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J Biomol Screen. 2016 Mar;21(3):243-51. doi: 10.1177/1087057115619597. Epub 2015 Dec 7.

An Alternative Thiol-Reactive Dye to Analyze Ligand Interactions with the Chemokine Receptor CXCR2 Using a New Thermal Shift Assay Format.

Author information

1
Novartis Institutes for BioMedical Research (NIBR), Center for Proteomic Chemistry, Switzerland christian.bergsdorf@novartis.com.
2
Novartis Institutes for BioMedical Research (NIBR), Center for Proteomic Chemistry, Switzerland Current affiliation: Heptares Therapeutics Ltd, BioPark, Welwyn Garden City, Hertfordshire, UK.
3
Novartis Institutes for BioMedical Research (NIBR), Respiratory Diseases, Horsham, UK Current affiliation: School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, UK.
4
Novartis Institutes for BioMedical Research (NIBR), Center for Proteomic Chemistry, Switzerland.
5
Novartis Institutes for BioMedical Research (NIBR), Center for Proteomic Chemistry, Switzerland Current affiliation: Novartis Pharma, Basel, Switzerland.

Abstract

Integral membrane proteins (IMPs) play an important role in many cellular events and are involved in numerous pathological processes. Therefore, understanding the structure and function of IMPs is a crucial prerequisite to enable successful targeting of these proteins with low molecular weight (LMW) ligands early on in the discovery process. To optimize IMP purification/crystallization and to identify/characterize LMW ligand-target interactions, robust, reliable, high-throughput, and sensitive biophysical methods are needed. Here, we describe a differential scanning fluorimetry (DSF) screening method using the thiol-reactive BODIPY FL-cystine dye to monitor thermal unfolding of the G-protein-coupled receptor (GPCR), CXCR2. To validate this method, the seven-transmembrane protein CXCR2 was analyzed with a set of well-characterized antagonists. This study showed that the new DSF assay assessed reliably the stability of CXCR2 in a 384-well format. The analysis of 14 ligands with a potency range over 4 log units demonstrated the detection/characterization of LMW ligands binding to the membrane protein target. Furthermore, DSF results cross-validated with the label-free differential static light scattering (DSLS) thermal denaturation method. These results underline the potential of the BODIPY assay format as a general tool to investigate membrane proteins and their interaction partners.

KEYWORDS:

ligand interaction; membrane protein; thermal shift assay; thiol-reactive dye

PMID:
26644402
DOI:
10.1177/1087057115619597
[Indexed for MEDLINE]

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