Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 3 de 3
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
SLAS Discov ; 26(8): 995-1003, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34049465

RESUMO

Surface-based biophysical methods for measuring binding kinetics of molecular interactions, such as surface plasmon resonance (SPR) or grating-coupled interferometry (GCI), are now well established and widely used in drug discovery. Increasing throughput is an often-cited need in the drug discovery process and this has been achieved with new instrument generations where multiple interactions are measured in parallel, shortening the total measurement times and enabling new application areas within the field. Here, we present the development of a novel technology called waveRAPID for a further-up to 10-fold-increase in throughput, consisting of an injection method using a single sample. Instead of sequentially injecting increasing analyte concentrations for constant durations, the analyte is injected at a single concentration in short pulses of increasing durations. A major advantage of the new method is its ability to determine kinetics from a single well of a microtiter plate, making it uniquely suitable for kinetic screening. We present the fundamentals of this approach using a small-molecule model system for experimental validation and comparing kinetic parameters to traditional methods. By varying experimental conditions, we furthermore assess the robustness of this new technique. Finally, we discuss its potential for improving hit quality and shortening cycle times in the areas of fragment screening, low-molecular-weight compound screening, and hit-to-lead optimization.


Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Cinética , Bibliotecas de Moléculas Pequenas , Ressonância de Plasmônio de Superfície/métodos
2.
Mol Cancer Ther ; 19(4): 1080-1088, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31871271

RESUMO

High-throughput construction of multivalent binders and subsequent screening for biological activity represent a fundamental challenge: A linear increase of monovalent components translates to the square of possible bivalent combinations. Even high-efficiency cloning and expression methods become limiting when thousands of bispecific binders need to be screened for activity. In this study, we present an in vitro method for the efficient production of flexibly linked bispecific binding agents from individually expressed and purified monovalent binders. We established a sortase A-mediated coupling reaction to generate bispecific designed ankyrin repeat proteins (DARPins), with an optimized reaction maximizing the bivalent coupling product with low levels of monovalent side-products. These one-pot reaction mixtures could be used directly, without further purification, in cell-based assays. We generated a matrix of 441 different bispecific DARPins against the extracellular domains of the cancer-associated receptors EGFR, ErbB2, ErbB3, ErbB4, EpCAM, and c-MET and screened on two different ErbB2-positive cancer cells lines for growth-inhibitory effects. We identified not only known but also novel biologically active biparatopic DARPins. Furthermore, we found that the cancer cell lines respond in a highly reproducible and defined manner to the treatment with the 441 different bivalent binding agents. The generated response profiles can thus be used for functional characterization of cell lines because they are strongly related to the cell line-specific surface receptor landscape. Thus, our method not only represents a robust tool for screening and lead identification of bispecific binding agents, but additionally offers an orthogonal approach for the functional characterization of cancer cell lines.


Assuntos
Aminoaciltransferases/metabolismo , Anticorpos Biespecíficos/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Neoplasias/metabolismo , Engenharia de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Aminoaciltransferases/genética , Repetição de Anquirina , Anticorpos Biespecíficos/genética , Proteínas de Bactérias/genética , Cisteína Endopeptidases/genética , Humanos , Neoplasias/genética , Neoplasias/patologia , Proteínas Recombinantes/genética , Células Tumorais Cultivadas
3.
J Mol Biol ; 431(10): 2020-2039, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30930049

RESUMO

MET, the product of the c-MET proto-oncogene, and its ligand hepatocyte growth factor/scatter factor (HGF/SF) control survival, proliferation and migration during development and tissue regeneration. HGF/SF-MET signaling is equally crucial for growth and metastasis of a variety of human tumors, but resistance to small-molecule inhibitors of MET kinase develops rapidly and therapeutic antibody targeting remains challenging. We made use of the designed ankyrin repeat protein (DARPin) technology to develop an alternative approach for inhibiting MET. We generated a collection of MET-binding DARPins covering epitopes in the extracellular MET domains and created comprehensive sets of bi-paratopic fusion proteins. This new class of molecules efficiently inhibited MET kinase activity and downstream signaling, caused receptor downregulation and strongly inhibited the proliferation of MET-dependent gastric carcinoma cells carrying MET locus amplifications. MET-specific bi-paratopic DARPins may represent a novel and potent strategy for therapeutic targeting of MET and other receptors, and this study has elucidated their mode of action.


Assuntos
Repetição de Anquirina , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/química , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-met/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...