RESUMO
We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.
Assuntos
Formação de Anticorpos/fisiologia , Antígenos/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Proteínas Recombinantes/imunologia , Clonagem Molecular , Humanos , Biblioteca de PeptídeosRESUMO
Antibodies are indispensable tools in biochemical research and play an expanding role as therapeutics. While hybridoma technology is the dominant method for antibody production, phage display is an emerging technology. Here, we developed and employed a high-throughput pipeline that enables selection of antibodies against hundreds of antigens in parallel. Binding selections using a phage-displayed synthetic antigen-binding fragment (Fab) library against 110 human SH3 domains yielded hundreds of Fabs targeting 58 antigens. Affinity assays demonstrated that representative Fabs bind tightly and specifically to their targets. Furthermore, we developed an efficient affinity maturation strategy adaptable to high-throughput, which increased affinity dramatically but did not compromise specificity. Finally, we tested Fabs in common cell biology applications and confirmed recognition of the full-length antigen in immunoprecipitation, immunoblotting and immunofluorescence assays. In summary, we have established a rapid and robust high-throughput methodology that can be applied to generate highly functional and renewable antibodies targeting protein domains on a proteome-wide scale.
Assuntos
Anticorpos/química , Técnicas de Visualização da Superfície Celular/métodos , Ensaios de Triagem em Larga Escala/métodos , Proteínas Recombinantes de Fusão/química , Domínios de Homologia de src/genética , Sequência de Aminoácidos , Anticorpos/genética , Anticorpos/metabolismo , Células HEK293 , Humanos , Dados de Sequência Molecular , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
Previous publications from our laboratory have introduced novel inhibitors of Polo-like kinase 4 (PLK4), a mitotic kinase identified as a potential target for cancer therapy. The search for potent and selective PLK4 inhibitors yielded (E)-3-((1Hindazol-6-yl)methylene)indolin-2-ones, which were superseded by the bioisosteric 2-(1H-indazol-6-yl)spiro[cyclopropane-1,3'-indolin]-2'-ones, e.g., 3. The later scaffold confers improved drug-like properties and incorporates two stereogenic centers. This work reports the discovery of a novel one-pot double SN2 displacement reaction for the stereoselective installation of the desired asymmetric centers and confirms the stereochemistry of the most potent stereoisomer, e.g., 44. Subsequent work keys on the optimization of the oral exposure of nanomolar PLK4 inhibitors with potent cancer cell growth inhibitory activity. A short list of compounds with superior potency and pharmacokinetic properties in rodents and dogs was studied in mouse models of tumor growth. We conclude with the identification of compound 48 (designated CFI-400945) as a novel clinical candidate for cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Indazóis/farmacologia , Indóis/farmacologia , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/análise , Administração Oral , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Descoberta de Drogas , Feminino , Células HCT116 , Humanos , Indazóis/química , Indazóis/farmacocinética , Indóis/química , Indóis/farmacocinética , Células MCF-7 , Masculino , Camundongos Nus , Camundongos SCID , Modelos Químicos , Estrutura Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
TTK kinase was identified by in-house siRNA screen and pursued as a tractable, novel target for cancer treatment. A screening campaign and systematic optimization, supported by computer modeling led to an indazole core with key sulfamoylphenyl and acetamido moieties at positions 3 and 5, respectively, establishing a novel chemical class culminating in identification of 72 (CFI-400936). This potent inhibitor of TTK (IC50=3.6nM) demonstrated good activity in cell based assay and selectivity against a panel of human kinases. A co-complex TTK X-ray crystal structure and results of a xenograft study with TTK inhibitors from this class are described.
Assuntos
Amidas/farmacologia , Benzenoacetamidas/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Descoberta de Drogas , Indazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Amidas/síntese química , Amidas/química , Benzenoacetamidas/síntese química , Benzenoacetamidas/química , Proteínas de Ciclo Celular/metabolismo , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Indazóis/síntese química , Indazóis/química , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Relação Estrutura-AtividadeRESUMO
Tumor cells gain a survival/growth advantage by adapting their metabolism to respond to environmental stress, a process known as metabolic transformation. The best-known aspect of metabolic transformation is the Warburg effect, whereby cancer cells up-regulate glycolysis under aerobic conditions. However, other mechanisms mediating metabolic transformation remain undefined. Here we report that carnitine palmitoyltransferase 1C (CPT1C), a brain-specific metabolic enzyme, may participate in metabolic transformation. CPT1C expression correlates inversely with mammalian target of rapamycin (mTOR) pathway activation, contributes to rapamycin resistance in murine primary tumors, and is frequently up-regulated in human lung tumors. Tumor cells constitutively expressing CPT1C show increased fatty acid (FA) oxidation, ATP production, and resistance to glucose deprivation or hypoxia. Conversely, cancer cells lacking CPT1C produce less ATP and are more sensitive to metabolic stress. CPT1C depletion via siRNA suppresses xenograft tumor growth and metformin responsiveness in vivo. CPT1C can be induced by hypoxia or glucose deprivation and is regulated by AMPKα. Cpt1c-deficient murine embryonic stem (ES) cells show sensitivity to hypoxia and glucose deprivation and altered FA homeostasis. Our results indicate that cells can use a novel mechanism involving CPT1C and FA metabolism to protect against metabolic stress. CPT1C may thus be a new therapeutic target for the treatment of hypoxic tumors.
Assuntos
Carnitina O-Palmitoiltransferase/metabolismo , Estresse Fisiológico/fisiologia , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Apoptose/fisiologia , Carnitina O-Palmitoiltransferase/deficiência , Carnitina O-Palmitoiltransferase/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Células Cultivadas , Resistencia a Medicamentos Antineoplásicos/genética , Células-Tronco Embrionárias/enzimologia , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Hipóxia/patologia , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Camundongos , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Estresse Fisiológico/genética , Serina-Treonina Quinases TOR/metabolismo , Transplante Heterólogo , Regulação para CimaRESUMO
Overexpression of fibroblast growth factor receptor 3 (FGFR3) is a hallmark of t(4;14) multiple myeloma (MM). To dissect the mechanism of FGFR3 oncogenesis in MM, we used 3 FGFR selective kinase inhibitors-CHIR258, PD173074, and SU5402-and FGFR3-specific siRNA to modulate FGFR3 activity. Conversely, the ligand FGF was used to stimulate FGFR3 function in human MM cells. The transcriptional response to FGFR3 modification was recorded, and gene expression changes common to all 5 modifiers were documented. Ten genes were commonly regulated. Macrophage inflammatory protein-1 alpha (MIP-1alpha) was the single most differentially altered gene. MIP-1 alpha promoter function, gene expression, and protein secretion were each down-regulated following inhibition of FGFR3 signaling. Down-regulation of MIP-1 alpha was not, however, observed following FGFR3 inhibition in MM cells with RAS mutations implicating RAS-MAPK in MIP-1 alpha regulation. As confirmation, inhibition of ERK1 also down-regulated MIP-1 alpha in FGFR3 inhibitor-resistant cells harboring RAS mutations. MIP-1 alpha is implicated in the survival and proliferation of MM cells and the pathogenesis of MM bone disease. Our observation is the first to directly link an initiating IgH translocation not only to MM-cell growth and survival but also to the disease-associated bone disease.
Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Inflamatórias de Macrófagos/metabolismo , Mieloma Múltiplo/etiologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Proteínas ras/metabolismo , Doenças Ósseas/etiologia , Quimiocina CCL3 , Quimiocina CCL4 , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Inflamatórias de Macrófagos/genética , Mieloma Múltiplo/complicações , Mieloma Múltiplo/genética , Mutação , Proteínas de Neoplasias , RNA Interferente Pequeno/farmacologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/fisiologia , Células Tumorais Cultivadas , Proteínas ras/genéticaRESUMO
We have created a molecular resource of genes expressed in primary malignant plasma cells using a combination of cDNA library construction, 5' end single-pass sequencing, bioinformatics, and microarray analysis. In total, we identified 9732 nonredundant expressed genes. This dataset is available as the Myeloma Gene Index (www.uhnres.utoronto.ca/akstewart_lab).Predictably, the sequenced profile of myeloma cDNAs mirrored the known function of immunoglobulin-producing, high-respiratory rate, low-cycling, terminally differentiated plasma cells. Nevertheless, approximately 10% of myeloma-expressed sequences matched only entries in the database of Expressed Sequence Tags (dbEST) or the high-throughput genomic sequence (htgs) database. Numerous novel genes of potential biologic significance were identified. We therefore spotted 4300 sequenced cDNAs on glass slides creating a myeloma-enriched microarray. Several of the most highly expressed genes identified by sequencing, such as a novel putative disulfide isomerase (MGC3178), tumor rejection antigen TRA1, heat shock 70-kDa protein 5, and annexin A2, were also differentially expressed between myeloma and B lymphoma cell lines using this myeloma-enriched microarray. Furthermore, a defined subset of 34 up-regulated and 18 down-regulated genes on the array were able to differentiate myeloma from nonmyeloma cell lines. These not only include genes involved in B-cell biology such as syndecan, BCMA, PIM2, MUM1/IRF4, and XBP1, but also novel uncharacterized genes matching sequences only in the public databases. In summary, our expressed gene catalog and myeloma-enriched microarray contains numerous genes of unknown function and may complement other commercially available arrays in defining the molecular portrait of this hematopoietic malignancy. GenBank Accession numbers include BF169967-BF176369, BF185966-BF185969, and BF177280-BF177455.