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Updated: Jan 19, 2026
NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode
Published on: June 4, 2021
Development of a Fragment-Based Screening Assay for the Focal Adhesion Targeting Domain Using SPR and NMR
Carlos Alvarado1, Erik Stahl2, Karissa Koessel3
1University of Arizona Cancer Center, 625 N. 6th Street, Phoenix, AZ 85004, USA. calvara6@email.arizona.edu.
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.
Insights
Fragment-based drug discovery successfully identified novel inhibitors for the Focal Adhesion Targeting (FAT) domain of Focal Adhesion Kinase (FAK), a key protein in cancer metastasis. This new method targets a challenging protein-protein interaction for cancer therapy development.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Focal Adhesion Kinase (FAK) is overexpressed in many cancers, promoting metastasis.
- The Focal Adhesion Targeting (FAT) domain of FAK interacts with paxillin, crucial for tumor growth and invasion.
- Targeting the FAT-paxillin interaction is challenging due to its large, complex binding interface.
Purpose of the Study:
- To develop and validate a novel fragment-based drug discovery (FBDD) pipeline for the FAK FAT domain.
- To identify initial fragment-like compounds that bind to the FAT domain.
Main Methods:
- Developed two fragment-based screening assays: Surface Plasmon Resonance (SPR) and heteronuclear single quantum coherence Nuclear Magnetic Resonance (HSQC-NMR).
- Optimized SPR conditions and obtained backbone assignments for the FAT domain for NMR.
- Screened a 189-compound fragment library using SPR, followed by secondary validation with NMR.
Main Results:
- The FBDD pipeline demonstrated feasibility for targeting the FAK FAT domain.
- Identified 19 initial hit compounds from the primary SPR screen.
- Validated 11 compounds as confirmed binders through secondary HSQC-NMR screening.
Conclusions:
- This study reports the first FBDD screen of the FAK FAT domain.
- The developed screening pipeline is a viable strategy for discovering inhibitors against this challenging drug target.
- These validated hits provide a foundation for further drug discovery efforts against FAK in cancer.
