RESUMO
Casein kinase 2 (CK2) is a highly pleiotropic serine-threonine kinase, which catalyzed phosphorylation of more than 300 proteins that are implicated in regulation of many cellular functions, such as signal transduction, transcriptional control, apoptosis and the cell cycle. On the other hand, CK2 is abnormally elevated in a variety of tumors, and is considered as a promising therapeutic target. The currently available ATP-competitive CK2 inhibitors, however, lack selectivity, which has impeded their development in cancer therapy. Because allosteric inhibitors can avoid the shortcomings of conventional kinase inhibitors, this study was aimed to discover a new allosteric site in CK2α and to investigate the effects of mutations in this site on the activity of CK2α. Using Allosite based on protein dynamics and structural alignment, we predicted a new allosteric site that was partly located in the αC helix of CK2α. Five residues exposed on the surface of this site were mutated to validate the prediction. Kinetic analyses were performed using a luminescent ADP detection assay by varying the concentrations of a peptide substrate, and the results showed that the mutations I78C and I78W decreased CK2α activity, whereas V31R, K75E, I82C and P109C increased CK2α activity. Potential allosteric pathways were identified using the Monte Carlo path generation approach, and the results of these predicted allosteric pathways were consistent with the mutation analysis. Multiple sequence alignments of CK2α with the other kinases in the family were conducted using the ClustalX method, which revealed the diversity of the residues in the site. In conclusion, we identified a new allosteric site in CK2α that can be altered to modulate the activity of the kinase. Because of the high diversity of the residues in the site, the site can be targeted using rational drug design of specific CK2α inhibitors for biological relevance.
Assuntos
Biologia Computacional , Algoritmos , Sítio Alostérico/efeitos dos fármacos , Sequência de Aminoácidos , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/química , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Moleculares , Alinhamento de SequênciaRESUMO
Deubiquitinating enzyme USP7 has been involved in the pathogenesis and progression of several cancers. Targeting USP7 is becoming an attractive strategy for cancer therapy. In this study, we identified synthetic triterpenoid C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid (CDDO-Me) as a novel inhibitor of USP7 but not of other cysteine proteases such as cathepsin B and cathepsin D. CDDO-Me inhibits USP7 activity via a mechanism that is independent of the presence of α, ß-unsaturated ketones. Molecular docking studies showed that CDDO-Me fits well in the ubiquitin carboxyl terminus-binding pocket on USP7. Given that CDDO-Me is known to be effective against ovarian cancer cells, we speculated that CDDO-Me may target USP7 in ovarian cancer cells. We demonstrated that ovarian cancer cells have higher USP7 expression than their normal counterparts. Knockdown of USP7 inhibits the proliferation of ovarian cancer cells both in vitro and in vivo. Using the cellular thermal shift assay and the drug affinity responsive target stability assay, we further demonstrated that CDDO-Me directly binds to USP7 in cells, which leads to the decrease of its substrates such as MDM2, MDMX and UHRF1. CDDO-Me suppresses ovarian cancer tumor growth in an xenograft model. In conclusion, we demonstrate that USP7 is a novel target of ovarian cancer cells; targeting USP7 may contribute to the anti-cancer effect of CDDO-Me. The development of novel USP7 selective compounds based on the CDDO-Me-scaffold warrants further investigation.
Assuntos
Inibidores Enzimáticos/administração & dosagem , Ácido Oleanólico/análogos & derivados , Neoplasias Ovarianas/tratamento farmacológico , Peptidase 7 Específica de Ubiquitina/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Simulação de Acoplamento Molecular , Ácido Oleanólico/administração & dosagem , Ácido Oleanólico/química , Ácido Oleanólico/farmacologia , Neoplasias Ovarianas/metabolismo , Peptidase 7 Específica de Ubiquitina/química , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Identifying novel targets to enhance leukemia-cell differentiation is an urgent requirement. We have recently proposed that inhibiting the antioxidant enzyme peroxiredoxin I (Prdx I) may induce leukemia-cell differentiation. However, this concept remains to be confirmed. In this work, we identified H7 as a novel Prdx I inhibitor through virtual screening, in vitro activity assay, and surface plasmon resonance assay. Cellular thermal shift assay showed that H7 directly bound to Prdx I but not to Prdxs II-V in cells. H7 treatment also increased reactive oxygen species (ROS) level and cell differentiation in leukemia cells, as reflected by the upregulation of the cell surface differentiation marker CD11b/CD14 and the morphological maturation of cells. The differentiation-induction effect of H7 was further observed in some non-acute promyelocytic leukemia (APL) and primary leukemia cells apart from APL NB4 cells. Moreover, the ROS scavenger N-acetyl cysteine significantly reversed the H7-induced cell differentiation. We demonstrated as well that H7-induced cell differentiation was associated with the activation of the ROS-Erk1/2-C/EBPß axis. Finally, we showed H7 treatment induced cell differentiation in an APL mouse model. All of these data confirmed that Prdx I was novel target for inducing leukemia-cell differentiation and that H7 was a novel lead compound for optimizing Prdx I inhibition.
Assuntos
Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Leucemia Mieloide Aguda/patologia , Leucemia Promielocítica Aguda/patologia , Peroxirredoxinas/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonamidas/farmacologia , Animais , Western Blotting , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/metabolismo , Camundongos , Camundongos Transgênicos , Ressonância de Plasmônio de Superfície , Células Tumorais CultivadasRESUMO
BACKGROUND: Annonaceous acetogenins are a family of natural products with antitumor activities. Annonaceous acetogenin mimic AA005 reportedly inhibits mammalian mitochondrial NADH-ubiquinone reductase (Complex I) and induces gastric cancer cell death. However, the mechanisms underlying its cell-death-inducing activity are unclear. METHODS: We used SW620 colorectal adenocarcinoma cells to study AA005 cytotoxic activity. Cell deaths were determined by Trypan blue assay and flow cytometry, and related proteins were characterized by western blot. Immunofluorescence and subcellular fractionation were used to evaluate AIF nuclear translocation. Reactive oxygen species were assessed by using redox-sensitive dye DCFDA. RESULTS: AA005 induces a unique type of cell death in colorectal adenocarcinoma cells, characterized by lack of caspase-3 activation or apoptotic body formation, sensitivity to poly (ADP-ribose) polymerase inhibitor Olaparib (AZD2281) but not pan-caspase inhibitor Z-VAD.fmk, and dependence on apoptosis-inducing factor (AIF). AA005 treatment also reduced expression of mitochondrial Complex I components, and leads to accumulation of intracellular reactive oxygen species (ROS) at the early stage. Blocking ROS formation significantly suppresses AA005-induced cell death in SW620 cells. Moreover, blocking activation of RIP-1 by necroptosis inhibitor necrotatin-1 inhibits AIF translocation and partially suppresses AA005-induced cell death in SW620 cells demonstrating that RIP-1 protein may be essential for cell death. CONCLUSIONS: AA005 may trigger the cell death via mediated by AIF through caspase-3 independent pathway. Our work provided new mechanisms for AA005-induced cancer cell death and novel clues for cancer treatment via AIF dependent cell death.
Assuntos
Acetogeninas/farmacologia , Fator de Indução de Apoptose/biossíntese , Caspase 3 , Álcoois Graxos/farmacologia , Lactonas/farmacologia , Acetogeninas/química , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Álcoois Graxos/química , Humanos , Lactonas/química , Espécies Reativas de Oxigênio/metabolismo , Células U937RESUMO
Sentrin/SUMO (small ubiquitin-like modifier)-specific proteases (SENPs) have been implicated in the development of prostate cancer. However, due to the low abundance of SUMO-modified proteins and high activity of SENPs, the SUMO substrates affected by SENPs in prostate cancer cells are largely unknown. Here, we identified SI2, a novel cell-permeable SENP-specific inhibitor, by high-throughput screening. Using SI2 as a way of inhibiting the activity of SENPs and the SUMO stably transfected PC3 cells as a prostate cancer model, in combination with the stable isotope labeling with amino acids (SILAC) quantitative proteomic technique, we identified more than 900 putative target proteins of SUMO, in which 231 proteins were further subjected to bioinformatic analysis. In the highly enriched spliceosome pathway, we validated that USP39, HSPA1A, and HSPA2 were novel target proteins of SUMO. Furthermore, we demonstrated that K6, K16, K29, K51, and K73 were the SUMOylation sites of USP39. Mutation of these SUMO modification sites of USP39 further promoted the proliferation-enhancing effect of USP39 on prostate cancer cells. This study provides the SUMOproteome of PC3 cells and reveals that SUMOylation of spliceosome factors may be implicated in the pathogenesis of prostate cancer. Optimization of SI2 for isotype-specific SENP inhibitors warrants further investigation.
Assuntos
Benzoatos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Endopeptidases/metabolismo , Neoplasias da Próstata/metabolismo , Inibidores de Proteases/farmacologia , Spliceossomos/metabolismo , Sumoilação/fisiologia , Benzoatos/química , Benzoatos/isolamento & purificação , Sítios de Ligação/genética , Western Blotting , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/isolamento & purificação , Catepsina B/metabolismo , Catepsina D/metabolismo , Cromatografia Líquida , Biologia Computacional , Cisteína Endopeptidases , Células HEK293 , Proteínas de Choque Térmico HSP70/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Imunoprecipitação , Concentração Inibidora 50 , Marcação por Isótopo , Masculino , Plasmídeos/genética , Inibidores de Proteases/química , Inibidores de Proteases/isolamento & purificação , Proteômica , Espectrometria de Massas em Tandem , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismoRESUMO
We report a capillary electrophoresis method in conjunction with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for screening of protein kinase inhibitors (PKIs) in natural extracts. Protein kinase A (PKA), substrate 5-carboxyfluorescein-labeled kemptide (CLK) and inhibitor H-89 were employed for the method development and validation. Enzymatic inhibition assay was performed with electrophoretically mediated microanalysis technique. Once the bioactivity of a natural extract was confirmed, an assay-guided isolation and structure elucidation using LC-MS/MS were accomplished to identify the compounds which are responsible for the observed bioactivity. Totally 33 natural extracts were screened with the method, and baicalin in the extract of Radix Scutellariae was identified to be a new PKI of PKA. This result demonstrated the practical applicability of our method in screening of PKIs from natural products.
Assuntos
Extratos Vegetais/química , Inibidores de Proteínas Quinases/análise , Cromatografia Líquida/métodos , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Eletroforese Capilar/métodos , Flavonoides/análise , Scutellaria baicalensis/química , Espectrometria de Massas em Tandem/métodosRESUMO
The proteolytic activation of protein kinase Cδ (PKCδ) generates a catalytic fragment called PKCδ-CF, which induces cell death. However, the mechanisms underlying PKCδ-CF-mediated cell death are largely unknown. On the basis of an engineering leukemic cell line with inducible expression of PKCδ-CF, here we employ SILAC-based quantitative phosphoproteomics to systematically and dynamically investigate the overall phosphorylation events during cell death triggered by PKCδ-CF expression. Totally, 3000 phosphorylation sites were analyzed. Considering the fact that early responses to PKCδ-CF expression initiate cell death, we sought to identify pathways possibly related directly with PKCδ by further analyzing the data set of phosphorylation events that occur in the initiation stage of cell death. Interacting analysis of this data set indicates that PKCδ-CF triggers complicated networks to initiate cell death, and motif analysis and biochemistry verification reveal that several kinases in the downstream of PKCδ conduct these networks. By analysis of the specific sequence motif of kinase-substrate, we also find 59 candidate substrates of PKCδ from the up-regulated phosphopeptides, of which 12 were randomly selected for in vitro kinase assay and 9 were consequently verified as substrates of PKCδ. To our greatest understanding, this study provides the most systematic analysis of phosphorylation events initiated by the cleaved activated PKCδ, which would vastly extend the profound understanding of PKCδ-directed signal pathways in cell death. The MS data have been deposited to the ProteomeXchange with identifier PXD000225.
Assuntos
Apoptose , Fosfoproteínas/metabolismo , Proteína Quinase C-delta/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Sequência Consenso , Proteínas Culina/metabolismo , Ontologia Genética , Células HEK293 , Humanos , Dados de Sequência Molecular , Fosfoproteínas/genética , Fosforilação , Mapas de Interação de Proteínas , Proteoma/genética , Proteômica , Transdução de SinaisRESUMO
A CE-based method for hexokinase inhibitor screening was developed in the present paper. In this method, hexokinase activity was assayed via electrophoretically mediated microanalysis (EMMA), which combines on-column hexokinase-mediated reaction and measurement of produced adenosine 5'-diphosphate (ADP) via electrophoretical separation and UV detection. Enzyme inhibition can be read out directly from the reduced peak area of ADP in comparison with a reference electropherogram obtained in the absence of any inhibitor. Conditions for on-column enzyme reaction and separation of adenosine 5'-triphosphate (ATP) and ADP were optimized. The optimal buffer composition for enzymatic reaction was 25 mM HEPES buffer (pH 7.5) containing 5 mM MgCl(2), whereas the optimal buffer composition for separation was 100 mM Tris-phosphate buffer (pH 5.5) containing 0.02% (m/v) hexadimethrine bromide (HDB). Fortunately, discontinuous buffer system can be adapted easily in the EMMA method. The time for separation was reduced dramatically to less than 3 min by reversing the direction of EOF via dynamically coating the capillary wall with the cationic polyelectrolyte HDB. Moreover, the peak tailing of ATP was also reduced by HDB coating. The Z' factor as high as 0.98 was obtained, indicating a high quality of the screening data. The present method is simple, robust and cost-effective.