title: Improving the apo-state detergent stability of NTS1 with CHESS for pharmacological and structural studies.
abstract: The largest single class of drug targets is the G protein-coupled receptor (GPCR) family. Modern high-throughput methods for drug discovery require working with pure protein, but this has been a challenge for GPCRs, and thus the success of screening campaigns targeting soluble, catalytic protein domains has not yet been realized for GPCRs. Therefore, most GPCR drug screening has been cell-based, whereas the strategy of choice for drug discovery against soluble proteins is HTS using purified proteins coupled to structure-based drug design. While recent developments are increasing the chances of obtaining GPCR crystal structures, the feasibility of screening directly against purified GPCRs in the unbound state (apo-state) remains low. GPCRs exhibit low stability in detergent micelles, especially in the apo-state, over the time periods required for performing large screens. Recent methods for generating detergent-stable GPCRs, however, offer the potential for researchers to manipulate GPCRs almost like soluble enzymes, opening up new avenues for drug discovery. Here we apply cellular high-throughput encapsulation, solubilization and screening (CHESS) to the neurotensin receptor 1 (NTS1) to generate a variant that is stable in the apo-state when solubilized in detergents. This high stability facilitated the crystal structure determination of this receptor and also allowed us to probe the pharmacology of detergent-solubilized, apo-state NTS1 using robotic ligand binding assays. NTS1 is a target for the development of novel antipsychotics, and thus CHESS-stabilized receptors represent exciting tools for drug discovery.
authors: Department of Biochemistry, The University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland; The Florey Institute of Neuroscience and Mental Health, and The Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors: Daniel J
authors: DJ
authors: Scott
authors: Department of Biochemistry, The University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors: Lutz
authors: L
authors: Kummer
authors: Department of Biochemistry, The University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors: Pascal
authors: P
authors: Egloff
authors: The Florey Institute of Neuroscience and Mental Health, and The Department of Biochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.
authors: Ross A D
authors: RA
authors: Bathgate
authors: Department of Biochemistry, The University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. Electronic address: plueckthun@bioc.uzh.ch.
authors: Andreas
authors: A
authors: Plückthun
keywords: Detergent; Directed evolution; Encapsulation; G protein-coupled receptor; Stabilization; Thermostability