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1.
Biol Proced Online ; 25(1): 4, 2023 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-36814189

RESUMO

BACKGROUND: Intraneuronal tau aggregation is the major pathological hallmark of neurodegenerative tauopathies. It is now generally acknowledged that tau aggregation also affects astrocytes in a cell non-autonomous manner. However, mechanisms involved are unclear, partly because of the lack of models that reflect the situation in the human tauopathy brain. To accurately model neuron-astrocyte interaction in tauopathies, there is a need for a model that contains both human neurons and human astrocytes, intraneuronal tau pathology and mimics the three-dimensional architecture of the brain. RESULTS: Here we established a novel 100-200 µm thick 3D human neuron/astrocyte co-culture model of tau pathology, comprising homogenous populations of hiPSC-derived neurons and primary human astrocytes in microwell format. Using confocal, electron and live microscopy, we validate the procedures by showing that neurons in the 3D co-culture form pre- and postsynapses and display spontaneous calcium transients within 4 weeks. Astrocytes in the 3D co-culture display bipolar and stellate morphologies with extensive processes that ensheath neuronal somas, spatially align with axons and dendrites and can be found perisynaptically. The complex morphology of astrocytes and the interaction with neurons in the 3D co-culture mirrors that in the human brain, indicating the model's potential to study physiological and pathological neuron-astrocyte interaction in vitro. Finally, we successfully implemented a methodology to introduce seed-independent intraneuronal tau aggregation in the 3D co-culture, enabling study of neuron-astrocyte interaction in early tau pathogenesis. CONCLUSIONS: Altogether, these data provide proof-of-concept for the utility of this rapid, miniaturized, and standardized 3D model for cell type-specific manipulations, such as the intraneuronal pathology that is associated with neurodegenerative disorders.

2.
J Pathol ; 236(3): 348-59, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25757065

RESUMO

Conventional high-grade osteosarcoma is the most common primary bone sarcoma, with relatively high incidence in young people. In this study we found that expression of Aven correlates inversely with metastasis-free survival in osteosarcoma patients and is increased in metastases compared to primary tumours. Aven is an adaptor protein that has been implicated in anti-apoptotic signalling and serves as an oncoprotein in acute lymphoblastic leukaemia. In osteosarcoma cells, silencing Aven triggered G2 cell-cycle arrest; Chk1 protein levels were attenuated and ATR-Chk1 DNA damage response signalling in response to chemotherapy was abolished in Aven-depleted osteosarcoma cells, while ATM, Chk2 and p53 activation remained intact. Osteosarcoma is notoriously difficult to treat with standard chemotherapy, and we examined whether pharmacological inhibition of the Aven-controlled ATR-Chk1 response could sensitize osteosarcoma cells to genotoxic compounds. Indeed, pharmacological inhibitors targeting Chk1/Chk2 or those selective for Chk1 synergized with standard chemotherapy in 2D cultures. Likewise, in 3D extracellular matrix-embedded cultures, Chk1 inhibition led to effective sensitization to chemotherapy. Together, these findings implicate Aven in ATR-Chk1 signalling and point towards Chk1 inhibition as a strategy to sensitize human osteosarcomas to chemotherapy.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Neoplasias Ósseas/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Membrana/genética , Osteossarcoma/genética , Proteínas Quinases/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antibióticos Antineoplásicos/farmacologia , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Dano ao DNA , Doxorrubicina/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular , Perfilação da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Osteossarcoma/tratamento farmacológico , Osteossarcoma/patologia , Fosforilação , Proteínas Quinases/metabolismo , Interferência de RNA , Transdução de Sinais , Tiofenos/farmacologia , Ureia/análogos & derivados , Ureia/farmacologia
3.
Mol Cancer ; 14: 147, 2015 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-26227951

RESUMO

BACKGROUND: Monolayer cultures of immortalised cell lines are a popular screening tool for novel anti-cancer therapeutics, but these methods can be a poor surrogate for disease states, and there is a need for drug screening platforms which are more predictive of clinical outcome. In this study, we describe a phenotypic antibody screen using three-dimensional cultures of primary cells, and image-based multi-parametric profiling in PC-3 cells, to identify anti-cancer biologics against new therapeutic targets. METHODS: ScFv Antibodies and designed ankyrin repeat proteins (DARPins) were isolated using phage display selections against primary non-small cell lung carcinoma cells. The selected molecules were screened for anti-proliferative and pro-apoptotic activity against primary cells grown in three-dimensional culture, and in an ultra-high content screen on a 3-D cultured cell line using multi-parametric profiling to detect treatment-induced phenotypic changes. The targets of molecules of interest were identified using a cell-surface membrane protein array. An anti-CUB domain containing protein 1 (CDCP1) antibody was tested for tumour growth inhibition in a patient-derived xenograft model, generated from a stage-IV non-small cell lung carcinoma, with and without cisplatin. RESULTS: Two primary non-small cell lung carcinoma cell models were established for antibody isolation and primary screening in anti-proliferative and apoptosis assays. These assays identified multiple antibodies demonstrating activity in specific culture formats. A subset of the DARPins was profiled in an ultra-high content multi-parametric screen, where 300 morphological features were measured per sample. Machine learning was used to select features to classify treatment responses, then antibodies were characterised based on the phenotypes that they induced. This method co-classified several DARPins that targeted CDCP1 into two sets with different phenotypes. Finally, an anti-CDCP1 antibody significantly enhanced the efficacy of cisplatin in a patient-derived NSCLC xenograft model. CONCLUSIONS: Phenotypic profiling using complex 3-D cell cultures steers hit selection towards more relevant in vivo phenotypes, and may shed light on subtle mechanistic variations in drug candidates, enabling data-driven decisions for oncology target validation. CDCP1 was identified as a potential target for cisplatin combination therapy.


Assuntos
Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Descoberta de Drogas/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Neoplasias , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas , Moléculas de Adesão Celular/antagonistas & inibidores , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Visualização da Superfície Celular , Cisplatino/farmacologia , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares , Camundongos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , Biblioteca de Peptídeos , Fenótipo , Anticorpos de Cadeia Única/farmacologia , Esferoides Celulares , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
4.
J Am Soc Nephrol ; 25(7): 1474-85, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24511123

RESUMO

Activation of Rap1 by exchange protein activated by cAMP (Epac) promotes cell adhesion and actin cytoskeletal polarization. Pharmacologic activation of Epac-Rap signaling by the Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP during ischemia-reperfusion (IR) injury reduces renal failure and application of 8-pCPT-2'-O-Me-cAMP promotes renal cell survival during exposure to the nephrotoxicant cisplatin. Here, we found that activation of Epac by 8-pCPT-2'-O-Me-cAMP reduced production of reactive oxygen species during reoxygenation after hypoxia by decreasing mitochondrial superoxide production. Epac activation prevented disruption of tubular morphology during diethyl maleate-induced oxidative stress in an organotypic three-dimensional culture assay. In vivo renal targeting of 8-pCPT-2'-O-Me-cAMP to proximal tubules using a kidney-selective drug carrier approach resulted in prolonged activation of Rap1 compared with nonconjugated 8-pCPT-2'-O-Me-cAMP. Activation of Epac reduced antioxidant signaling during IR injury and prevented tubular epithelial injury, apoptosis, and renal failure. Our data suggest that Epac1 decreases reactive oxygen species production by preventing mitochondrial superoxide formation during IR injury, thus limiting the degree of oxidative stress. These findings indicate a new role for activation of Epac as a therapeutic application in renal injury associated with oxidative stress.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/fisiologia , Túbulos Renais Proximais/metabolismo , Estresse Oxidativo , Urotélio/metabolismo , Animais , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Fatores de Troca do Nucleotídeo Guanina/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Urotélio/efeitos dos fármacos
5.
Arch Toxicol ; 88(5): 1083-95, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24599296

RESUMO

Immortalized hepatocyte cell lines show only a weak resemblance to primary hepatocytes in terms of gene expression and function, limiting their value in predicting drug-induced liver injury (DILI). Furthermore, primary hepatocytes cultured on two-dimensional tissue culture plastic surfaces rapidly dedifferentiate losing their hepatocyte functions and metabolic competence. We have developed a three-dimensional in vitro model using extracellular matrix-based hydrogel for long-term culture of the human hepatoma cell line HepG2. HepG2 cells cultured in this model stop proliferating, self-organize and differentiate to form multiple polarized spheroids. These spheroids re-acquire lost hepatocyte functions such as storage of glycogen, transport of bile salts and the formation of structures resembling bile canaliculi. HepG2 spheroids also show increased expression of albumin, urea, xenobiotic transcription factors, phase I and II drug metabolism enzymes and transporters. Consistent with this, cytochrome P450-mediated metabolism is significantly higher in HepG2 spheroids compared to monolayer cultures. This highly differentiated phenotype can be maintained in 384-well microtiter plates for at least 28 days. Toxicity assessment studies with this model showed an increased sensitivity in identifying hepatotoxic compounds with repeated dosing regimens. This simple and robust high-throughput-compatible methodology may have potential for use in toxicity screening assays and mechanistic studies and may represent an alternative to animal models for studying DILI.


Assuntos
Células Hep G2/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Testes de Toxicidade/métodos , Albuminas/metabolismo , Canalículos Biliares/efeitos dos fármacos , Canalículos Biliares/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , Humanos , Inativação Metabólica/genética , Fígado/metabolismo , Esferoides Celulares , Ureia/metabolismo
6.
J Pharmacol Exp Ther ; 342(1): 119-30, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22490379

RESUMO

Microtubule-destabilizing agents, such as vinca alkaloids (VAs), are part of the treatment currently applied in patients with high-risk neuroblastoma (NB). However, the development of drug resistance and toxicity make NB difficult to treat with these drugs. In this study we explore the combination of VAs (vincristine or vinblastine) with knockdown of the microtubule-associated proteins encoded by the doublecortin-like kinase (DCLK) gene by using short interference RNA (siRNA). We examined the effect of VAs and DCLK knockdown on the microtubule network by immunohistochemistry. We performed dose-response studies on cell viability and proliferation. By combining VA with DCLK knockdown we observed a strong reduction in the EC(50) to induce cell death: up to 7.3-fold reduction of vincristine and 21.1-fold reduction of vinblastine. Using time-lapse imaging of phosphatidylserine translocation and a terminal deoxynucleotidyl transferase dUTP nick-end labeling-based assay, we found a significant increase of apoptosis by the combined treatment. Induction of caspase-3 activity, as detected via cleavage of N-acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin, showed a 3.3- to 12.0-fold increase in the combined treatment. We detected significant increases in caspase-8 activity as well. Moreover, the multidrug dose effect calculated by using the median effect method showed a strong synergistic inhibition of proliferation and induction of apoptosis at most of the combined concentrations of siRNAs and VAs. Together, our data demonstrate that the silencing of DCLK sensitizes NB cells to VAs, resulting in a synergetic apoptotic effect.


Assuntos
Apoptose/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Alcaloides de Vinca/farmacologia , Animais , Apoptose/genética , Caspase 3/genética , Caspase 3/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Quinases Semelhantes a Duplacortina , Sinergismo Farmacológico , Inativação Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/genética , Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fosfatidilserinas/genética , Fosfatidilserinas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Vimblastina/farmacologia , Vincristina/farmacologia
7.
Am J Pathol ; 179(6): 2766-78, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21982831

RESUMO

Renal ischemia/reperfusion (I/R) injury is associated with cell matrix and focal adhesion remodeling. Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that localizes at focal adhesions and regulates their turnover. Here, we investigated the role of FAK in renal I/R injury, using a novel conditional proximal tubule-specific fak-deletion mouse model. Tamoxifen treatment of FAK(loxP/loxP)//γGT-Cre-ER(T2) mice caused renal-specific fak recombination (FAK(ΔloxP/ΔloxP)) and reduction of FAK expression in proximal tubules. In FAK(ΔloxP/ΔloxP) mice compared with FAK(loxP/loxP) controls, unilateral renal ischemia followed by reperfusion resulted in less tubular damage with reduced tubular cell proliferation and lower expression of kidney injury molecule-1, which was independent from the postischemic inflammatory response. Oxidative stress is involved in the pathophysiology of I/R injury. Primary cultured mouse renal cells were used to study the role of FAK deficiency for oxidative stress in vitro. The conditional fak deletion did not affect cell survival after hydrogen peroxide-induced cellular stress, whereas it impaired the recovery of focal adhesions that were disrupted by hydrogen peroxide. This was associated with reduced c-Jun N-terminal kinase-dependent phosphorylation of paxillin at serine 178 in FAK-deficient cells, which is required for focal adhesion turnover. Our findings support a role for FAK as a novel factor in the initiation of c-Jun N-terminal kinase-mediated cellular stress response during renal I/R injury and suggest FAK as a target in renal injury protection.


Assuntos
Injúria Renal Aguda/enzimologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Traumatismo por Reperfusão/enzimologia , Transdução de Sinais/fisiologia , Animais , Adesão Celular/fisiologia , Citocinas/biossíntese , Inibidores Enzimáticos/farmacologia , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Proteína-Tirosina Quinases de Adesão Focal/deficiência , Peróxido de Hidrogênio/farmacologia , Túbulos Renais Proximais/enzimologia , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Camundongos , Camundongos Knockout , Nefrite/enzimologia , Oxidantes/farmacologia , Estresse Oxidativo/fisiologia , Tamoxifeno/farmacologia
8.
J Am Soc Nephrol ; 22(5): 859-72, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21493776

RESUMO

Renal ischemia-reperfusion injury is associated with the loss of tubular epithelial cell-cell and cell-matrix interactions which contribute to renal failure. The Epac-Rap signaling pathway is a potent regulator of cell-cell and cell-matrix adhesion. The cyclic AMP analogue 8-pCPT-2'-O-Me-cAMP has been shown to selectively activate Epac, whereas the addition of an acetoxymethyl (AM) ester to 8-pCPT-2'-O-Me-cAMP enhanced in vitro cellular uptake. Here we demonstrate that pharmacological activation of Epac-Rap signaling using acetoxymethyl-8-pCPT-2'-O-Me-cAMP preserves cell adhesions during hypoxia in vitro, maintaining the barrier function of the epithelial monolayer. Intrarenal administration in vivo of 8-pCPT-2'-O-Me-cAMP also reduced renal failure in a mouse model for ischemia-reperfusion injury. This was accompanied by decreased expression of the tubular cell stress marker clusterin-α, and lateral expression of ß-catenin after ischemia indicative of sustained tubular barrier function. Our study emphasizes the undervalued importance of maintaining tubular epithelial cell adhesion in renal ischemia and demonstrates the potential of pharmacological modulation of cell adhesion as a new therapeutic strategy to reduce the extent of injury in kidney disease and transplantation.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/fisiologia , Isquemia/complicações , Rim/irrigação sanguínea , Insuficiência Renal/etiologia , Transdução de Sinais/fisiologia , Estresse Fisiológico , Proteínas rap1 de Ligação ao GTP/fisiologia , Junções Aderentes/fisiologia , Animais , Adesão Celular , Hipóxia Celular , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Adesões Focais , Túbulos Renais Proximais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL
9.
Nat Cancer ; 3(4): 418-436, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35469014

RESUMO

Patient-derived organoids (PDOs) recapitulate tumor architecture, contain cancer stem cells and have predictive value supporting personalized medicine. Here we describe a large-scale functional screen of dual-targeting bispecific antibodies (bAbs) on a heterogeneous colorectal cancer PDO biobank and paired healthy colonic mucosa samples. More than 500 therapeutic bAbs generated against Wingless-related integration site (WNT) and receptor tyrosine kinase (RTK) targets were functionally evaluated by high-content imaging to capture the complexity of PDO responses. Our drug discovery strategy resulted in the generation of MCLA-158, a bAb that specifically triggers epidermal growth factor receptor degradation in leucine-rich repeat-containing G-protein-coupled receptor 5-positive (LGR5+) cancer stem cells but shows minimal toxicity toward healthy LGR5+ colon stem cells. MCLA-158 exhibits therapeutic properties such as growth inhibition of KRAS-mutant colorectal cancers, blockade of metastasis initiation and suppression of tumor outgrowth in preclinical models for several epithelial cancer types.


Assuntos
Anticorpos Biespecíficos , Neoplasias Epiteliais e Glandulares , Anticorpos Biespecíficos/farmacologia , Receptores ErbB/metabolismo , Humanos , Imidazóis , Neoplasias Epiteliais e Glandulares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Organoides , Pirazinas , Receptores Acoplados a Proteínas G/metabolismo
10.
Sci Rep ; 11(1): 7259, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33790333

RESUMO

Screening for effective candidate drugs for breast cancer has shifted from two-dimensional (2D) to three-dimensional (3D) cultures. Here we systematically compared the transcriptomes of these different culture conditions by RNAseq of 14 BC cell lines cultured in both 2D and 3D conditions. All 3D BC cell cultures demonstrated increased mitochondrial metabolism and downregulated cell cycle programs. Luminal BC cells in 3D demonstrated overall limited reprogramming. 3D basal B BC cells showed increased expression of extracellular matrix (ECM) interaction genes, which coincides with an invasive phenotype not observed in other BC cells. Genes downregulated in 3D were associated with metastatic disease progression in BC patients, including cyclin dependent kinases and aurora kinases. Furthermore, the overall correlation of the cell line transcriptome to the BC patient transcriptome was increased in 3D cultures for all TNBC cell lines. To define the most optimal culture conditions to study the oncogenic pathway of interest, an open source bioinformatics strategy was established.


Assuntos
Neoplasias da Mama , Reprogramação Celular , Sistemas de Liberação de Medicamentos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos
11.
Sci Rep ; 10(1): 1672, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015419

RESUMO

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common monogenic disorders, characterized by the progressive formation of fluid-filled cysts. Tolvaptan is an approved drug for ADPKD patients, but is also associated with multiple side effects. The peroxisome proliferator-activator receptor gamma (PPARγ) agonist pioglitazone slows disease progression in the PCK rat model for PKD. Here, we tested whether a combination treatment of relevant doses of tolvaptan and pioglitazone leads to improved efficacy in an adult-onset PKD mouse model. Tolvaptan indeed slowed PKD progression, but the combination treatment was not more effective than tolvaptan alone. In addition, although pioglitazone raised plasma levels of its surrogate drug marker adiponectin, the drug unexpectedly failed to slow PKD progression. The pioglitazone target PPARγ was expressed at surprisingly low levels in mouse, rat and human kidneys. Other pioglitazone targets were more abundantly expressed, but this pattern was comparable across various species. The data suggest that several potential pharmacokinetic and pharmacodynamic (PK/PD) differences between different species may underlie whether or not pioglitazone is able to slow PKD progression. The ongoing phase II clinical trial with low-dose pioglitazone treatment (NCT02697617) will show whether pioglitazone is a suitable drug candidate for ADPKD treatment.


Assuntos
Cistos/tratamento farmacológico , Rim/efeitos dos fármacos , Pioglitazona/farmacologia , Rim Policístico Autossômico Dominante/tratamento farmacológico , Tolvaptan/farmacologia , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos/farmacologia , Técnicas de Cultura de Células/métodos , Terapia Combinada/métodos , Cistos/metabolismo , Progressão da Doença , Humanos , Rim/metabolismo , Masculino , Camundongos , PPAR gama/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Ratos , Ratos Wistar
12.
EBioMedicine ; 51: 102585, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31879244

RESUMO

BACKGROUND: Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common causes of end-stage renal failure, caused by mutations in PKD1 or PKD2 genes. Tolvaptan, the only drug approved for ADPKD treatment, results in serious side-effects, warranting the need for novel drugs. METHODS: In this study, we applied RNA-sequencing of Pkd1cko mice at different disease stages, and with/without drug treatment to identify genes involved in ADPKD progression that were further used to identify novel drug candidates for ADPKD. We followed an integrative computational approach using a combination of gene expression profiling, bioinformatics and cheminformatics data. FINDINGS: We identified 1162 genes that had a normalized expression after treating the mice with drugs proven effective in preclinical models. Intersecting these genes with target affinity profiles for clinically-approved drugs in ChEMBL, resulted in the identification of 116 drugs targeting 29 proteins, of which several are previously linked to Polycystic Kidney Disease such as Rosiglitazone. Further testing the efficacy of six candidate drugs for inhibition of cyst swelling using a human 3D-cyst assay, revealed that three of the six had cyst-growth reducing effects with limited toxicity. INTERPRETATION: Our data further establishes drug repurposing as a robust drug discovery method, with three promising drug candidates identified for ADPKD treatment (Meclofenamic Acid, Gamolenic Acid and Birinapant). Our strategy that combines multiple-omics data, can be extended for ADPKD and other diseases in the future. FUNDING: European Union's Seventh Framework Program, Dutch Technology Foundation Stichting Technische Wetenschappen and the Dutch Kidney Foundation.


Assuntos
Perfilação da Expressão Gênica , Rim Policístico Autossômico Dominante/tratamento farmacológico , Rim Policístico Autossômico Dominante/genética , Animais , Progressão da Doença , Regulação da Expressão Gênica , Rim/metabolismo , Rim/patologia , Camundongos , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Transdução de Sinais/efeitos dos fármacos
13.
J Mol Cell Biol ; 12(8): 644-653, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31065693

RESUMO

Polycystic kidney disease (PKD) is a prevalent genetic disorder, characterized by the formation of kidney cysts that progressively lead to kidney failure. The currently available drug tolvaptan is not well tolerated by all patients and there remains a strong need for alternative treatments. The signaling rewiring in PKD that drives cyst formation is highly complex and not fully understood. As a consequence, the effects of drugs are sometimes difficult to predict. We previously established a high throughput microscopy phenotypic screening method for quantitative assessment of renal cyst growth. Here, we applied this 3D cyst growth phenotypic assay and screened 2320 small drug-like molecules, including approved drugs. We identified 81 active molecules that inhibit cyst growth. Multi-parametric phenotypic profiling of the effects on 3D cultured cysts discriminated molecules that showed preferred pharmacological effects above genuine toxicological properties. Celastrol, a triterpenoid from Tripterygium Wilfordii, was identified as a potent inhibitor of cyst growth in vitro. In an in vivo iKspCre-Pkd1lox,lox mouse model for PKD, celastrol inhibited the growth of renal cysts and maintained kidney function.


Assuntos
Avaliação Pré-Clínica de Medicamentos , Triterpenos Pentacíclicos/uso terapêutico , Doenças Renais Policísticas/tratamento farmacológico , Animais , Cistos/patologia , Cistos/fisiopatologia , Testes de Função Renal , Camundongos , Triterpenos Pentacíclicos/farmacologia , Fenótipo , Doenças Renais Policísticas/patologia , Doenças Renais Policísticas/fisiopatologia , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico
14.
PLoS One ; 15(8): e0235319, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32810173

RESUMO

Aberrant activation of the Wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers. The development of inhibitors of Wnt signalling has been the focus of multiple drug discovery programs targeting colorectal cancer and other malignancies associated with aberrant pathway activation. However, progression of new clinical entities targeting the Wnt pathway has been slow. One challenge lies with the limited predictive power of 2D cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity. In particular, the relationship between 2D cancer cell biology and cancer stem cell function is poorly understood. By contrast, 3D tumour organoids provide a platform in which complex cell-cell interactions can be studied. However, complex 3D models provide a challenging platform for the quantitative analysis of drug responses of therapies that have differential effects on tumour cell subpopulations. Here, we generated tumour organoids from colorectal cancer patients and tested their responses to inhibitors of Tankyrase (TNKSi) which are known to modulate Wnt signalling. Using compounds with 3 orders of magnitude difference in cellular mechanistic potency together with image-based assays, we demonstrate that morphometric analyses can capture subtle alterations in organoid responses to Wnt inhibitors that are consistent with activity against a cancer stem cell subpopulation. Overall our study highlights the value of phenotypic readouts as a quantitative method to asses drug-induced effects in a relevant preclinical model.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Organoides/efeitos dos fármacos , Tanquirases/antagonistas & inibidores , Adulto , Animais , Antineoplásicos/uso terapêutico , Neoplasias Colorretais/patologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Organoides/patologia
15.
J Cell Biol ; 160(4): 487-93, 2003 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-12578910

RESUMO

cAMP controls many cellular processes mainly through the activation of protein kinase A (PKA). However, more recently PKA-independent pathways have been established through the exchange protein directly activated by cAMP (Epac), a guanine nucleotide exchange factor for the small GTPases Rap1 and Rap2. In this report, we show that cAMP can induce integrin-mediated cell adhesion through Epac and Rap1. Indeed, when Ovcar3 cells were treated with cAMP, cells adhered more rapidly to fibronectin. This cAMP effect was insensitive to the PKA inhibitor H-89. A similar increase was observed when the cells were transfected with Epac. Both the cAMP effect and the Epac effect on cell adhesion were abolished by the expression of Rap1-GTPase-activating protein, indicating the involvement of Rap1 in the signaling pathway. Importantly, a recently characterized cAMP analogue, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate, which specifically activates Epac but not PKA, induced Rap-dependent cell adhesion. Finally, we demonstrate that external stimuli of cAMP signaling, i.e., isoproterenol, which activates the G alpha s-coupled beta 2-adrenergic receptor can induce integrin-mediated cell adhesion through the Epac-Rap1 pathway. From these results we conclude that cAMP mediates receptor-induced integrin-mediated cell adhesion to fibronectin through the Epac-Rap1 signaling pathway.


Assuntos
Adesão Celular/fisiologia , AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Integrinas/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/metabolismo , Agonistas Adrenérgicos beta/metabolismo , Carcinoma , AMP Cíclico/análogos & derivados , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Fibronectinas/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Isoproterenol/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias Ovarianas , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas , Proteínas rap1 de Ligação ao GTP/genética
16.
SLAS Discov ; 24(6): 615-627, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30817892

RESUMO

The introduction of more relevant cell models in early preclinical drug discovery, combined with high-content imaging and automated analysis, is expected to increase the quality of compounds progressing to preclinical stages in the drug development pipeline. In this review we discuss the current switch to more relevant 3D cell culture models and associated challenges for high-throughput screening and high-content analysis. We propose that overcoming these challenges will enable front-loading the drug discovery pipeline with better biology, extracting the most from that biology, and, in general, improving translation between in vitro and in vivo models. This is expected to reduce the proportion of compounds that fail in vivo testing due to a lack of efficacy or to toxicity.


Assuntos
Técnicas de Cultura de Células , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala , Antineoplásicos/farmacologia , Automação Laboratorial , Linhagem Celular Tumoral , Descoberta de Drogas/normas , Avaliação Pré-Clínica de Medicamentos/normas , Ensaios de Triagem em Larga Escala/métodos , Ensaios de Triagem em Larga Escala/normas , Humanos , Processamento de Imagem Assistida por Computador , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Microscopia Confocal , Controle de Qualidade
17.
Antibodies (Basel) ; 7(2)2018 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-31544867

RESUMO

Antibody-drug conjugates (ADCs) offer a combination of antibody therapy and specific delivery of potent small-molecule payloads to target cells. The properties of the ADC molecule are determined by the balance of its components. The efficacy of the payload component increases with higher drug-to-antibody ratio (DAR), while homogeneous DAR = 8 ADCs are easily prepared by conjugation to the four accessible antibody hinge cystines. However, use of hydrophobic payloads has permitted only DAR = 2-4, due to poor pharmacokinetics and aggregation problems. Here, we describe generation and characterization of homogeneous DAR = 8 ADCs carrying a novel auristatin ß-D-glucuronide, MMAU. The glycoside payload contributed to overall hydrophilicity of the ADC reducing aggregation. Compared to standard DAR = 2-4 ADCs, cytotoxicity of the homogeneous DAR = 8 ADCs was improved to low-picomolar IC50 values against cancer cells in vitro. Bystander efficacy was restored after ADC internalization and subsequent cleavage of the glycoside, although unconjugated MMAU was relatively non-toxic to cells. DAR = 8 MMAU ADCs were effective against target antigen-expressing xenograft tumors. The ADCs were also studied in 3D in vitro patient-derived xenograft (PDX) assays where they outperformed clinically used ADC. In conclusion, increased hydrophilicity of the payload contributed to the ADC's hydrophilicity, stability and safety to non-target cells, while significantly improving cytotoxicity and enabling bystander efficacy.

18.
SLAS Discov ; 22(8): 974-984, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28644734

RESUMO

Polycystic kidney disease (PKD) is a prevalent disorder characterized by renal cysts that lead to kidney failure. Various signaling pathways have been targeted to stop disease progression, but most interventions still focus on alleviating PKD-associated symptoms. The mechanistic complexity of the disease, as well as the lack of functional in vitro assays for compound testing, has made drug discovery for PKD challenging. To identify modulators of PKD, Pkd1-/- kidney tubule epithelial cells were applied to a scalable and automated 3D cyst culture model for compound screening, followed by phenotypic profiling to determine compound efficacy. We used this screening platform to screen a library of 273 kinase inhibitors to probe various signaling pathways involved in cyst growth. We show that inhibition of several targets, including aurora kinase, CDK, Chk, IGF-1R, Syk, and mTOR, but, surprisingly, not PI3K, prevented forskolin-induced cyst swelling. Additionally, we show that multiparametric phenotypic classification discriminated potentially undesirable (i.e., cytotoxic) compounds from molecules inducing the desired phenotypic change, greatly facilitating hit selection and validation. Our findings show that a pathophysiologically relevant 3D cyst culture model of PKD coupled to phenotypic profiling can be used to identify potentially therapeutic compounds and predict and validate molecular targets for PKD.


Assuntos
Ensaios de Triagem em Larga Escala/métodos , Terapia de Alvo Molecular , Doenças Renais Policísticas/tratamento farmacológico , Inibidores de Proteínas Quinases/análise , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Linhagem Celular , Colforsina , Hidrogel de Polietilenoglicol-Dimetacrilato , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/patologia , Camundongos , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Doenças Renais Policísticas/patologia , Inibidores de Proteínas Quinases/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo
19.
Biochem J ; 389(Pt 2): 465-9, 2005 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-15725075

RESUMO

Ephrins are cell surface ligands that activate Eph receptor tyrosine kinases. This ligand-receptor interaction plays a central role in the sorting of cells. We have previously shown that the ephrinB-EphB signalling pathway is also involved in the migration of intestinal precursor cells along the crypts. Using the colon cell line DLD1 expressing the EphB2 receptor, we showed that stimulation of these cells with soluble ephrinB1 results in a rapid retraction of cell extensions and a detachment of cells. On ephrinB1 stimulation, the small GTPases Rho and Ras are activated and Rap1 is inactivated. Importantly, when a constitutively active Rap1 mutant was introduced into these cells, ephrinB1-induced retraction was inhibited. From these results, we conclude that down-regulation of Rap1 is a prerequisite for ephrin-induced cell retraction in colon cells.


Assuntos
Regulação para Baixo , Efrina-B1/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Forma Celular , Colo/metabolismo , Colo/patologia , Regulação da Expressão Gênica , Humanos , Receptor EphB2/metabolismo , Proteínas rap1 de Ligação ao GTP/genética , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
20.
J Biomol Screen ; 21(9): 912-22, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27412535

RESUMO

3D tissue cultures provide a more physiologically relevant context for the screening of compounds, compared with 2D cell cultures. Cells cultured in 3D hydrogels also show complex phenotypes, increasing the scope for phenotypic profiling. Here we describe a high-content screening platform that uses invasive human prostate cancer cells cultured in 3D in standard 384-well assay plates to study the activity of potential therapeutic small molecules and antibody biologics. Image analysis tools were developed to process 3D image data to measure over 800 phenotypic parameters. Multiparametric analysis was used to evaluate the effect of compounds on tissue morphology. We applied this screening platform to measure the activity and selectivity of inhibitors of the c-Met and epidermal growth factor (EGF) receptor (EGFR) tyrosine kinases in 3D cultured prostate carcinoma cells. c-Met and EGFR activity was quantified based on the phenotypic profiles induced by their respective ligands, hepatocyte growth factor and EGF. The screening method was applied to a novel collection of 80 putative inhibitors of c-Met and EGFR. Compounds were identified that induced phenotypic profiles indicative of selective inhibition of c-Met, EGFR, or bispecific inhibition of both targets. In conclusion, we describe a fully scalable high-content screening platform that uses phenotypic profiling to discriminate selective and nonselective (off-target) inhibitors in a physiologically relevant 3D cell culture setting.


Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios de Triagem em Larga Escala/métodos , Neoplasias da Próstata/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Proliferação de Células/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Humanos , Hidrogéis/química , Masculino , Neoplasias da Próstata/genética , Inibidores de Proteínas Quinases/isolamento & purificação , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/genética , Bibliotecas de Moléculas Pequenas/uso terapêutico
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