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1.
Front Pediatr ; 8: 536, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33014932

RESUMO

Functional profiling of CFTR-directed therapeutics offers the potential to provide significant benefits to young people with cystic fibrosis (CF). However, the development of 2D airway epithelial cell models for individual response tests in CF children remains a central task. The objective of this study was to determine the utility of EpiXTM technology for expansion of nasal epithelial cells for use in electrophysiological CFTR function measurements. An initial harvest of as few as 20,000 cells was sufficient to expand up to 50 million cells that were used to generate air-liquid interface (ALI) cultures for ion transport studies with the Ussing assay. CFTR function was assessed by measuring responses to forskolin and the CFTR potentiator VX-770 (ivacaftor) in ALI cultures generated from passage 3 and 4 cells. Short-circuit current (Isc) measurements of blocked CFTR currents (ΔICFTRinh) discriminated CFTR function between healthy control (wild type, WT) and patients with intermediate (F508del/R117H-7T: 56% WT) and severe (F508del/F508del: 12% WT) CF disease. For the mixed genotypes, CFTR activity for F508del/c.850dupA was 12% WT, R334W/406-1G>A was 24% WT, and CFTRdele2,3(21 kb)/CFTRdele2,3(21 kb) was 9% WT. The CFTR correctors VX-809 (lumacaftor) and VX-661 (tezacaftor) significantly increased CFTR currents for F508del/R117H to 73 and 67% WT, respectively. Cultures with the large deletion mutation CFTRdele2,3(21 kb) unexpectedly responded to VX-661 treatment (20% WT). Amiloride-sensitive sodium currents were robust and ranged between 20-80 µA/cm2 depending on the subject. In addition to characterizing the electrophysiological profile of mutant CFTR activity in cultures for five genotypes, our study exemplifies the promising paradigm of bed-to-bench side cooperation and personalized medicine.

2.
Cell Rep ; 25(3): 598-610.e5, 2018 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-30332641

RESUMO

Despite substantial self-renewal capability in vivo, epithelial stem and progenitor cells located in various tissues expand for a few passages in vitro in feeder-free condition before they succumb to growth arrest. Here, we describe the EpiX method, which utilizes small molecules that inhibit PAK1-ROCK-Myosin II and TGF-ß signaling to achieve over one trillion-fold expansion of human epithelial stem and progenitor cells from skin, airway, mammary, and prostate glands in the absence of feeder cells. Transcriptomic and epigenomic studies show that this condition helps epithelial cells to overcome stresses for continuous proliferation. EpiX-expanded basal epithelial cells differentiate into mature epithelial cells consistent with their tissue origins. Whole-genome sequencing reveals that the cells retain remarkable genome integrity after extensive in vitro expansion without acquiring tumorigenicity. EpiX technology provides a solution to exploit the potential of tissue-resident epithelial stem and progenitor cells for regenerative medicine.


Assuntos
Células Epiteliais/citologia , Miosina Tipo II/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Células-Tronco/citologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Associadas a rho/antagonistas & inibidores , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Alimentadoras/citologia , Células Alimentadoras/efeitos dos fármacos , Células Alimentadoras/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo , Quinases Associadas a rho/genética , Quinases Associadas a rho/metabolismo
3.
Mol Cancer ; 16(1): 177, 2017 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-29212548

RESUMO

Efforts to develop effective cancer therapeutics have been hindered by a lack of clinically predictive preclinical models which recapitulate this complex disease. Patient derived xenograft (PDX) models have emerged as valuable tools for translational research but have several practical limitations including lack of sustained growth in vitro. In this study, we utilized Conditional Reprogramming (CR) cell technology- a novel cell culture system facilitating the generation of stable cultures from patient biopsies- to establish PDX-derived cell lines which maintain the characteristics of the parental PDX tumor. Human lung and ovarian PDX tumors were successfully propagated using CR technology to create stable explant cell lines (CR-PDX). These CR-PDX cell lines maintained parental driver mutations and allele frequency without clonal drift. Purified CR-PDX cell lines were amenable to high throughput chemosensitivity screening and in vitro genetic knockdown studies. Additionally, re-implanted CR-PDX cells proliferated to form tumors that retained the growth kinetics, histology, and drug responses of the parental PDX tumor. CR technology can be used to generate and expand stable cell lines from PDX tumors without compromising fundamental biological properties of the model. It offers the ability to expand PDX cells in vitro for subsequent 2D screening assays as well as for use in vivo to reduce variability, animal usage and study costs. The methods and data detailed here provide a platform to generate physiologically relevant and predictive preclinical models to enhance drug discovery efforts.


Assuntos
Linhagem Celular Tumoral/citologia , Técnicas de Reprogramação Celular/métodos , Neoplasias Pulmonares/patologia , Neoplasias Ovarianas/patologia , Animais , Linhagem Celular Tumoral/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Neoplasias Pulmonares/genética , Masculino , Camundongos , Mutação , Neoplasias Ovarianas/genética , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Oncotarget ; 6(30): 30194-211, 2015 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-26327203

RESUMO

The metalloproteinase SAS1B [ovastacin, ASTL, astacin-like] was immunolocalized on the oolemma of ovulated human oocytes and in normal ovaries within the pool of growing oocytes where SAS1B protein was restricted to follicular stages spanning the primary-secondary follicle transition through ovulation. Gene-specific PCR and immunohistochemical studies revealed ASTL messages and SAS1B protein in both endometrioid [74%] and malignant mixed Mullerian tumors (MMMT) [87%] of the uterus. A MMMT-derived cell line, SNU539, expressed cell surface SAS1B that, after binding polyclonal antibodies, internalized into EEA1/LAMP1-positive early and late endosomes. Treatment of SNU539 cells with anti-SAS1B polyclonal antibodies caused growth arrest in the presence of active complement. A saporin-immunotoxin directed to SAS1B induced growth arrest and cell death. The oocyte restricted expression pattern of SAS1B among adult organs, cell-surface accessibility, internalization into the endocytic pathway, and tumor cell growth arrest induced by antibody-toxin conjugates suggest therapeutic approaches that would selectively target tumors while limiting adverse drug effects in healthy cells. The SAS1B metalloproteinase is proposed as a prototype cancer-oocyte tumor surface neoantigen for development of targeted immunotherapeutics with limited on-target/off tumor effects predicted to be restricted to the population of growing oocytes.


Assuntos
Anticorpos/farmacologia , Antígenos de Neoplasias , Imunoconjugados/farmacologia , Imunoterapia/métodos , Metaloproteases/antagonistas & inibidores , Tumor Mulleriano Misto/tratamento farmacológico , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Neoplasias Uterinas/tratamento farmacológico , Sequência de Aminoácidos , Anticorpos/metabolismo , Anticorpos/toxicidade , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Endocitose , Feminino , Humanos , Imunoconjugados/metabolismo , Imunoconjugados/toxicidade , Imunoterapia/efeitos adversos , Metaloproteases/genética , Metaloproteases/imunologia , Metaloproteases/metabolismo , Tumor Mulleriano Misto/enzimologia , Tumor Mulleriano Misto/genética , Tumor Mulleriano Misto/imunologia , Tumor Mulleriano Misto/patologia , Dados de Sequência Molecular , Terapia de Alvo Molecular , Oócitos/efeitos dos fármacos , Oócitos/enzimologia , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/toxicidade , Saporinas , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Neoplasias Uterinas/enzimologia , Neoplasias Uterinas/genética , Neoplasias Uterinas/imunologia , Neoplasias Uterinas/patologia
6.
ACS Chem Biol ; 3(11): 711-22, 2008 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-18839960

RESUMO

Spinal muscular atrophy (SMA) is caused by deletion or mutation of both copies of the SMN1 gene, which produces an essential protein known as SMN. The severity of SMA is modified by variable copy number of a second gene,SMN2, which produces an mRNA that is incorrectly spliced with deletion of the last exon. We described previously the discovery of potent C5-substituted quinazolines that increase SMN2 gene expression by 2-fold. Discovery of potent SMN2 promoter inducers relied on a cellular assay without knowledge of the molecular target. Using protein microarray scanning with a radiolabeled C5-substituted quinazoline probe, we identified the scavenger decapping enzyme, DcpS, as a potential binder. We show that the C5-substituted quinazolines potently inhibit DcpS decapping activity and that the potency of inhibition correlates with potency forSMN2 promoter induction. Binding of C5-substituted quinazolines to DcpS holds the enzyme in an open, catalytically incompetent conformation. DcpS is a nuclear shuttling protein that binds and hydrolyzes the m(7)GpppN mRNA cap structure and a modulator of RNA metabolism. Therefore DcpS represents a novel therapeutic target for modulating gene expression by a small molecule.


Assuntos
Endorribonucleases/antagonistas & inibidores , Atrofia Muscular Espinal/tratamento farmacológico , Quinazolinas/farmacologia , Sistemas de Liberação de Medicamentos , Humanos , Ligação Proteica , Conformação Proteica/efeitos dos fármacos
7.
Assay Drug Dev Technol ; 6(2): 213-23, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18471075

RESUMO

The life-threatening consequences of acquired, or drug-induced, long QT syndrome due to block of the human ether-a-go-go-related gene (hERG) channel are well appreciated and have been the cause of several drugs being removed from the market in recent years because of patient death. In the last decade, the propensity for block of the hERG channel by a diverse and expanding set of compounds has led to the requirement that all new drugs be tested for hERG channel block in a functional patch-clamp assay. Because of the need to identify potential hERG blockers early in the discovery process, radiometric hERG binding assays are preferred over patch-clamp assays for compound triage, because of relative advantages in speed and cost. Even so, these radiometric binding assays are laborious and require dedicated instrumentation and infrastructure to cope with the regulatory and safety issues associated with the use of radiation. To overcome these limitations, we developed a homogeneous, fluorescence polarization-based assay to identify and characterize the affinity of small molecules for the hERG channel and have demonstrated tight correlation with data obtained from either radioligand binding or patch-clamp assays. Key to the development of this assay was a cell line that expressed highly elevated levels of hERG protein, which was generated by coupling expression of the hERG channel to that of a selectable cell surface marker. A high-expressing clone was isolated by flow cytometry and used to generate membrane preparations that contained >50-fold the typical density of hERG channels measured by [(3)H]astemizole binding. This strategy enabled the Predictor (Invitrogen, Carlsbad, CA) hERG fluorescence polarization assay and should be useful in the development of other fluorescence polarization-based assays that use membrane proteins.


Assuntos
Canais de Potássio Éter-A-Go-Go/metabolismo , Polarização de Fluorescência/métodos , Antígenos CD8/fisiologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Interpretação Estatística de Dados , Avaliação Pré-Clínica de Medicamentos/métodos , Eletrofisiologia , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Citometria de Fluxo , Corantes Fluorescentes , Engenharia Genética , Humanos , Imuno-Histoquímica , Potenciais da Membrana/fisiologia , Proteínas de Membrana/fisiologia , Técnicas de Patch-Clamp , Ensaio Radioligante
8.
Expert Opin Drug Discov ; 3(1): 115-29, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23480143

RESUMO

Over the past decade, a variety of technologies for the identification and characterization of protein kinase inhibitors have been implemented in the laboratories of nearly every major pharmaceutical and biotechnology company. Although the majority of these assay technologies are highly robust, the ability of many assays to identify compounds that target the kinase of interest in a true biological context remains questionable. Because every in vitro assay represents a trade-off between biological relevancy and factors such as cost, throughput and accuracy, it is important to acknowledge and balance these trade-offs when interrogating a kinase target in such an assay. This review addresses some of the factors that should be considered when developing protein kinase assays, as well as strategies used to address those factors.

10.
Hum Mol Genet ; 14(14): 2003-18, 2005 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-15944201

RESUMO

We have exploited the existence of a second copy of the human SMN gene (SMN2) to develop a high-throughput screening strategy to identify potential small molecule therapeutics for the genetic disease spinal muscular atrophy (SMA), which is caused by the loss of the SMN1 gene. Our screening process was designed to identify synthetic compounds that increase the total amount of full-length SMN messenger RNA and protein arising from the SMN2 gene, thereby suppressing the deleterious effects of losing SMN1. A cell-based bioassay was generated that detects SMN2 promoter activity, on which greater than 550,000 compounds was tested. This resulted in the identification of 17 distinct compounds with confirmed biological activity on the cellular primary assay, belonging to nine different structural families. Six of the nine scaffolds were chosen on the basis of their drug-like features to be tested for their ability to modulate SMN gene expression in SMA patient-derived fibroblasts. Five of the six compound classes altered SMN mRNA levels or mRNA splicing patterns in SMA patient-derived fibroblasts. Two of the compound classes, a quinazoline compound series and an indole compound, also increased SMN protein levels and nuclear gem/Cajal body numbers in patient-derived cells. In addition, these two distinct scaffolds showed additive effects when used in combination, suggesting that they may act on different molecular targets. The work described here has provided the foundation for a successful medicinal chemistry effort to further advance these compounds as potential small molecule therapeutics for SMA.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Atrofia Muscular Espinal/genética , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a RNA/genética , Sequência de Bases , Western Blotting , Células Cultivadas , Corpos Enovelados/metabolismo , Primers do DNA , Relação Dose-Resposta a Droga , Humanos , Imuno-Histoquímica , Atrofia Muscular Espinal/tratamento farmacológico , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas do Complexo SMN , Proteína 1 de Sobrevivência do Neurônio Motor , Proteína 2 de Sobrevivência do Neurônio Motor
11.
J Am Chem Soc ; 126(9): 2670-1, 2004 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-14995162

RESUMO

Sortase (SrtA), a transpeptidase from Staphylococcus aureus, catalyzes a cell-wall sorting reaction at an LPXTG motif by cleaving between threonine and glycine and subsequently joining the carboxyl group of threonine to an amino group of pentaglycine on the cell wall peptidoglycan. We have applied this transpeptidyl activity of sortase to in vitro protein ligation. We found that in the presence of sortase, protein/peptide with an LPXTG motif can be specifically ligated to an aminoglycine protein/peptide via an amide bond. Additionally, sortase can even conjugate substrates such as (d)-peptides, synthetic branched peptides, and aminoglycine-derivatized small molecules to the C terminus of a recombinant protein. The sortase-mediate protein ligation is robust, specific, and easy to perform, and can be widely applied to specific protein conjugation with polypeptides or molecules of unique biochemical and biophysical properties.


Assuntos
Aminoaciltransferases/química , Peptídeos/química , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases , Proteínas de Fluorescência Verde , Proteínas Luminescentes/química , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
12.
Assay Drug Dev Technol ; 1(6): 755-65, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15090222

RESUMO

Gene targeting allows for precise genomic engineering and has been used extensively to generate both loss-of-function and gain-of-function models in mice. Similar manipulation of the genome of somatic cell lines holds high value in basic and applied research, but has been hampered by low recombination frequencies and the subsequent labor-intensive analysis of a large number of cell clones. By combining gene targeting methods with fluorescence-activated cell sorting, gain-of-function cell lines were generated and identified based on a functional readout. To demonstrate the general applicability of this approach to drug discovery, we generated targeted promoter insertion cell lines for two key drug target classes -- the G protein-coupled receptor melanocortin-receptor 4 and the nuclear receptor peroxisome proliferator-activated receptor-gamma. Molecular analysis of the engineered cell clones confirmed the predicted integration of a constitutive promoter into an endogenous allele, and the appropriate pharmacology for these targets validated the use of these gain-of-function cell lines in drug discovery applications, including high-throughput compound screening.


Assuntos
Células Híbridas/fisiologia , Recombinação Genética/genética , Tecnologia Farmacêutica/métodos , alfa-MSH/análogos & derivados , Linhagem Celular , Relação Dose-Resposta a Droga , Marcação de Genes/métodos , Vetores Genéticos , Humanos , Células Híbridas/efeitos dos fármacos , Receptor Tipo 4 de Melanocortina/genética , Receptores Citoplasmáticos e Nucleares/genética , Recombinação Genética/efeitos dos fármacos , Fatores de Transcrição/genética , alfa-MSH/farmacologia
13.
J Biomol Screen ; 7(1): 45-55, 2002 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11897055

RESUMO

Pleckstrin homology (PH) domains are present in key proteins involved in many vital cell processes. For example, the PH domain of Bruton's tyrosine kinase (Btk) binds to phosphatidylinositol triphosphate (PIP(3)) in the plasma membrane after stimulation of the B-cell receptor in B cells. Mutations in the Btk PH domain result in changes in its affinity for PIP(3), with higher binding leading to cell transformation in vitro and lower binding leading to antibody deficiencies in both humans and mice. We describe here a fluorescence resonance energy transfer (FRET)-based biochemical assay that directly monitors the interaction of a PH domain with PIP(3) at a membrane surface. We overexpressed a fusion protein consisting of an enhanced green fluorescent protein (GFP) and the N-terminal 170 amino acids of a Tec family kinase that contains its PH domain (PH170). Homogeneous unilamellar vesicles were made that contained PIP(3) and octadecylrhodamine (OR), a lipophilic FRET acceptor for GFP. After optimization of both protein and vesicle components, we found that binding of the GFP-PH170 protein to PIP3 in vesicles that contain OR results in about a 90% reduction of GFP fluorescence. Using this assay to screen 1440 compounds, we identified three that efficiently inhibited binding of GFP-PH170 to PIP(3) in vesicles. This biochemical assay readily miniaturized to 1.8-microl reaction volumes and was validated in a 3456-well screening format.


Assuntos
Proteínas Sanguíneas/química , Membrana Celular/metabolismo , Avaliação Pré-Clínica de Medicamentos , Fosfatidilinositóis/metabolismo , Fosfoproteínas/química , Espectrometria de Fluorescência/métodos , Tirosina Quinase da Agamaglobulinemia , Animais , Ligação Competitiva , Relação Dose-Resposta a Droga , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde , Humanos , Cinética , Metabolismo dos Lipídeos , Proteínas Luminescentes/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo
14.
Assay Drug Dev Technol ; 1(1 Pt 1): 9-19, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15090152

RESUMO

Protein phosphorylation is one of the major regulatory mechanisms involved in signal-induced cellular events, including cell proliferation, apoptosis, and metabolism. Because many facets of biology are regulated by protein phosphorylation, aberrant kinase and/or phosphatase activity forms the basis for many different types of pathology. The disease relevance of protein kinases and phosphatases has led many pharmaceutical and biotechnology companies to expend significant resources in lead discovery programs for these two target classes. The existence of >500 kinases and phosphatases encoded by the human genome necessitates development of methodologies for the rapid screening for novel and specific compound inhibitors. We describe here a fluorescence-based, molecular assay platform that is compatible with robotic, ultra-high throughput screening systems and can be applied to virtually all tyrosine and serine/threonine protein kinases and phosphatases. The assay has a coupled-enzyme format, utilizing the differential protease sensitivity of phosphorylated versus nonphosphorylated peptide substrates. In addition to screening individual kinases, the assay can be formatted such that kinase pathways are re-created in vitro to identify compounds that specifically interact with inactive kinases. Miniaturization of this assay format to the 1-microl scale allows for the rapid and accurate compound screening of a host of kinase and phosphatase targets, thereby facilitating the hunt for new leads for these target classes.


Assuntos
Bioensaio/instrumentação , Avaliação Pré-Clínica de Medicamentos/instrumentação , Biblioteca de Peptídeos , Fosfoproteínas Fosfatases/química , Proteínas Quinases/química , Sulfonamidas , Trifosfato de Adenosina/fisiologia , Animais , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Fluorescência , Humanos , Isoquinolinas/farmacologia , Toxinas Marinhas , Microcistinas , Peptídeos Cíclicos/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Inibidores de Proteínas Quinases , Proteínas Quinases/fisiologia , Transdução de Sinais/fisiologia , Estaurosporina/farmacologia , Vanadatos/farmacologia
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