Technology for Discovery of Antifibrotic Drugs: Phenotypic Screening for LARP6 Inhibitors Using Inverted Yeast Three Hybrid System.
Assay Drug Dev Technol
; 17(3): 116-127, 2019 04.
Article
em En
| MEDLINE
| ID: mdl-30901265
ABSTRACT
Fibrosis is defined by excessive production of type I collagen in various organs. Excessive type I collagen production in fibrosis is stimulated by binding of RNA protein LARP6 to the structural element of collagen mRNAs, the 5' stem loop (5'SL). The LARP6-dependent regulation is specific for type I collagen and critical for fibrosis development. Inhibitors of LARP6 binding have potential to be specific antifibrotic drugs, as evidenced by the discovery of one such inhibitor. To create technology for phenotypic screening of additional compounds we developed an inverted yeast three hybrid system. The system is based on expression of human LARP6 and a short RNA containing the 5'SL of human collagen α1(I) mRNA in Saccharomyces cerevisiae cells. The cells were engineered in such a way that when LARP6 is bound to 5'SL RNA they fail to grow in a specific synthetic medium. Dissociation of LARP6 from 5'SL RNA permits the cell growth, allowing identification of the inhibitors of LARP6 binding. The assay simply involves measuring optical density of cells growing in multiwall plates and is pertinent for high throughput applications. We describe the specificity of the system and its characteristics for high throughput screening. As a proof of principle, the result of one screen using collection of FDA approved drugs is also presented. This screen demonstrates that using this technology discovery of novel LARP6 inhibitors is possible.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Ribonucleoproteínas
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Saccharomyces cerevisiae
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Técnicas do Sistema de Duplo-Híbrido
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Descoberta de Drogas
Tipo de estudo:
Diagnostic_studies
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Prognostic_studies
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Screening_studies
Limite:
Humans
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article