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StaR engineering: GPCR stabilisation for structure‐based drug design
Author(s) -
Wiggin Giselle,
Patel Jayesh C,
Kean James,
Khan Samir Ali,
Mould Richard,
Brown Jason,
Ng Irene W,
Errey James C,
Dore Andrew S,
Zhukov Andrei,
Tehan Benjamin,
Christopher John A,
Jazayeri Ali,
Marshall Fiona H
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.lb547
Subject(s) - g protein coupled receptor , receptor , surface plasmon resonance , ligand (biochemistry) , drug discovery , chemistry , orexin , agonist , biophysics , computational biology , biology , biochemistry , nanotechnology , materials science , neuropeptide , nanoparticle
G‐protein‐coupled receptors (GPCRs) represent one of the largest and most important classes of membrane proteins for drug discovery. However, due to their inherent flexibility and instability outside of the membrane, they are challenging targets for structural and biophysical studies. Here we demonstrate how these challenges have been overcome through protein engineering, by identifying a minimal number of thermostabilising mutations that can lock a receptor in a single conformation and enhance its survival in a detergent environment. This has been applied to GPCRs from families A, B and C, with peptide or small molecule ligands, and to orphan receptors with no known ligand. The resulting stabilised receptors (StaRs) are more amenable to purification, crystallisation and biophysical analysis of ligand binding. Using this approach, StaRs were generated for Orexin 1 and Orexin 2, two peptide GPCRs involved in regulating the sleep‐wake cycle. The StaRs show decreased agonist signalling, whilst maintaining antagonist binding, consistent with a shift to the antagonist conformation. Structural determination, together with biophysical mapping and fragment screening using surface plasmon resonance (SPR), has led to the rapid identification of highly optimised dual Orexin receptor antagonists (DORAs) with very high affinity, good pharmacokinetics and excellent in vivo activity in models of insomnia.