Allosteric Inhibitor of KRas Identified Using a Barcoded Assay Microchip Platform.
Anal Chem
; 90(15): 8824-8830, 2018 08 07.
Article
em En
| MEDLINE
| ID: mdl-29979578
ABSTRACT
Protein catalyzed capture agents (PCCs) are synthetic antibody surrogates that can target a wide variety of biologically relevant proteins. As a step toward developing a high-throughput PCC pipeline, we report on the preparation of a barcoded rapid assay platform for the analysis of hits from PCC library screens. The platform is constructed by first surface patterning a micrometer scale barcode composed of orthogonal ssDNA strands onto a glass slide. The slide is then partitioned into microwells, each of which contains multiple copies of the full barcode. Biotinylated candidate PCCs from a click screen are assembled onto the barcode stripes using a complementary ssDNA-encoded cysteine-modified streptavidin library. This platform was employed to evaluate candidate PCC ligands identified from an epitope targeted in situ click screen against the two conserved allosteric switch regions of the Kirsten rat sarcoma (KRas) protein. A single microchip was utilized for the simultaneous evaluation of 15 PCC candidate fractions under more than a dozen different assay conditions. The platform also permitted more than a 10-fold savings in time and a more than 100-fold reduction in biological and chemical reagents relative to traditional multiwell plate assays. The best ligand was shown to exhibit an in vitro inhibition constant (IC50) of â¼24 µM.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA de Cadeia Simples
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Proteínas Proto-Oncogênicas p21(ras)
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Análise em Microsséries
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Inibidores Enzimáticos
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Regulação Alostérica
Limite:
Humans
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Estados Unidos