Development of a Fragment-Based Screening Assay for the Focal Adhesion Targeting Domain Using SPR and NMR.
Molecules
; 24(18)2019 Sep 14.
Article
en En
| MEDLINE
| ID: mdl-31540099
ABSTRACT
The Focal Adhesion Targeting (FAT) domain of Focal Adhesion Kinase (FAK) is a promising drug target since FAK is overexpressed in many malignancies and promotes cancer cell metastasis. The FAT domain serves as a scaffolding protein, and its interaction with the protein paxillin localizes FAK to focal adhesions. Various studies have highlighted the importance of FAT-paxillin binding in tumor growth, cell invasion, and metastasis. Targeting this interaction through high-throughput screening (HTS) provides a challenge due to the large and complex binding interface. In this report, we describe a novel approach to targeting FAT through fragment-based drug discovery (FBDD). We developed two fragment-based screening assays-a primary SPR assay and a secondary heteronuclear single quantum coherence nuclear magnetic resonance (HSQC-NMR) assay. For SPR, we designed an AviTag construct, optimized SPR buffer conditions, and created mutant controls. For NMR, resonance backbone assignments of the human FAT domain were obtained for the HSQC assay. A 189-compound fragment library from Enamine was screened through our primary SPR assay to demonstrate the feasibility of a FAT-FBDD pipeline, with 19 initial hit compounds. A final total of 11 validated hits were identified after secondary screening on NMR. This screening pipeline is the first FBDD screen of the FAT domain reported and represents a valid method for further drug discovery efforts on this difficult target.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Resonancia Magnética Nuclear Biomolecular
/
Resonancia por Plasmón de Superficie
/
Adhesiones Focales
/
Quinasa 1 de Adhesión Focal
Tipo de estudio:
Diagnostic_studies
/
Screening_studies
Límite:
Humans
Idioma:
En
Revista:
Molecules
Asunto de la revista:
BIOLOGIA
Año:
2019
Tipo del documento:
Article
País de afiliación:
Estados Unidos