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Development of a Fragment-Based Screening Assay for the Focal Adhesion Targeting Domain Using SPR and NMR
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 foca...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6766811/ https://www.ncbi.nlm.nih.gov/pubmed/31540099 http://dx.doi.org/10.3390/molecules24183352 |
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author | Alvarado, Carlos Stahl, Erik Koessel, Karissa Rivera, Andrew Cherry, Brian R. Pulavarti, Surya V.S.R.K. Szyperski, Thomas Cance, William Marlowe, Timothy |
author_facet | Alvarado, Carlos Stahl, Erik Koessel, Karissa Rivera, Andrew Cherry, Brian R. Pulavarti, Surya V.S.R.K. Szyperski, Thomas Cance, William Marlowe, Timothy |
author_sort | Alvarado, Carlos |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-6766811 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-67668112019-10-02 Development of a Fragment-Based Screening Assay for the Focal Adhesion Targeting Domain Using SPR and NMR Alvarado, Carlos Stahl, Erik Koessel, Karissa Rivera, Andrew Cherry, Brian R. Pulavarti, Surya V.S.R.K. Szyperski, Thomas Cance, William Marlowe, Timothy Molecules Article 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. MDPI 2019-09-14 /pmc/articles/PMC6766811/ /pubmed/31540099 http://dx.doi.org/10.3390/molecules24183352 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Alvarado, Carlos Stahl, Erik Koessel, Karissa Rivera, Andrew Cherry, Brian R. Pulavarti, Surya V.S.R.K. Szyperski, Thomas Cance, William Marlowe, Timothy Development of a Fragment-Based Screening Assay for the Focal Adhesion Targeting Domain Using SPR and NMR |
title | Development of a Fragment-Based Screening Assay for the Focal Adhesion Targeting Domain Using SPR and NMR |
title_full | Development of a Fragment-Based Screening Assay for the Focal Adhesion Targeting Domain Using SPR and NMR |
title_fullStr | Development of a Fragment-Based Screening Assay for the Focal Adhesion Targeting Domain Using SPR and NMR |
title_full_unstemmed | Development of a Fragment-Based Screening Assay for the Focal Adhesion Targeting Domain Using SPR and NMR |
title_short | Development of a Fragment-Based Screening Assay for the Focal Adhesion Targeting Domain Using SPR and NMR |
title_sort | development of a fragment-based screening assay for the focal adhesion targeting domain using spr and nmr |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6766811/ https://www.ncbi.nlm.nih.gov/pubmed/31540099 http://dx.doi.org/10.3390/molecules24183352 |
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