RESUMEN
Data included are related to the research article "Isolation of biofunctional bovine immunoglobulin G from milk- and colostral whey with mixed-mode chromatography at lab and pilot scale" (Heidebrecht et al., 2018) [1]. Data show individual bovine whey proteins in flow-through and elution fractions using different chromatographic resins as well as different binding and elution conditions. The relevant analytical methods for individual protein detection were SDS-PAGE and reversed phase- high performance liquid chromatography. The focus of the data is on the two mixed mode materials MEP HyperCel™ and Capto™-multimodal chromatography. Resins were used individually, in series and at different scale. Data provide information at which binding and elution conditions it is possible to isolate bovine IgG from milk and colostral whey and at which purity.
RESUMEN
The aim of the present work was to develop a new scalable and cost-efficient process to isolate bovine immunoglobulin G from colostral whey with high purity and minimal loss of activity. The mixed mode material Mercapto-Ethyl-Pyridine-Hypercel™ was identified appropriate for direct capture of immunoglobulin G. The binding mechanism is primarily based on hydrophobic interactions at physiological conditions. As compared to immunoglobulin G, all other low molecular whey proteins such as α-Lactalbumin or ß-Lactoglobulin, except lactoperoxidase, are more hydrophilic and were therefore found in the flow-through fraction. In order to remove lactoperoxidase as an impurity the column was combined in series with a second mixed mode material (Capto™- with N-benzoyl-homocysteine as ligand) using the same binding conditions. At pH 7.5 the carboxyl group of this ligand is negatively charged and can hence bind the positively charged lactoperoxidase, whose isoelectric point is at pH 9.6. After sample application, the columns were eluted separately. By combining the two columns it was possible to obtain immunoglobulin G with a purity of >96.1% and yield of 65-80%. The process development was carried out using 1â¯mL columns and upscaling was performed in three steps up to a column volume of 8800â¯mL for the Hypercel™ column and 3000â¯mL for the Capto™- column. At this scale it is possible to obtain 130-150â¯g pure immunoglobulin G from 3â¯L colostrum within five hours, including the regeneration of both columns. Additionally, the impact of freeze-drying on the isolated immunoglobulin G was studied. The nativity of the freeze dried immunoglobulin was above 95%, which was proven by reversed phase liquid chromatography and validated by differential scanning calorimetry. The activity of immunoglobulin G was preserved over the isolation process and during drying as measured by enzyme-linked immunosorbent assay. In conclusion, by applying the proposed isolation process, it becomes feasible to obtain pure, active and stable imunnunoglobulin G at large scale.
Asunto(s)
Técnicas de Química Analítica/métodos , Cromatografía , Calostro/química , Inmunoglobulina G/aislamiento & purificación , Leche/química , Suero Lácteo/química , Animales , Bovinos , Ensayo de Inmunoadsorción Enzimática , Proteínas de la Leche/análisisRESUMEN
Constitutively activating internal tandem duplication (ITD) alterations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) are common in acute myeloid leukemia (AML) and classifies FLT3 as an attractive therapeutic target. So far, applications of FLT3 small molecule inhibitors have been investigated primarily in FLT3-ITD+ patients. Only recently, a prolonged event-free survival has been observed in AML patients who were treated with the multikinase inhibitor sorafenib in addition to standard therapy. Here, we studied the sorafenib effect on proliferation in a panel of 13 FLT3-ITD- and FLT3-ITD+ AML cell lines. Sorafenib IC50 values ranged from 0.001 to 5.6 µm, whereas FLT3-ITD+ cells (MOLM-13, MV4-11) were found to be more sensitive to sorafenib than FLT3-ITD- cells. However, we identified two FLT3-ITD- cell lines (MONO-MAC-1 and OCI-AML-2) which were also sorafenib sensitive. Phosphoproteome analyses revealed that the affected pathways differed in sorafenib sensitive FLT3-ITD- and FLT3-ITD+ cells. In MV4-11 cells sorafenib suppressed mTOR signaling by direct inhibition of FLT3. In MONO-MAC-1 cells sorafenib inhibited the MEK/ERK pathway. These data suggest that the FLT3 status in AML patients might not be the only factor predicting response to treatment with sorafenib.
Asunto(s)
Leucemia Mieloide Aguda/metabolismo , Mutación , Niacinamida/análogos & derivados , Compuestos de Fenilurea/farmacología , Fosfoproteínas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Tirosina Quinasa 3 Similar a fms/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Niacinamida/farmacología , Fosfoproteínas/análisis , Proteómica/métodos , SorafenibRESUMEN
Infection with human cytomegalovirus (HCMV) is a serious medical problem, particularly in immunocompromised individuals and neonates. The success of standard antiviral therapy is hampered by low drug compatibility and induction of viral resistance. A novel strategy is based on the exploitation of cell-directed signaling inhibitors. The broad antiinfective drug artesunate (ART) offers additional therapeutic options such as oral bioavailability and low levels of toxic side-effects. Here, novel ART-derived compounds including dimers and trimers were synthesized showing further improvements over the parental drug. Antiviral activity and mechanistic aspects were determined leading to the following statements: (i) ART exerts antiviral activity towards human and animal herpesviruses, (ii) no induction of ART-resistant HCMV mutants occurred in vitro, (iii) chemically modified derivatives of ART showed strongly enhanced anti-HCMV efficacy, (iv) NF-κB reporter constructs, upregulated during HCMV replication, could be partially blocked by ART treatment, (v) ART activity analyzed in stable reporter cell clones indicated an inhibition of stimulated NF-κB but not CREB pathway, (vi) solid-phase immobilized ART was able to bind to NF-κB RelA/p65, and (vii) peptides within NF-κB RelA/p65 represent candidates of ART binding as analyzed by in silico docking and mass spectrometry. These novel findings open new prospects for the future medical use of ART and ART-related drug candidates.
Asunto(s)
Artemisininas/farmacología , Citomegalovirus/efectos de los fármacos , Citomegalovirus/metabolismo , FN-kappa B/metabolismo , Factor de Transcripción ReIA/metabolismo , Antivirales/química , Antivirales/farmacología , Artemisininas/química , Artesunato , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Citomegalovirus/genética , Farmacorresistencia Viral , Células HEK293 , Herpesviridae/efectos de los fármacos , Humanos , Mutación , FN-kappa B/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Activación Transcripcional , Regulación hacia ArribaRESUMEN
Human cytomegalovirus infection can lead to life-threatening clinical manifestations particularly in the immunocompromised host. Current therapy options face severe limitations leading to a continued search for alternative drug candidates. Viral replication is dependent on a balanced interaction between viral and cellular proteins. Especially protein kinases are important regulators of virus-host interaction indicated by remarkable kinome alterations induced upon HCMV infection. Here we report a novel approach of kinome profiling with an outcome that suggests an important role of specific cellular protein kinases, such as AMPK, ABL2 and Aurora A. Inhibition of AMPK and ABL kinases showed a significant reduction, whereas inhibition of Aurora A kinase led to a slight activation of HCMV replication, as measured in a GFP reporter-based replication assay. Furthermore, analysis of the mode of antiviral action suggested a substantial benefit for the efficiency of viral replication at the immediate early (AMPK) or early-late (ABL) phases of HCMV gene expression. In contrast, inhibition of Aurora A kinase promoted an enhancement of viral early-late gene expression, suggesting a putative role of Aurora A signaling in host defense. Thus, the combined data provide new information on host cell kinases involved in viral replication and uncovered potential targets for future antiviral strategies.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Aurora Quinasa A/metabolismo , Infecciones por Citomegalovirus/enzimología , Citomegalovirus/fisiología , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Antivirales/farmacología , Antivirales/uso terapéutico , Aurora Quinasa A/antagonistas & inhibidores , Benzamidas/farmacología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citomegalovirus/genética , Infecciones por Citomegalovirus/virología , Regulación Viral de la Expresión Génica/efectos de los fármacos , Humanos , Mesilato de Imatinib , Péptidos y Proteínas de Señalización Intracelular , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Inhibidores de la Síntesis del Ácido Nucleico/uso terapéutico , Piperazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Pirimidinas/farmacología , Serina-Treonina Quinasa 3 , Replicación Viral/efectos de los fármacosRESUMEN
ST1926 is an atypical retinoid and a promising anti-tumour agent with selective apoptotic activity on the leukaemic blast. The anti-tumour activity of the compound has been associated with its capacity to induce DNA double stranded breaks. Target profiling by affinity chromatography coupled to mass spectrometry led to the identification of histone H2A.Z as a protein capable of binding ST1926 specifically. The result was confirmed by studies involving Surface Plasmon Resonance (SPR). This indicates that H2A.Z is a primary target of ST1926 and links the perturbations of the histone pathway observed by microarray analysis to the DNA damage and apoptotic responses caused by the atypical retinoid. Comparison of the whole-genome gene-expression profiles of the ST1926-sensitive NB4 and the ST1926-resistant NB4.437r cell lines demonstrated differential expression of numerous genes. Network analysis of the data indicated enrichment of the cellular pathways controlling cAMP (cyclic adenosine-monophosphate)-dependent signal transduction, proteasome-dependent protein degradation and nuclear histones in NB4.437r cells. Pharmacological inhibition of cAMP-dependent protein kinase A with H89 partially reverted resistance of NB4.437r cells to ST1926. Conversely, inhibition of the proteasome with MG132 or bortezomib blocked the apoptotic response afforded by ST1926 in the NB4 cell line. This last effect was associated with a dramatic reduction in the DNA damage caused by the atypical retinoid. The results corroborate the idea that DNA damage is an important determinant of ST1926 apoptotic activity. More importantly, they demonstrate a proactive role of the proteasome in the DNA damaging and ensuing apoptotic response observed upon the challenge of acute myeloid leukaemia cells with ST1926.
Asunto(s)
Adamantano/análogos & derivados , Apoptosis/efectos de los fármacos , Cinamatos/farmacología , Enfermedad Aguda , Adamantano/metabolismo , Adamantano/farmacología , Apoptosis/genética , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Cinamatos/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Histonas/genética , Histonas/metabolismo , Humanos , Isoquinolinas/farmacología , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Leupeptinas/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sulfonamidas/farmacología , Resonancia por Plasmón de SuperficieRESUMEN
Targeted drugs are less toxic than traditional chemotherapeutic therapies; however, the proportion of patients that benefit from these drugs is often smaller. A marker that confidently predicts patient response to a specific therapy would allow an individual therapy selection most likely to benefit the patient. Here, we used quantitative mass spectrometry to globally profile the basal phosphoproteome of a panel of non-small cell lung cancer cell lines. The effect of the kinase inhibitor dasatinib on cellular growth was tested against the same panel. From the phosphoproteome profiles, we identified 58 phosphorylation sites, which consistently differ between sensitive and resistant cell lines. Many of the corresponding proteins are involved in cell adhesion and cytoskeleton organization. We showed that a signature of only 12 phosphorylation sites is sufficient to accurately predict dasatinib sensitivity. Four of the phosphorylation sites belong to integrin ß4, a protein that mediates cell-matrix or cell-cell adhesion. The signature was validated in cross-validation and label switch experiments and in six independently profiled breast cancer cell lines. The study supports that the phosphorylation of integrin ß4, as well as eight further proteins comprising the signature, are candidate biomarkers for predicting response to dasatinib in solid tumors. Furthermore, our results show that identifying predictive phosphorylation signatures from global, quantitative phosphoproteomic data is possible and can open a new path to discovering molecular markers for response prediction.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Fosfoproteínas/análisis , Pirimidinas/farmacología , Tiazoles/farmacología , Biomarcadores de Tumor , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Adhesión Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dasatinib , Resistencia a Antineoplásicos , Femenino , Perfilación de la Expresión Génica , Humanos , Integrina beta4/química , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Espectrometría de Masas , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteoma/análisisRESUMEN
A placebo-controlled phase 3 trial demonstrated that the epidermal growth factor receptor (EGFR) inhibitor erlotinib in combination with gemcitabine was especially efficient in a pancreatic ductal adenocarcinoma (PDAC) subgroup of patients developing skin toxicity. However, EGFR expression was not predictive for response, and markers to characterize an erlotinib-responding PDAC group are currently missing. In this work, we observed high erlotinib IC50 values in a panel of human and murine PDAC cell lines. Using EGFR small interfering RNA, we detected that the erlotinib response was marginally influenced by EGFR. To find novel EGFR targets, we used an unbiased chemical proteomics approach for target identification and quality-controlled target affinity determination combined with quantitative mass spectrometry based on stable isotope labeling by amino acids in cell culture. In contrast to gefitinib, we observed a broad target profile of erlotinib in PDAC cells by quantitative proteomics. Six protein kinases bind to erlotinib with similar or higher affinity (K(d) = 0.09-0.358 µM) than the EGFR (K(d) 0.434 µM). We provide evidence that one of the novel erlotinib targets, ARG, contributes in part to the erlotinib response in a PDAC cell line. Our data show that erlotinib is a multikinase inhibitor, which can act independent of EGFR in PDAC. These findings may help to monitor future erlotinib trials in the clinic.
Asunto(s)
Adenocarcinoma/patología , Carcinoma Ductal Pancreático/patología , Neoplasias Pancreáticas/patología , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animales , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Evaluación Preclínica de Medicamentos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Clorhidrato de Erlotinib , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/efectos adversos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quinazolinas/administración & dosificación , Quinazolinas/efectos adversos , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/farmacología , Transfección , Células Tumorales CultivadasRESUMEN
BACKGROUND: Various high throughput methods are available for detecting regulations at the level of transcription, translation or posttranslation (e.g. phosphorylation). Integrating these data with protein networks should make it possible to identify subnetworks that are significantly regulated. Furthermore, such integration can support identification of regulated entities from often noisy high throughput data. In particular, processing mass spectrometry-based phosphoproteomic data in this manner may expose signal transduction pathways and, in the case of experiments with drug-treated cells, reveal the drug's mode of action. RESULTS: Here, we introduce SubExtractor, an algorithm that combines phosphoproteomic data with protein network information from STRING to identify differentially regulated subnetworks and individual proteins. The method is based on a Bayesian probabilistic model combined with a genetic algorithm and rigorous significance testing. The Bayesian model accounts for information about both differential regulation and network topology. The method was tested with artificial data and subsequently applied to a comprehensive phosphoproteomics study investigating the mode of action of sorafenib, a small molecule kinase inhibitor. CONCLUSIONS: SubExtractor reliably identifies differentially regulated subnetworks from phosphoproteomic data by integrating protein networks. The method can also be applied to gene or protein expression data.
Asunto(s)
Algoritmos , Proteómica/métodos , Teorema de Bayes , Perfilación de la Expresión Génica , Modelos Estadísticos , Fosforilación , Transducción de SeñalRESUMEN
Knowledge about molecular drug action is critical for the development of protein kinase inhibitors for cancer therapy. Here, we establish a chemical proteomic approach to profile the anticancer drug SU6668, which was originally designed as a selective inhibitor of receptor tyrosine kinases involved in tumor vascularization. By employing immobilized SU6668 for the affinity capture of cellular drug targets in combination with mass spectrometry, we identified previously unknown targets of SU6668 including Aurora kinases and TANK-binding kinase 1. Importantly, a cell cycle block induced by SU6668 could be attributed to inhibition of Aurora kinase activity. Moreover, SU6668 potently suppressed antiviral and inflammatory responses by interfering with TANK-binding kinase 1-mediated signal transmission. These results show the potential of chemical proteomics to provide rationales for the development of potent kinase inhibitors, which combine rather unexpected biological modes of action by simultaneously targeting defined sets of both serine/threonine and tyrosine kinases involved in cancer progression.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Indoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirroles/farmacología , Animales , Aurora Quinasas , Células COS , División Celular/efectos de los fármacos , Chlorocebus aethiops , Células HeLa , Humanos , Oxindoles , Propionatos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , TransfecciónRESUMEN
Targeted inhibition of protein kinases with small molecule drugs has evolved into a viable approach for anticancer therapy. However, the true selectivity of these therapeutic agents has remained unclear. Here, we used a proteomic method to profile the cellular targets of the clinical epidermal growth factor receptor kinase inhibitor gefitinib. Our data suggest alternative cellular modes of action for gefitinib and provide rationales for the development of related drugs.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Animales , Células COS , Chlorocebus aethiops , Receptores ErbB/antagonistas & inhibidores , Gefitinib , Células HeLa , Humanos , Proteómica/métodos , Relación Estructura-ActividadRESUMEN
Small molecule inhibitors belonging to the pyrido[2,3-d]pyrimidine class of compounds were developed as antagonists of protein tyrosine kinases implicated in cancer progression. Derivatives from this compound class are effective against most of the imatinib mesylate-resistant BCR-ABL mutants isolated from advanced chronic myeloid leukemia patients. Here, we established an efficient proteomics method employing an immobilized pyrido[2,3-d]pyrimidine ligand as an affinity probe and identified more than 30 human protein kinases affected by this class of compounds. Remarkably, in vitro kinase assays revealed that the serine/threonine kinases Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK) and p38alpha were among the most potently inhibited kinase targets. Thus, pyrido[2,3-d]pyrimidines did not discriminate between tyrosine and serine/threonine kinases. Instead, we found that these inhibitors are quite selective for protein kinases possessing a conserved small amino acid residue such as threonine at a critical site of the ATP binding pocket. We further demonstrated inhibition of both p38 and RICK kinase activities in intact cells upon pyrido[2,3-d]pyrimidine inhibitor treatment. Moreover, the established functions of these two kinases as signal transducers of inflammatory responses could be correlated with a potent in vivo inhibition of cytokine production by a pyrido[2,3-d]pyrimidine compound. Thus, our data demonstrate the utility of proteomic methods employing immobilized kinase inhibitors for identifying new targets linked to previously unrecognized therapeutic applications.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteómica/métodos , Piridinas/farmacología , Pirimidinas/farmacología , Adenosina Trifosfato/química , Secuencia de Aminoácidos , Animales , Antineoplásicos/química , Apoptosis , Sitios de Unión , Células COS , Línea Celular Tumoral , Células Cultivadas , Cromatografía Liquida , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/química , Células HeLa , Humanos , Inflamación , Concentración 50 Inhibidora , Ligandos , Lipopolisacáridos/química , Espectrometría de Masas , Modelos Químicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos/metabolismo , Polietilenglicoles/química , Inhibidores de Proteínas Quinasas/química , Piridinas/química , Pirimidinas/química , Homología de Secuencia de Aminoácido , Transducción de Señal , Factores de TiempoRESUMEN
Small-molecule inhibitors of protein kinases constitute a novel class of drugs for therapeutic intervention in a variety of human diseases. Most of these agents target the relatively conserved ATP-binding site of protein kinases and have only been tested against a rather small subset of all human protein kinases. Therefore, the selectivity of protein kinase inhibitors has remained a widely underestimated, but highly important issue in drug development programs. In this review, we focus on the recent advancement of chemical proteomic methods to evaluate drug selectivity in an unbiased, comprehensive way. Efficient affinity purification procedures using immobilized kinase inhibitors combined with the sensitivity of mass spectrometry detection permit the mapping of drug targets on a proteome-wide scale. Data from this type of assessment can be used to set up tailor-made selectivity panels, which guide compound development in the context of the most relevant off-targets during lead optimization. In cases in which identified alternative targets are of validated clinical relevance, chemical proteomics provides the opportunity to repeatedly exploit a once established kinase inhibitor principle for additional target kinases and can thereby dramatically shorten the time toward highly selective, preclinical candidates. Moreover, the identification of alternative targets for preclinical or clinical drugs can provide new insights into their cellular modes of action, which might help to define those disease settings in which the most beneficial therapeutic effect is likely to occur.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Inhibidores de Proteínas Quinasas , Proteómica/métodos , Animales , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/química , Enzimas Inmovilizadas/química , Humanos , Proteínas Quinasas/metabolismoRESUMEN
Bisindolylmaleimide compounds such as GF109203X are potent inhibitors of protein kinase C (PKC) activity. Although bisindolylmaleimides are not entirely selective for PKC and are known to inhibit a few other protein kinases, these reagents have been extensively used to study the functional roles of PKC family enzymes in cellular signal transduction for more than a decade. Here, we establish a proteomics approach to gain further insights into the cellular effects of this compound class. Functional immobilization of suitable bisindolylmaleimide analogues in combination with the specific purification of cellular binding proteins by affinity chromatography led to the identification of several known and previously unknown enzyme targets. Subsequent in vitro binding and activity assays confirmed the protein kinases Ste20-related kinase and cyclin-dependent kinase 2 (CDK2) and the non-protein kinases adenosine kinase and quinone reductase type 2 as novel targets of bisindolylmaleimide inhibitors. As observed specifically for CDK2, minor chemical variation of the ligand by immobilizing the closely related bisindolylmaleimides III, VIII, and X dramatically affected target binding. These observed changes in affinity correlated with both the measured IC(50) values for in vitro CDK2 inhibition and results from molecular docking into the CDK2 crystal structure. Moreover, the conditions for affinity purification could be adapted in a way that immobilized bisindolylmaleimide III selectively interacted with either PKC alpha or ribosomal S6 protein kinase 1 only after activation of these kinases. Thus, we have established an efficient technique for the rapid identification of cellular bisindolylmaleimide targets and further demonstrate the comparative selectivity profiling of closely related kinase inhibitors within a cellular proteome.
Asunto(s)
Quinasas CDC2-CDC28/metabolismo , Indoles/farmacología , Maleimidas/farmacología , Proteína Quinasa C/metabolismo , Proteínas Quinasas/metabolismo , Proteómica , Animales , Quinasas CDC2-CDC28/antagonistas & inhibidores , Células COS , Células Cultivadas , Chlorocebus aethiops , Cromatografía de Afinidad , Quinasa 2 Dependiente de la Ciclina , Inhibidores Enzimáticos/farmacología , Células HeLa , Humanos , Modelos Moleculares , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C-alfa , Inhibidores de Proteínas Quinasas , Quinona Reductasas/antagonistas & inhibidores , Quinona Reductasas/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismoRESUMEN
Small molecule inhibitors of protein kinases have become highly popular tools in signal transduction research, despite the fact that rather limited data about their respective selectivities have been available. We established an efficient chemical proteomics method to characterize the cellular targets of the widely used inhibitor SB 203580, which was deemed to be rather specific for p38 kinase. Our results revealed several protein kinases as high affinity targets of SB 203580 and therefore imply a far more complicated cellular mode of action of this inhibitor than previously assumed. This raises the important question whether a lack of selectivity is inherent to many other "specific" inhibitors of protein kinases and warrants their evaluation employing experimental approaches adapted from our described proteomic technique.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Piridinas/farmacología , Animales , Antiinflamatorios no Esteroideos/farmacología , Cromatografía de Afinidad , Drosophila , Humanos , Fosforilación , Proteoma , Transducción de Señal , Rayos Ultravioleta , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Small molecule inhibitors of protein kinases are widely used in signal transduction research and are emerging as a major class of drugs. Although interpretation of biological results obtained with these reagents critically depends on their selectivity, efficient methods for proteome-wide assessment of kinase inhibitor selectivity have not yet been reported. Here, we address this important issue and describe a method for identifying targets of the widely used p38 kinase inhibitor SB 203580. Immobilization of a suitable SB 203580 analogue and thoroughly optimized biochemical conditions for affinity chromatography permitted the dramatic enrichment and identification of several previously unknown protein kinase targets of SB 203580. In vitro kinase assays showed that cyclin G-associated kinase (GAK) and CK1 were almost as potently inhibited as p38alpha whereas RICK [Rip-like interacting caspase-like apoptosis-regulatory protein (CLARP) kinase/Rip2/CARDIAK] was even more sensitive to inhibition by SB 203580. The cellular kinase activity of RICK, a known signal transducer of inflammatory responses, was already inhibited by submicromolar concentrations of SB 203580 in intact cells. Therefore, our results warrant a reevaluation of the vast amount of data obtained with SB 203580 and might have significant implications on the development of p38 inhibitors as antiinflammatory drugs. Based on the procedures described here, efficient affinity purification techniques can be developed for other protein kinase inhibitors, providing crucial information about their cellular modes of action.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/química , Inhibidores de Proteínas Quinasas , Proteómica , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Espectrometría de Masas , Proteínas Quinasas Activadas por Mitógenos/aislamiento & purificación , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Transfección , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Faced with the current wealth of genomic data, it is essential to have robust and reliable methods of converting DNA sequences into their functional gene products. We demonstrate here that when conditions are established that take advantage of the replication-associated virus amplification, the virus-induced shutdown of host protein synthesis as well as the activation of signalling pathways that normally occur during virus replication, adenovirus biology can be exploited to generate a potent kinase expression system. Residual virus in the protein production has always been a limitation for adenovirus systems and we describe a DNA intercalator/ultraviolet light treatment that eliminates residual adenovirus in protein preparations that has no deleterious effect on enzyme activity. The use of mammalian cells in combination with adenovirus generated a variety of active enzymes which could not be produced in Escherichia coli or baculovirus-infected insect cells. Thus, the utility of adenovirus-mediated enzyme expression as a versatile alternative to established protein production technologies is demonstrated.
Asunto(s)
Adenoviridae/fisiología , Mamíferos , Proteínas Quinasas/biosíntesis , Proteínas Quinasas/genética , Replicación Viral , Adenoviridae/efectos de los fármacos , Adenoviridae/genética , Adenoviridae/efectos de la radiación , Animales , Baculoviridae/genética , Baculoviridae/fisiología , Línea Celular , Cromatografía de Afinidad , Contaminación de Medicamentos , Activación Enzimática , Ingeniería Genética , Humanos , Sustancias Intercalantes/farmacología , Mamíferos/virología , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/aislamiento & purificación , Complejos Multienzimáticos/metabolismo , Proteínas Quinasas/aislamiento & purificación , Proteínas Quinasas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transfección , Rayos UltravioletaRESUMEN
The N terminus of the human MUC2 mucin (amino acids 1-1397) has been expressed as a recombinant tagged protein in Chinese hamster ovary cells. The intracellular form was found to be an endoglycosidase H-sensitive monomer, whereas the secreted form was an oligomer that gave monomers upon disulfide bond reduction. The secreted MUC2 N terminus contained a trypsin-resistant core fragment. Edman sequencing and mass spectrometry of the peptides obtained localized this core fragment to the C-terminal end of the recombinant protein. This core retained its oligomeric nature with an apparent mass of approximately 240 kDa. Upon reduction, peptides of approximately 85 kDa were found, suggesting that the N terminus forms trimers. This interpretation was also supported by gel electrophoresis and gel filtration of the intact MUC2 N terminus. Electron microscopy revealed three globular domains each linked via an extended and flexible region to a central part in a trefoil-like manner. Immunostaining with gold-labeled antibodies localized the N-terminal end to the three globular structures, and the antibodies directed against the Myc and green fluorescent protein tags attached at the C terminus localized these to the stalk side of the central trefoil. The N terminus of the MUC2 mucin is thus assembled into trimers that contain proteolytically stable parts, suggesting that MUC2 can only be partly degraded by intestinal proteases and thus is able to maintain a mucin network protecting the intestine.
Asunto(s)
Mucinas/química , Secuencia de Aminoácidos , Animales , Células CHO , Cromatografía en Gel , Cricetinae , Dimerización , Disulfuros , Electroforesis en Gel de Poliacrilamida , Vectores Genéticos , Glicosilación , Ácido Fluorhídrico/farmacología , Immunoblotting , Microscopía Electrónica , Datos de Secuencia Molecular , Mucina 2 , Mucinas/metabolismo , Mutagénesis Sitio-Dirigida , Péptidos/química , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tripsina/farmacologíaRESUMEN
The alga Volvox carteri is one of the simplest multicellular organisms, yet it has a surprisingly complex extracellular matrix (ECM), making Volvox suitable as a model system in which to study ECM self-assembly. Here, we analyze the primary structures and post-translational modifications of two main ECM components synthesized in response to sexual induction as well as wounding. These proteins are members of the pherophorin family with as yet unknown properties. They contain polyhydroxyproline spacers as long as 500 and 2750 residues. Even the highly purified proteins retain the capacity to self-assemble and cross-link, producing an insoluble fibrous network in an apparently autocatalytic reaction. This pherophorin-based network is located within the deep zone of the ECM. A molecular genetic search for additional members of the pherophorin family indicates that at least nine different pherophorin species can be expected to serve as precursors for ECM substructures. Therefore, the highly diversified members of the pherophorin family represent region-specific morphological building blocks for ECM assembly and cross-linking.