Development of a fragment-based screening assay for the focal adhesion targeting domain using SPR and NMR

Carlos Alvarado, Erik Stahl, Karissa Koessel, Andrew Rivera, Brian R. Cherry, Surya V.S.R.K. Pulavarti, Thomas Szyperski, William Cance, Timothy Marlowe

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

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.

Original languageEnglish (US)
Article number3352
JournalMolecules
Volume24
Issue number18
DOIs
StatePublished - Sep 14 2019

Keywords

  • FAT domain
  • Focal adhesion kinase
  • Fragment-based drug discovery
  • Nuclear magnetic resonance
  • Surface plasmon resonance

ASJC Scopus subject areas

  • Analytical Chemistry
  • Chemistry (miscellaneous)
  • Molecular Medicine
  • Pharmaceutical Science
  • Drug Discovery
  • Physical and Theoretical Chemistry
  • Organic Chemistry

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