title: An Alternative Thiol-Reactive Dye to Analyze Ligand Interactions with the Chemokine Receptor CXCR2 Using a New Thermal Shift Assay Format.
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.
authors.affiliation: Novartis Institutes for BioMedical Research (NIBR), Center for Proteomic Chemistry, Switzerland christian.bergsdorf@novartis.com.
authors.forename: Christian
authors.initials: C
authors.lastname: Bergsdorf
authors.affiliation: Novartis Institutes for BioMedical Research (NIBR), Center for Proteomic Chemistry, Switzerland Current affiliation: Heptares Therapeutics Ltd, BioPark, Welwyn Garden City, Hertfordshire, UK.
authors.forename: Cédric
authors.initials: C
authors.lastname: Fiez-Vandal
authors.affiliation: 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.
authors.forename: David A
authors.initials: DA
authors.lastname: Sykes
authors.affiliation: Novartis Institutes for BioMedical Research (NIBR), Center for Proteomic Chemistry, Switzerland.
authors.forename: Pascal
authors.initials: P
authors.lastname: Bernet
authors.affiliation: Novartis Institutes for BioMedical Research (NIBR), Center for Proteomic Chemistry, Switzerland Current affiliation: Novartis Pharma, Basel, Switzerland.
authors.forename: Sonia
authors.initials: S
authors.lastname: Aussenac
authors.affiliation: 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.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Charlton
authors.affiliation: Novartis Institutes for BioMedical Research (NIBR), Center for Proteomic Chemistry, Switzerland.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schopfer
authors.affiliation: Novartis Institutes for BioMedical Research (NIBR), Center for Proteomic Chemistry, Switzerland.
authors.forename: Johannes
authors.initials: J
authors.lastname: Ottl
authors.affiliation: Novartis Institutes for BioMedical Research (NIBR), Center for Proteomic Chemistry, Switzerland.
authors.forename: Myriam
authors.initials: M
authors.lastname: Duckely
mesh_terms: D002152:Calorimetry, Differential Scanning / Q000379:methods; D004396:Coloring Agents; D055808:Drug Discovery / Q000379:methods*; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D055550:Protein Stability; D058767:Protein Unfolding; D023063:Receptors, Interleukin-8B / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D054852:Small Molecule Libraries; D013438:Sulfhydryl Compounds
chemical_list: D004396:Coloring Agents; D008024:Ligands; D008565:Membrane Proteins; D023063:Receptors, Interleukin-8B; D011993:Recombinant Fusion Proteins; D054852:Small Molecule Libraries; D013438:Sulfhydryl Compounds
keywords: ligand interaction; membrane protein; thermal shift assay; thiol-reactive dye