Crystal structures of G protein-coupled receptor (GPCR) ligand complexes allow a rational design of novel molecular probes and drugs. Here we report the structure-guided design, chemical synthesis and biological investigations of bivalent ligands for dopamine D2 receptor/neurotensin NTS1 receptor (D2R/NTS1R) heterodimers. The compounds of types 1–3 consist of three different D2R pharmacophores bound to an affinity-generating lipophilic appendage, a polyethylene glycol-based linker and the NTS1R agonist NT(8-13). The bivalent ligands show binding affinity in the picomolar range for cells coexpressing both GPCRs and unprecedented selectivity (up to three orders of magnitude), compared with cells that only express D2Rs. A functional switch is observed for the bivalent ligands 3b,c inhibiting cAMP formation in cells singly expressing D2Rs but stimulating cAMP accumulation in D2R/NTS1R-coexpressing cells. Moreover, the newly synthesized bivalent ligands show a strong, predominantly NTS1R-mediated β-arrestin-2 recruitment at the D2R/NTS1R-coexpressing cells.

Dopamine is a neurotransmitter of great physiological relevance. Disorders in dopaminergic signal transduction are associated with psychiatric and neurological pathologies such as parkinson's disease, schizophrenia and substance abuse. therefore, a detailed understanding of dopaminergic neurotransmission may provide access to novel therapeutic strategies for the treatment of these diseases. caged compounds with photoremovable groups represent molecular tools to investigate a biological target with high spatiotemporal resolution. Based on the crystal structure of the D 3 receptor in complex with eticlopride, we have developed caged D 2 /D 3 receptor ligands by rational design. We initially found that eticlopride, a widely used D 2 /D 3 receptor antagonist, was photolabile and therefore is not suitable for caging. Subtle structural modification of the pharmacophore led us to the photostable antagonist dechloroeticlopride, which was chemically transformed into caged ligands. Among those, the 2-nitrobenzyl derivative 4 (MG307) showed excellent photochemical stability, pharmacological behavior and decaging properties when interacting with dopamine receptor-expressing cells. Photopharmacology substantially contributes to our understanding of receptor function, potentially paving the way for new therapeutics 1. Hence, photoswitchable small molecules and neuropeptides have facilitated optical control of GPCR function 2. Moreover, photoactivable agonists and antagonists (caged ligands) have been developed 3. The rapid spatiotemporal control of such ligands upon photo-uncaging provides valuable insights into kinetics of association, dissociation as well as receptor-induced signalling. In vivo photopharmacology has been a significant challenge, because delivery of UV light to deep tissue infusion is technically challenging. However, new wireless devices being able to co-deliver light and drug or prodrug simultaneously may be a major breakthrough 4. Caged compounds consist of a biologically active molecule masked by a photolabile protective group, to prevent target binding and thus attenuate biological activity. Upon suitable illumination, photolytic cleavage of the cage leads to rapid release of the active molecule towards cellular targets via concentration jumps, ideally within the time span of a light pulse 5,6. Most prominent photosensitive masking groups are nitrobenzyl derivatives. These well-established cages have previously been introduced to a wide range of functionalities including ions 7,8 , phosphates 9 , phenols 10-13 , amines 13 and carboxylic acids 14. Photolytic cleavage of nitrobenzyl-type cages proceeds via a radical mechanism and is triggered by UV illumination with excitatory wavelengths ranging from 300 to 400 nm 15. Simple structural modifications involving formal introduction of two methoxy substituents allowed a cleavage with light of longer wavelengths 13,15,16. The neurotransmitter dopamine is critically involved in the regulation of movement, fine-motor control, emotions and behavior. Its physiolog...

Dopamine D2 receptors (D2Rs) are major targets in the treatment of psychiatric and neurodegenerative diseases. As with many other G protein-coupled receptors (GPCRs), D2Rs interact within the cellular membrane, leading to a transient receptor homo- or heterodimerization. These interactions are known to alter ligand binding, signaling, and receptor trafficking. Bivalent ligands are ideally suited to target GPCR dimers and are composed of two pharmacophores connected by a spacer element. If properly designed, bivalent ligands are able to engange the two orthosteric binding sites of a GPCR dimer simultaneously. Taking advantage of previously developed ligands for heterodimers of D2R and the neurotensin receptor 1 (NTSR1), we synthesized homobivalent ligands targeting D2R. Employing bioluminescence resonance energy transfer, we found that the bivalent ligands 3b and 4b comprising a 92-atom spacer are able to foster D2R-homodimerization while simultaneously reducing interactions of D2R with NTSR1. Both receptors are coexpressed in the central nervous system and involved in important physiological processes. The newly developed bivalent ligands are excellent tools to further understand the pharmacological consequences of D2R homo- and heterodimerization. Not limited to the dopaminergic system, modifying class A GPCRs’ dynamic equilibrium between monomers, homomers, and heteromers with bivalent ligands may represent a novel pharmacological concept paving the way toward innovative drugs.