Inhibitor Library Screening of SH2 Domains Through Denaturation-Based Assays.

Ligands src Homology Domains Gene Library Blotting, Western Drug Discovery Fluorescent Dyes
Cellular thermal shift assay Fluorescence Inhibitor Phosphopeptide SH2 domain Thermal shift assay

Journal

Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969

Informations de publication

Date de publication:
2023
Historique:
medline: 6 9 2023
pubmed: 5 9 2023
entrez: 5 9 2023
Statut: ppublish

Résumé

Screening of inhibitor libraries for candidate ligands is an important step in the drug discovery process. Thermal denaturation-based screening strategies are built on the premise that a protein-ligand complex has an altered stability profile compared to the protein alone. As such, these assays provide an accessible and rapid methodology for stratifying ligands that directly engage with the protein target of interest. Here, we describe three denaturation-based strategies for examining protein-inhibitor binding, in the context of SH2 domains. This includes conventional dye-based Thermal Shift Assays (TSA), nonconventional labeled ligand-based TSA, and Cellular Thermal Shift Assays (CETSA). Conventional dye-based TSA reports on the fluorescence of an external hydrophobic dye as it interacts with heat-exposed nonpolar protein surfaces as the temperature is incrementally increased. By contrast, nonconventional-labeled ligand TSA involves a fluorescence-tagged probe (phosphopeptide for SH2 domains) that is quenched as it dissociates from the protein during the denaturation process. CETSA involves monitoring the presence of the protein via Western blotting as the temperature is increased. In all three approaches, performing the assay in the presence of a candidate ligand can alter the melting profile of the protein. These assays offer primary screening tools to examine SH2 domain inhibitors libraries with varying chemical motifs, and a subset of the advantages and limitations of each approach is also discussed.

Identifiants

DOI: 10.1007/978-1-0716-3393-9_11 PMID: 37668976
pubmed: 37668976
doi: 10.1007/978-1-0716-3393-9_11
doi:

Substances chimiques

Ligands 0
Fluorescent Dyes 0

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Pagination

213-223

Informations de copyright

© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Références

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Auteurs

Elvin D de Araujo (ED)

Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, ON, Canada.

Anna Orlova (A)

Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria.

Qirat F Ashraf (QF)

Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, ON, Canada.
Department of Chemistry, University of Toronto, Toronto, ON, Canada.

Richard Moriggl (R)

Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria.

Patrick T Gunning (PT)

Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, ON, Canada. patrick.gunning@utoronto.ca.
Department of Chemistry, University of Toronto, Toronto, ON, Canada. patrick.gunning@utoronto.ca.

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Classifications MeSH

questionsmedicales.fr › Actions chimiques et utilisations › Utilisations spécialisées de produits chimiques › Produits chimiques de laboratoire › Ligands › Inhibitor Library Screening of SH2 Domains...
questionsmedicales.fr › Phénomènes chimiques › Phénomènes biochimiques › Conformation moléculaire › Conformation des protéines › Éléments structuraux des protéines › Motifs et domaines d'intéraction protéique › Domaine d'homologie SRC › Inhibitor Library Screening of SH2 Domains...
questionsmedicales.fr › Phénomènes génétiques › Structures génétiques › Banque de gènes › Inhibitor Library Screening of SH2 Domains...
questionsmedicales.fr › Techniques d'investigation › Techniques de sonde moléculaire › Dosage immunologique › Immunotransfert › Technique de Western › Inhibitor Library Screening of SH2 Domains...
questionsmedicales.fr › Techniques d'investigation › Développement de médicament › Découverte de médicament › Inhibitor Library Screening of SH2 Domains...
questionsmedicales.fr › Actions chimiques et utilisations › Utilisations spécialisées de produits chimiques › Produits chimiques de laboratoire › Indicateurs et réactifs › Luminescents › Colorants fluorescents › Inhibitor Library Screening of SH2 Domains...