title: Functional Evaluation of Bivalent Ligands at Heterodimers of Dopamine D3 and Neurotensin 1 Receptors
Creators: name: Vogt, Hannah
Publisher: name: Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)
Year: 2025

description: G protein-coupled receptors (GPCRs) comprise the largest family of cell surface receptors with over 800 proteins in humans. GPCRs are involved in and control numerous physiological processes, like neuronal transmission, sensory transduction, hormonal signaling, and cell-cell communication, and are activated by several stimuli, like peptide and non-peptide neurotransmitters, odorant molecules, hormones, small molecule metabolites, growth factors, and light. GPCRs are therefore ideal targets for drug development. More than 30% of the FDA-approved drugs on the market are targeting GPCRs, however, only 10% of the whole GPCR family is addressed. The co-expression of NTSR1 and D2R and the allosteric modulation of NTSR1 on D2R within a D2R-NTSR1 heterodimer opened a new strategy to treat CNS disorders. Besides targeting D2R-NTSR1 heterodimers to potentially improve the therapy of Parkinson’s disease and schizophrenia, D3R-NTSR1 heterodimers could be addressed in addition for addiction and inflammatory diseases as well as pathological gambling. Bivalent ligands present extremely powerful tools for the study of these GPCR heterodimers and may potentially serve as next generation therapeutics with reduced side effects. In this thesis, the ability of bivalent ligands to bind to and foster heterodimerization of D3R with NTSR1 was analyzed using new potentially D3R-selective bivalent ligands. Functional analyses were carried out to characterize their pharmacological behavior and revealed an individual signaling profile of bivalently bound D3R-NTSR1 heterodimers. First, the effects of the new potentially D3R-selective bivalent ligands on heterodimer formation were investigated using BRET saturation assays (chapter 3) and compared to the effects on D2R-NTSR1 heterodimer formation. Since bivalent ligands often possess a higher binding aﬀinity to the dimer compared to the monomeric parent receptors, radioligand binding studies were employed to analyze the aﬀinities of the new bivalent ligands and to investigate their ability to bivalently bind to the heterodimer (chapter 4). In addition, binding studies to D2R and D2R-NTSR1 demonstrated their binding selectivity for D3R and D3R-NTSR1. Further, heterodimerization can lead to altered signal transduction and receptor traﬀicking. Thus, the bivalent ligand-induced G protein activation, mini G protein and β-arrestin-2 recruitment to the heterodimer in comparison to the monomeric receptors, as well as the induced traﬀicking route of the receptors was investigated in the chapters 5 and 6. Therefore, enzyme complementation assays and several BRET-based approaches were utilized to investigate the bivalent ligand-induced potencies and eﬀicacies, as well as kinetic studies, which were performed during my research stay in the laboratory of Prof. Davide Calebiro (University of Birmingham). In addition, D3R-NTSR1 heterodimerization were explored in CHO cells. These cells are well established for microscopic studies, especially using the high-resolution TIRF technology. The chapter 7 focuses on the evaluation of suitable conditions and receptor constructs for the microscopic investigation of D3R-NTSR1 on the plasma membrane of living cells. These experiments are also part of the research stay in the laboratory of Davide Calebiro at the University of Birmingham. Furthermore, a recently described allosteric modulator for NTSR1 (SBI-553) is considered as potential pharmacophore for novel hetero-bivalent ligands. An allosteric ligand could have the advantage to bypass the side effects of orthosteric NTSR1 ligands by modulating specific signaling pathways and inhibiting others. The focus of chapter 8 was the characterization of SBI-553 derivatives as allosteric NTSR1 pharmacophores, as well as the analysis of the most promising candidate incorporated into a bivalent ligand (synthesis by Patrick Shinkwin, Chair of Medicinal Chemistry, Gmeiner laboratory, FAU) for its effects on dopamine and neurotensin receptor heterodimer formation and ligand binding. Overall, the results clearly demonstrate the D3R over D2R selectivity of the new potentially D3R-selective bivalent ligands in mono- and co-expression systems. The D3R-selective bivalent ligands with a spacer length of 44-atoms or more were able to bivalently bind to D3R-NTSR1 heterodimers and enhance the D3R-NTSR1 interaction. The pharmacological properties of D3R-NTSR1 are altered compared to monomeric D3 receptors: The bivalent ligands were able to induce mini Gq, mini Gs, mini Gi and ß-arrestin-2 recruitment to the heterodimer as well as the internalization of the heterodimer-bivalent ligand complex. These results indicate the unique pharmacological properties of the bivalent ligands and demonstrate that the D3R-NTSR1 heterodimer serves as a novel functional entity. Treatment of these heterodimers with bivalent ligands may lead to an innovative tissue selective treatment option due to the high affinity of the bivalent ligands to the heterodimer over monomeric D3R.