Author Correction: Structural insights into µ-opioid receptor activation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structure-based discovery of opioid analgesics with reduced side effects.

Morphine is an alkaloid from the opium poppy used to treat pain. The potentially lethal side effects of morphine and related opioids-which include fatal respiratory depression-are thought to be mediated by µ-opioid-receptor (µOR) signalling through the ß-arrestin pathway or by actions at other receptors. Conversely, G-protein µOR signalling is thought to confer analgesia. Here we computationally dock over 3 million molecules against the µOR structure and identify new scaffolds unrelated to known opioids. Structure-based optimization yields PZM21-a potent Gi activator with exceptional selectivity for µOR and minimal ß-arrestin-2 recruitment. Unlike morphine, PZM21 is more efficacious for the affective component of analgesia versus the reflexive component and is devoid of both respiratory depression and morphine-like reinforcing activity in mice at equi-analgesic doses. PZM21 thus serves as both a probe to disentangle µOR signalling and a therapeutic lead that is devoid of many of the side effects of current opioids.

Structural insights into µ-opioid receptor activation.

Activation of the µ-opioid receptor (µOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for µOR activation, here we report a 2.1 Å X-ray crystal structure of the murine µOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the µOR binding pocket are subtle and differ from those observed for agonist-bound structures of the ß2-adrenergic receptor (ß2AR) and the M2 muscarinic receptor. Comparison with active ß2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the µOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.

Basal Histamine H4 Receptor Activation: Agonist Mimicry by the Diphenylalanine Motif.

Histamine H4 receptor (H4 R) orthologues are G-protein-coupled receptors (GPCRs) that exhibit species-dependent basal activity. In contrast to the basally inactive mouse H4 R (mH4 R), human H4 R (hH4 R) shows a high degree of basal activity. We have performed long-timescale molecular dynamics simulations and rigidity analyses on wild-type hH4 R, the experimentally characterized hH4 R variants S179M, F169V, F169V+S179M, F168A, and on mH4 R to investigate the molecular nature of the differential basal activity. H4 R variant-dependent differences between essential motifs of GPCR activation and structural stabilities correlate with experimentally determined basal activities and provide a molecular explanation for the differences in basal activation. Strikingly, during the MD simulations, F16945.55 dips into the orthosteric binding pocket only in the case of hH4 R, thus adopting the role of an agonist and contributing to the stabilization of the active state. The results shed new light on the molecular mechanism of basal H4 R activation that are of importance for other GPCRs.

NTS2-selective neurotensin mimetics with tetrahydrofuran amino acids.

Stimulation of the NTS2 neurotensin receptor causes antipsychotic effects and leads to a promotion of the µ-opioid-independent antinociception, which is important in the modulation of tonic pain sensitivity. We report the synthesis and properties of a small library of peptidic agonists based on the active neurotensin fragment NT(8-13). Two tetrahydrofuran amino acid derivatives were synthesized to replace Tyr11 in NT(8-13). Additionally, Arg8, Arg9, and Ile12 of the lead peptide were exchanged by Lys, Lys, and Gly, respectively. The new compounds showed substantial NTS2 binding affinity and up to 1000-fold selectivity over NTS1. The highest selectivity (Ki(NTS2): 29nM, Ki(NTS1): 35,000nM) was observed for the peptide analog 17Rtrans.

Identification of Two Distinct Sites for Antagonist and Biased Agonist Binding to the Human Chemokine Receptor CXCR3.

The chemokine receptor CXCR3 is a G protein-coupled receptor that conveys extracellular signals into cells by changing its conformation upon ligand binding. We previously hypothesized that small-molecule allosteric CXCR3-agonists do not bind to the same allosteric binding pocket as 8-azaquinazolinone-based negative allosteric modulators. We have now performed molecular-dynamics (MD) simulations with metadynamics enhanced sampling on the CXCR3 system to refine structures and binding modes and to predict the CXCR3-binding affinities of the biased allosteric agonist FAUC1036 and the negative allosteric modulator RAMX3. We have identified two distinct binding sites; a "shallow" and a second "deeper" pocket to which the biased allosteric agonist FAUC1036 and negative allosteric modulator RAMX3 bind, respectively.

GPCR crystal structures: Medicinal chemistry in the pocket.

Recent breakthroughs in GPCR structural biology have significantly increased our understanding of drug action at these therapeutically relevant receptors, and this will undoubtedly lead to the design of better therapeutics. In recent years, crystal structures of GPCRs from classes A, B, C and F have been solved, unveiling a precise snapshot of ligand-receptor interactions. Furthermore, some receptors have been crystallized in different functional states in complex with antagonists, partial agonists, full agonists, biased agonists and allosteric modulators, providing further insight into the mechanisms of ligand-induced GPCR activation. It is now obvious that there is enormous diversity in the size, shape and position of the ligand binding pockets in GPCRs. In this review, we summarise the current state of solved GPCR structures, with a particular focus on ligand-receptor interactions in the binding pocket, and how this can contribute to the design of GPCR ligands with better affinity, subtype selectivity or efficacy.

1,4-Disubstituted aromatic piperazines with high 5-HT2A/D2 selectivity: Quantitative structure-selectivity investigations, docking, synthesis and biological evaluation.

Simultaneous targeting of dopamine D2 and 5-HT2A receptors for the treatment of schizophrenia is one key feature of typical and atypical antipsychotics. In most of the top-selling antipsychotic drugs like aripiprazole and risperidone, high affinity to both receptors can be attributed to the presence of 1,4-disubstituted aromatic piperazines or piperidines as primary receptor recognition elements. Taking advantage of our in-house library of phenylpiperazine-derived dopamine receptor ligands and experimental data, we established highly significant CoMFA and CoMSIA models for the prediction of 5-HT2A over D2 selectivity. Subsequently, the models were applied to identify the selective candidates 55-57 from our newly synthesized library of GPCR ligands comprising a pyrazolo[1,5-a]pyridine head group and a 1,2,3-triazole based linker unit. The test compound 57 showed subnanomolar a Ki value (0.64 nM) for 5-HT2A and more than 10- and 30-fold selectivity over the dopamine receptor isoforms D2S and D2L, respectively.

Comparative MD Simulations Indicate a Dual Role for Arg1323.50 in Dopamine-Dependent D2R Activation.

Residue Arg3.50 belongs to the highly conserved DRY-motif of class A GPCRs, which is located at the bottom of TM3. On the one hand, Arg3.50 has been reported to help stabilize the inactive state of GPCRs, but on the other hand has also been shown to be crucial for stabilizing active receptor conformations and mediating receptor-G protein coupling. The combined results of these studies suggest that the exact function of Arg3.50 is likely to be receptor-dependent and must be characterized independently for every GPCR. Consequently, we now present comparative molecular-dynamics simulations that use our recently described inactive-state and Gα-bound active-state homology models of the dopamine D2 receptor (D2R), which are either bound to dopamine or ligand-free, performed to identify the function of Arg1323.50 in D2R. Our results are consistent with a dynamic model of D2R activation in which Arg1323.50 adopts a dual role, both by stabilizing the inactive-state receptor conformation and enhancing dopamine-dependent D2R-G protein coupling.

Development of Covalent Ligand-Receptor Pairs to Study the Binding Properties of Nonpeptidic Neurotensin Receptor 1 Antagonists.

The neurotensin receptor NTS1 has been suggested to be of pharmaceutical relevance, as it was found to exert modulatory effects on dopaminergic signal transduction and to be involved in tumor progression. Rational drug design of NTS1 receptor ligands requires molecular insights into the binding behavior of a particular lead compound. Although crystal structures of NTS1 have revealed the molecular determinants of peptide-agonist interactions, the binding mode of small-molecule antagonists remains largely unknown. Employing a disulfide-based tethering approach, we developed covalently binding molecular probes. The ligands 1 and 2 are based on the pharmacophore of the nonpeptidic NTS1 antagonist SR142948A, allowing the formation of a disulfide bond to an engineered cysteine residue of NTS1. The position of the covalent bond between Cys127(2.65) and the ligand was used to predict the binding mode of the covalent antagonist 1 and its parent compound SR142948A by molecular dynamics simulations.

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.

Active-state model of a dopamine D2 receptor-Gαi complex stabilized by aripiprazole-type partial agonists.

Partial agonists exhibit a submaximal capacity to enhance the coupling of one receptor to an intracellular binding partner. Although a multitude of studies have reported different ligand-specific conformations for a given receptor, little is known about the mechanism by which different receptor conformations are connected to the capacity to activate the coupling to G-proteins. We have now performed molecular-dynamics simulations employing our recently described active-state homology model of the dopamine D2 receptor-Gαi protein-complex coupled to the partial agonists aripiprazole and FAUC350, in order to understand the structural determinants of partial agonism better. We have compared our findings with our model of the D2R-Gαi-complex in the presence of the full agonist dopamine. The two partial agonists are capable of inducing different conformations of important structural motifs, including the extracellular loop regions, the binding pocket and, in particular, intracellular G-protein-binding domains. As G-protein-coupling to certain intracellular epitopes of the receptor is considered the key step of allosterically triggered nucleotide-exchange, it is tempting to assume that impaired coupling between the receptor and the G-protein caused by distinct ligand-specific conformations is a major determinant of partial agonist efficacy.

Functionally selective dopamine D2, D3 receptor partial agonists.

Dopamine D2 receptor-promoted activation of Gα(o) over Gα(i) may increase synaptic plasticity and thereby might improve negative symptoms of schizophrenia. Heterocyclic dopamine surrogates comprising a pyrazolo[1,5-a]pyridine moiety were synthesized and investigated for their binding properties when low- to subnanomolar K(i) values were determined for D(2L), D(2S), and D3 receptors. Measurement of [(35)S]GTPγS incorporation at D(2S) coexpressed with G-protein subunits indicated significant bias for promotion of Gα(o1) over Gα(i2) coupling for several test compounds. Functionally selective D(2S) activation was most striking for the carbaldoxime 8b (Gα(o1), pEC50 = 8.87, E(max) = 65%; Gα(i2), pEC50 = 6.63, E(max) = 27%). In contrast, the investigated 1,4-disubstituted aromatic piperazines (1,4-DAPs) behaved as antagonists for ß-arrestin-2 recruitment, implying significant ligand bias for G-protein activation over ß-arrestin-2 recruitment at D(2S) receptors. Ligand efficacy and selectivity between D(2S) and D3 activation were strongly influenced by regiochemistry and the nature of functional groups attached to the pyrazolo[1,5-a]pyridine moiety.

Active-state models of ternary GPCR complexes: determinants of selective receptor-G-protein coupling.

Based on the recently described crystal structure of the ß2 adrenergic receptor--Gs-protein complex, we report the first molecular-dynamics simulations of ternary GPCR complexes designed to identify the selectivity determinants for receptor-G-protein binding. Long-term molecular dynamics simulations of agonist-bound ß2AR-Gαs and D2R-Gαi complexes embedded in a hydrated bilayer environment and computational alanine-scanning mutagenesis identified distinct residues of the N-terminal region of intracellular loop 3 to be crucial for coupling selectivity. Within the G-protein, specific amino acids of the α5-helix, the C-terminus of the Gα-subunit and the regions around αN-ß1 and α4-ß6 were found to determine receptor recognition. Knowledge of these determinants of receptor-G-protein binding selectivity is essential for designing drugs that target specific receptor/G-protein combinations.

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.