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Open AccessArticle

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

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University of Arizona Cancer Center, 625 N. 6th Street, Phoenix, AZ 85004, USA
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University of Arizona College of Pharmacy-Phoenix, 650 E. Van Buren Street, Phoenix, AZ 85004, USA
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Molecular Discovery Core, University of Arizona College of Medicine-Phoenix, 475 N. 5th Street, Phoenix, AZ 85004, USA
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The Magnetic Resonance Research Center, Arizona State University, Tempe, AZ 85287, USA
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Department of Chemistry, State University of New York at Buffalo, Buffalo, NY 14260, USA
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Interdisciplinary Oncology, University of Arizona College of Medicine-Phoenix, 475 N. 5th Street, Phoenix, AZ 85004, USA
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Pharmacology and Toxicology, University of Arizona College of Medicine-Phoenix, P.O. Box 210207, Tucson, AZ 85721, USA
*
Author to whom correspondence should be addressed.
Academic Editor: Brian J. Stockman
Molecules 2019, 24(18), 3352; https://doi.org/10.3390/molecules24183352
Received: 25 August 2019 / Revised: 11 September 2019 / Accepted: 13 September 2019 / Published: 14 September 2019
(This article belongs to the Special Issue Fragment Based Drug Discovery)
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. View Full-Text
Keywords: focal adhesion kinase; FAT domain; fragment-based drug discovery; surface plasmon resonance; nuclear magnetic resonance focal adhesion kinase; FAT domain; fragment-based drug discovery; surface plasmon resonance; nuclear magnetic resonance
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MDPI and ACS Style

Alvarado, C.; Stahl, E.; Koessel, K.; Rivera, A.; Cherry, B.R.; Pulavarti, S.V.; Szyperski, T.; Cance, W.; Marlowe, T. Development of a Fragment-Based Screening Assay for the Focal Adhesion Targeting Domain Using SPR and NMR. Molecules 2019, 24, 3352.

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