Development of a Fragment-Based Screening Assay for the Focal Adhesion Targeting Domain Using SPR and NMR

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.

Assays and Technologies for Developing Proteolysis Targeting Chimera Degraders

“…Nevertheless, BLI and SPR are superior to the other technologies discussed previously in that they can determine binding kinetics and binding mechanisms in real-time without the use of fluorescent probes. In the past two decades we have witnessed an increasing application of SPR technology in every aspect of pharmaceutical analyses, from fragment-based drug screening [77,78] and HTS [79] to pharmacokinetic drug profiling [80], early absorption, distribution, metabolism, excretion and toxicity studies [81] and quality control [82]. In recent years, biosensor technology also finds its application in PROTACs, not only for binary binding determination [17,61,83] but also for ternary complex characterization [11,84].…”

Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution

“…SEMA3A-Fc proteins (either human or mouse) were flowed over the immobilized mouse NRP1 to measure binding affinities. OneStep 98 kinetics was used for affinity measurements, wherein the analyte was injected at a single fixed concentration (100 nM mouse SEMA3A Fc or 50 nM human SEMA3A Fc; 75 μl/min flowrate). Binding analysis and affinity calculations were performed using Pioneer QDAT software (Version 3.41).…”

Application of Fragment-Based Drug Discovery to Versatile Targets

“…MS and X-ray Sijbesma et al, 2019 The RNA-dependent RNA polymerase X-ray Riccio et al, 2019 Apical membrane antigen 1 PRE, NMR Akter et al, 2019 Glyoxalase 1 Computational approach Perez et al, 2019 Focal Adhesion Kinase SPR and NMR Alvarado et al, 2019 E. coli DsbA NMR/X-ray Duncan et al, 2019 PDEδ-RAS (PPI) STD, CMPG-NMR Chen et al, 2019 *This table lists some studies using FBDD. Only a few studies published in 2019 were list for elucidating the application of FBDD to multiple targets.…”