Ligand binding, activation, and stabilization of the orphan G protein-coupled receptor GPR17

Abstract

G protein-coupled receptors (GPCRs) are one of the largest protein families in the human genome with more than 800 different sequences. They are transmembrane proteins that mediate the transduction of signals from extracellular to intracellular compartments. GPCRs are one of the most important classes of drug targets. More than 30 % of all approved therapeutics activate or inhibit GPCRs. However, drugs that are used to treat human diseases target only about 10 % of human GPCRs. Orphan GPCRs, for which the physiological agonist is still unknown or unconfirmed, represent an untapped group of potential drug targets. <br/> The orphan GPCR GPR17 belongs to the δ-branch of class A (rhodopsin-like) receptors and is primarily expressed in the central nervous system, particularly in oligodendrocyte progenitor cells and pre-oligodendrocytes. It has emerged as a promising therapeutic target for a range of diseases including multiple sclerosis, neurodegenerative disorders, diabetes, glioblastoma, and ischemic stroke. <br/> However, GPR17 is still underresearched. This dissertation presents research results from several subprojects related to the receptor. The first major part (Chapters 4–6) focuses on the modulation of GPR17 by agonists and antagonists, which were tested in calcium mobilization and radioligand binding assays. Since the endogenous agonist of GPR17 has not been identified yet, efforts to deorphanize GPR17 have been pursued, including the evaluation of previously proposed endogenous ligand 24(<em>S</em>)-hydroxycholesterol and further candidates. A virtual screening approach was employed to identify potential endogenous ligands based on molecular docking utilizing a homology model of the receptor. In parallel, novel synthetic ligands were investigated. A previous collaboration led to the discovery and optimization of anthranilic acid-based GPR17 antagonists. Through systematic structure–activity relationship studies, potent and selective compounds were identified, including PSB-22269 and PSB-24040, which showed nanomolar inhibitory activity and high binding affinity. Further GPR17 antagonists were also investigated, including 3-(3-carboxypropyl)indole-based, indolylsulfonamide-based, and peptide-mimetic scaffolds. These tool compounds will facilitate future studies and may serve as lead structures for therapeutic development. <br/> The second major part (Chapter 7) addresses the stabilization of GPR17 for structural studies to identify and characterize the ligand binding site as a basis for further drug development. Multiple strategies were employed, including point mutations, truncations, junction site optimization of the fusion protein BRIL, varying expression conditions in <em> Sf9</em> and <em>Tni</em> insect cells, and varying purification conditions. These efforts significantly improved receptor yield and thermostability, laying the foundation for its future structural elucidation by X-ray crystallography or cryo-electron microscopy.