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.

Highlights

  • Focal Adhesion Kinase (FAK) is a 125 kDa non-receptor tyrosine kinase overexpressed at the protein and mRNA levels in over 80% of solid malignant tumors [1,2,3]

  • FAK is critical in cancer development due to its function as a central component to multiple oncogenic signaling pathways [4,5,6,7]

  • This article reports an alternative approach in the discovery of FAK inhibitors, in targeting the Focal Adhesion Targeting (FAT)-Paxillin interaction

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Summary

Introduction

Focal Adhesion Kinase (FAK) is a 125 kDa non-receptor tyrosine kinase overexpressed at the protein and mRNA levels in over 80% of solid malignant tumors [1,2,3]. Numerous studies have provided validation that FAK is critical for human cancer progression due to its role in cellular adhesion, motility, invasion, metastasis, and angiocrine signaling [4,5,6,7]. Knockdown studies of FAK in transgenic mouse models has shown decrease in tumor growth, invasion, and metastasis [8,9,10,11]. FAK is composed of three primary domains: the N-terminal 4.1 ezrin, radizin, moesin (FERM) domain, the central kinase domain, and the C-terminal focal adhesion targeting (FAT) domain. The FERM and FAT domains allow FAK to function as a scaffolding protein in conjunction with its kinase activity [13,14].

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