Molecular determinants of biased agonism at the dopamine D2 receptor.

The development of biased (functionally selective) ligands provides a formidable challenge in medicinal chemistry. In an effort to learn to design functionally selective molecular tools for the highly therapeutically relevant dopamine D2 receptor, we synthesized a collection of agonists based on structurally distinct head groups derived from canonical or atypical dopaminergic pharmacophores. The test compounds feature a long lipophilic appendage that was shown to mediate biased signaling. By employing functional assays and molecular dynamics simulations, we could show that atypical dopamine surrogates of type 1 and 2 promote biased signaling, while ligands built from classical dopaminergic head groups (type 3 and 4) typically elicit more balanced signaling profiles. Besides this, we found a strong influence of the stereochemistry of type 4 aminotetraline-derived agonists on functional selectivity at D2 receptors. Whereas the (S)-enantiomer behaved as a full agonist, the biased ligand (R)-4 induced poor G protein coupling but substantial ß-arrestin recruitment.

Covalent agonists for studying G protein-coupled receptor activation.

Structural studies on G protein-coupled receptors (GPCRs) provide important insights into the architecture and function of these important drug targets. However, the crystallization of GPCRs in active states is particularly challenging, requiring the formation of stable and conformationally homogeneous ligand-receptor complexes. Native hormones, neurotransmitters, and synthetic agonists that bind with low affinity are ineffective at stabilizing an active state for crystallogenesis. To promote structural studies on the pharmacologically highly relevant class of aminergic GPCRs, we here present the development of covalently binding molecular tools activating Gs-, Gi-, and Gq-coupled receptors. The covalent agonists are derived from the monoamine neurotransmitters noradrenaline, dopamine, serotonin, and histamine, and they were accessed using a general and versatile synthetic strategy. We demonstrate that the tool compounds presented herein display an efficient covalent binding mode and that the respective covalent ligand-receptor complexes activate G proteins comparable to the natural neurotransmitters. A crystal structure of the ß2-adrenoreceptor in complex with a covalent noradrenaline analog and a conformationally selective antibody (nanobody) verified that these agonists can be used to facilitate crystallogenesis.

Functionally selective dopamine D2/D3 receptor agonists comprising an enyne moiety.

Dopaminergics of types 1 and 2 incorporating a conjugated enyne as an atypical catechol-simulating moiety were synthesized in enantiomerically pure form and investigated for their metabolic stability. Radioligand binding studies indicated high affinity to D2-like receptors. The test compounds were evaluated for their ability to differentially activate distinct signaling pathways. Measurement of D(2L)- and D(2S)-mediated [(35)S]GTPγS incorporation in the presence of coexpressed Gα(o) and Gα(i) subunits showed significantly biased receptor activation for several test compounds. Thus, the 2-azaindolylcarboxamide (S)-2a exhibited substantial functional selectivity for D(2S)-promoted G(o) activation over G(i) coupling. The most significant bias was determined for the triazolylalkoxy-substituted benzamide (S)-2c that displayed higher potency for G(o) activation than for G(i) coupling at the D(2L) subtype. Functional selectivity for ß-arrestin recruitment over G(i) activation was observed for the biphenylcarboxamide (R)-1 and the 2-benzothiophenylcarboxamide (S)-2d, whereas the 2-substituted azaindole (S)-2a preferred ß-arrestin recruitment compared to G(o) coupling.

Class A G-protein-coupled receptor (GPCR) dimers and bivalent ligands.

G-protein-coupled receptors (GPCRs) represent the largest family of membrane proteins involved in cellular signal transduction and are activated by various different ligand types including photons, peptides, proteins, but also small molecules like biogenic amines. Therefore, GPCRs are involved in diverse physiological processes and provide valuable drug targets for numerous diseases. Emerging body of evidence suggests that GPCRs exist as monomers or cross-react forming dimers and higher-ordered oligomers. In this Perspective we will review current biochemical and biophysical techniques to visualize GPCR dimerization, functional consequences of homo- and heterodimers, and approaches of medicinal chemists to target these receptor complexes with homo- and heterobivalent ligands.