that target a novel pocket structure within NP.
Subject(s)
Antiviral Agents/pharmacology , Influenza A virus/metabolism , Protein Multimerization , RNA-Binding Proteins/metabolism , Viral Core Proteins/metabolism , Active Transport, Cell Nucleus/drug effects , Animals , Humans , Influenza A virus/drug effects , Mice , Nucleocapsid Proteins , RNA, Viral/drug effects , RNA, Viral/metabolism , Structure-Activity RelationshipABSTRACT
Grb2 is an adaptor protein that mediates cellular signal transduction. Grb2 contains an SH2 domain that interacts with phosphotyrosine-containing sequences in EGFR and other signaling molecules, and it is a promising molecular target for anticancer agents. To identify novel inhibitors of the Grb2 SH2 domain from natural products and their mirror-image isomers, screening systems using both enantiomers of a synthetic Grb2 SH2 domain protein were established. A pair of synthetic procedures for the proteins were investigated: one employed a single native chemical ligation (NCL) of two segment peptides, and the other used the N-to-C-directed NCL of three segment peptides for easier preparation. Labeling at the N-terminus or the Ala115 residue of the Grb2 SH2 domain provided functional probes to detect binding to a phosphotyrosine-containing peptide. The resulting synthetic-protein-based probes were applied to bioassays, including chemical array analysis and enzyme-linked immunosorbent assays.
Subject(s)
Drug Discovery/methods , GRB2 Adaptor Protein/chemical synthesis , src Homology Domains/drug effects , Amino Acid Sequence , Enzyme-Linked Immunosorbent Assay , GRB2 Adaptor Protein/antagonists & inhibitors , GRB2 Adaptor Protein/chemistry , GRB2 Adaptor Protein/metabolism , Humans , Models, Molecular , Peptides/chemistry , Peptides/pharmacologyABSTRACT
Mirror-image screening using d-proteins is a powerful approach to provide mirror-image structures of chiral natural products for drug screening. During the course of our screening study for novel MDM2-p53 interaction inhibitors, we identified that NPD6878 (R-(-)-apomorphine) inhibited both the native l-MDM2-l-p53 interaction and the mirror-image d-MDM2-d-p53 interaction at equipotent doses. In addition, both enantiomers of apomorphine showed potent inhibitory activity against the native MDM2-p53 interaction. In this study, we investigated the inhibitory mechanism of both enantiomers of apomorphine against the MDM2-p53 interaction. Achiral oxoapomorphine, which was converted from chiral apomorphines under aerobic conditions, served as the reactive species to form a covalent bond at Cys77 of MDM2, leading to the inhibitory effect against the binding to p53.
Subject(s)
Apomorphine/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/metabolism , Apomorphine/chemistry , Cell Line, Tumor , Humans , Protein Binding , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Stereoisomerism , Structure-Activity Relationship , Surface Plasmon Resonance , Tumor Suppressor Protein p53/antagonists & inhibitorsABSTRACT
Trichothecene mycotoxins often accumulate in apparently normal grains of cereal crops. In an effort to develop an agricultural chemical to reduce trichothecene contamination, we screened trichothecene production inhibitors from the compounds on the chemical arrays. By using the trichodiene (TDN) synthase tagged with hexahistidine (rTRI5) as a target protein, 32 hit compounds were obtained from chemical library of the RIKEN Natural Product Depository (NPDepo) by chemical array screening. At 10µgmL-1, none of the 32 chemicals inhibited trichothecene production by Fusarium graminearum in liquid culture. Against the purified rTRI5 enzyme, however, NPD10133 [progesterone 3-(O-carboxymethyl)oxime amide-bonded to phenylalanine] showed weak inhibitory activity at 10µgmL-1 (18.7µM). For the screening of chemicals inhibiting trichothecene accumulation in liquid culture, 20 analogs of NPD10133 selected from the NPDepo chemical library were assayed. At 10µM, only NPD352 [testosterone 3-(O-carboxymethyl)oxime amide-bonded to phenylalanine methyl ester] inhibited rTRI5 activity and trichothecene production. Kinetic analysis suggested that the enzyme inhibition was of a mixed-type. The identification of NPD352 as a TDN synthase inhibitor lays the foundation for the development of a more potent inhibitor via systematic introduction of wide structural diversity on the gonane skeleton and amino acid residues.
Subject(s)
Carbon-Carbon Lyases/antagonists & inhibitors , Fungicides, Industrial/chemistry , Fungicides, Industrial/pharmacology , Fusarium/metabolism , Trichothecenes/antagonists & inhibitors , Enzyme Inhibitors , Small Molecule LibrariesABSTRACT
Abscisic acid (ABA) signaling is involved in multiple processes in plants, such as water stress control and seed dormancy. Major regulators of ABA signaling are the PYR/PYL/RCAR family receptor proteins, groupâ A protein phosphatasesâ 2C (PP2Cs), and subclassâ III of SNF1-related protein kinaseâ 2 (SnRK2). Novel ABA agonists and antagonists to modulate the functions of these proteins would not only contribute to clarification of the signaling mechanisms but might also be used to improve crop yields. To obtain small molecules that interact with Arabidopsis ABA receptor PYR1, we screened 24 275 compounds from a chemical library at the RIKEN Natural Products Depository by using a chemical array platform. Subsequent SnRK2 and PP2C assays narrowed down the candidates to two molecules. One antagonized ABA in a competitive manner and inhibited the formation of the PYR1-ABA-PP2C ternary complex. These compounds might have potential as bioprobes to analyze ABA signaling.
Subject(s)
Arabidopsis Proteins/antagonists & inhibitors , Abscisic Acid/chemistry , Abscisic Acid/pharmacology , Arabidopsis/growth & development , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Germination/drug effects , High-Throughput Screening Assays , Kinetics , Membrane Transport Proteins/metabolism , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoprotein Phosphatases/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Pyrans/chemistry , Seeds/growth & development , Seeds/metabolism , Signal Transduction/drug effects , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacologyABSTRACT
Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade many extracellular matrix components and that have been implicated in the pathogenesis of various human diseases including cancer metastasis. Here, we screened MMP-9 inhibitors using photo-cross-linked chemical arrays, which can detect small-molecule ligand-protein interactions on a chip in a high-throughput manner. The array slides were probed sequentially with His-MMP-9, anti-His antibody, and a Cy5-labeled secondary antibody and then scanned with a microarray scanner. We obtained 27 hits among 24,275 compounds from the NPDepo library; 2 of the identified compounds (isoxazole compound 1 and naphthofluorescein) inhibited MMP-9 enzyme activity in vitro. We further explored 17 analogs of 1 and found that compound 18 had the strongest inhibitory activity. Compound 18 also inhibited other MMPs, including MMP-2, MMP-12, and MMP-13 and significantly inhibited cell migration in human fibrosarcoma HT1080 cells. These results suggest that 18 is a broad-spectrum MMP inhibitor.
Subject(s)
Fibroblasts/drug effects , Fluoresceins/pharmacology , Isoxazoles/pharmacology , Matrix Metalloproteinase 9/chemistry , Matrix Metalloproteinase Inhibitors/pharmacology , Small Molecule Libraries/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Drug Discovery , Extracellular Matrix/drug effects , Fibroblasts/enzymology , Fibroblasts/pathology , Fluoresceins/chemistry , Gene Expression , High-Throughput Screening Assays , Histidine/genetics , Histidine/metabolism , Humans , Isoxazoles/chemistry , Matrix Metalloproteinase 12/chemistry , Matrix Metalloproteinase 12/genetics , Matrix Metalloproteinase 12/metabolism , Matrix Metalloproteinase 13/chemistry , Matrix Metalloproteinase 13/genetics , Matrix Metalloproteinase 13/metabolism , Matrix Metalloproteinase 2/chemistry , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Matrix Metalloproteinase Inhibitors/chemistry , Microarray Analysis , Oligopeptides/genetics , Oligopeptides/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Small Molecule Libraries/chemistryABSTRACT
Induction of excessive levels of reactive oxygen species (ROS) by small-molecule compounds has been considered a potentially effective therapeutic strategy against cancer cells, which are often subjected to chronic oxidative stress. However, to elucidate the mechanisms of action of bioactive compounds is generally a time-consuming process. We have recently identified NPD926, a small molecule that induces rapid cell death in cancer cells. Using a combination of two comprehensive and complementary approaches, proteomic profiling and affinity purification, together with the subsequent biochemical assays, we have elucidated the mechanism of action underlying NPD926-induced cell death: conjugation with glutathione mediated by GST, depletion of cellular glutathione and subsequent ROS generation. NPD926 preferentially induced effects in KRAS-transformed fibroblast cells, compared with their untransformed counterparts. Furthermore, NPD926 sensitized cells to inhibitors of system x(c)â», a cystine-glutamate antiporter considered to be a potential therapeutic target in cancers including cancer stem cells. These data show the effectiveness of a newly identified ROS inducer, which targets glutathione metabolism, in cancer treatment.
Subject(s)
Antineoplastic Agents , Glutathione/metabolism , Neoplastic Stem Cells/metabolism , Reactive Oxygen Species/metabolism , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Antiporters/antagonists & inhibitors , Antiporters/metabolism , Cell Death/drug effects , Cell Line, Transformed , HL-60 Cells , Humans , Jurkat Cells , K562 Cells , Neoplastic Stem Cells/pathology , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins p21(ras) , U937 Cells , ras Proteins/metabolismABSTRACT
Ras proteins are of importance in cell proliferation, and hence their mutated forms play causative roles in many kinds of cancer in different tissues. Inhibition of the Ras-depalmitoylating enzyme acyl protein thioesterases APT1 and -2 is a new approach to modulating the Ras cycle. Here we present boronic and borinic acid derivatives as a new class of potent and nontoxic APT inhibitors. These compounds were detected by extensive library screening using chemical arrays and turned out to inhibit human APT1 and -2 in a competitive mode. Furthermore, one of the molecules was demonstrated to inhibit Erk1/2 phosphorylation significantly.
Subject(s)
Boron/chemistry , Boron/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Thiolester Hydrolases/antagonists & inhibitors , Animals , Boron/toxicity , Dogs , Drug Evaluation, Preclinical , Enzyme Inhibitors/toxicity , Humans , Lipoylation/drug effects , Madin Darby Canine Kidney Cells , ras Proteins/antagonists & inhibitors , ras Proteins/metabolismABSTRACT
MDM2 and MDMX are oncoproteins that negatively regulate the activity and stability of the tumor suppressor protein p53. The inhibitors of protein-protein interactions (PPIs) of MDM2-p53 and MDMX-p53 represent potential anticancer agents. In this study, a novel approach for identifying MDM2-p53 and MDMX-p53 PPI inhibitor candidates by affinity-based screening using a chemical array has been established. A number of compounds from an in-house compound library, which were immobilized onto a chemical array, were screened for interaction with fluorescence-labeled MDM2 and MDMX proteins. The subsequent fluorescent polarization assay identified several compounds that inhibited MDM2-p53 and MDMX-p53 interactions.
Subject(s)
High-Throughput Screening Assays , Nuclear Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Tumor Suppressor Protein p53/antagonists & inhibitors , Cell Cycle Proteins , Dose-Response Relationship, Drug , Humans , Models, Molecular , Molecular Structure , Nuclear Proteins/metabolism , Protein Binding/drug effects , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Structure-Activity Relationship , Tumor Suppressor Protein p53/metabolismABSTRACT
Trichothecene 3-O-acetyltransferase (TRI101) is an indispensable enzyme for the biosynthesis of trichothecenes, a group of mycotoxins produced by Fusarium graminearum. In this study, an inhibitor of TRI101 was identified by chemical array analysis using compounds from the RIKEN Natural Products Depository (NPDepo) library. Although the addition of the identified enzyme inhibitor to the fungal culture did not inhibit trichothecene production, it can serve as a candidate lead compound in the development of a mycotoxin inhibitor that inactivates fungal defense mechanisms.
Subject(s)
Acetyltransferases/antagonists & inhibitors , Drug Evaluation, Preclinical/methods , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Time Factors , Valerates/chemistry , Valerates/pharmacologyABSTRACT
The E3 ubiquitin ligase RFFL is an apoptotic inhibitor highly expressed in cancers and its knockdown suppresses cancer cell growth and sensitizes to chemotherapy. RFFL also participates in peripheral protein quality control which removes the functional cell surface ΔF508-CFTR channel and reduces the efficacy of pharmaceutical therapy for cystic fibrosis (CF). Although RFFL inhibitors have therapeutic potential for both cancer and CF, they remain undiscovered. Here, a chemical array screening has identified α-tocopherol succinate (αTOS) as an RFFL ligand. NMR analysis revealed that αTOS directly binds to RFFL's substrate-binding region without affecting the E3 enzymatic activity. Consequently, αTOS inhibits the RFFL-substrate interaction, ΔF508-CFTR ubiquitination and elimination from the plasma membrane of epithelial cells, resulting in the increased functional CFTR channel. Among the α-tocopherol (αTOL) analogs we tested, only αTOS inhibited the RFFL-substrate interaction and increased the cell surface ΔF508-CFTR, depending on RFFL expression. Similarly, the unique proapoptotic effect of αTOS was dependent on RFFL expression. Thus, unlike other αTOL analogs, αTOS acts as an RFFL protein-protein interaction inhibitor which may explain its unique biological properties among αTOL analogs. Moreover, αTOS may act as a CFTR stabilizer, a novel class of drugs that extend cell surface ΔF508-CFTR lifetime.