Structure-based discovery of CFTR potentiators and inhibitors.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, whereas its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here, we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify CFTR modulators. We docked ∼155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered mid-nanomolar potentiators, as well as inhibitors, that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.

Time-resolved cryo-EM of G-protein activation by a GPCR.

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit1. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G-protein complex. By monitoring the transitions of the stimulatory Gs protein in complex with the ß2-adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor. Twenty structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of main events driving G-protein activation in response to GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα switch regions and the α5 helix that weaken the G-protein-receptor interface. Molecular dynamics simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP on closure of the α-helical domain against the nucleotide-bound Ras-homology domain correlates with α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signalling events.

Conformational Complexity and Dynamics in a Muscarinic Receptor Revealed by NMR Spectroscopy.

The M2 muscarinic acetylcholine receptor (M2R) is a prototypical GPCR that plays important roles in regulating heart rate and CNS functions. Crystal structures provide snapshots of the M2R in inactive and active states, but the allosteric link between the ligand binding pocket and cytoplasmic surface remains poorly understood. Here we used solution NMR to examine the structure and dynamics of the M2R labeled with 13CH3-ε-methionine upon binding to various orthosteric and allosteric ligands having a range of efficacy for both G protein activation and arrestin recruitment. We observed ligand-specific changes in the NMR spectra of 13CH3-ε-methionine probes in the M2R extracellular domain, transmembrane core, and cytoplasmic surface, allowing us to correlate ligand structure with changes in receptor structure and dynamics. We show that the M2R has a complex energy landscape in which ligands with different efficacy profiles stabilize distinct receptor conformations.

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.

Therapeutic antagonism of the neurokinin 1 receptor in endosomes provides sustained pain relief.

The hypothesis that sustained G protein-coupled receptor (GPCR) signaling from endosomes mediates pain is based on studies with endocytosis inhibitors and lipid-conjugated or nanoparticle-encapsulated antagonists targeted to endosomes. GPCR antagonists that reverse sustained endosomal signaling and nociception are needed. However, the criteria for rational design of such compounds are ill-defined. Moreover, the role of natural GPCR variants, which exhibit aberrant signaling and endosomal trafficking, in maintaining pain is unknown. Herein, substance P (SP) was found to evoke clathrin-mediated assembly of endosomal signaling complexes comprising neurokinin 1 receptor (NK1R), Gαq/i, and ßarrestin-2. Whereas the FDA-approved NK1R antagonist aprepitant induced a transient disruption of endosomal signals, analogs of netupitant designed to penetrate membranes and persist in acidic endosomes through altered lipophilicity and pKa caused sustained inhibition of endosomal signals. When injected intrathecally to target spinal NK1R+ve neurons in knockin mice expressing human NK1R, aprepitant transiently inhibited nociceptive responses to intraplantar injection of capsaicin. Conversely, netupitant analogs had more potent, efficacious, and sustained antinociceptive effects. Mice expressing C-terminally truncated human NK1R, corresponding to a natural variant with aberrant signaling and trafficking, displayed attenuated SP-evoked excitation of spinal neurons and blunted nociceptive responses to SP. Thus, sustained antagonism of the NK1R in endosomes correlates with long-lasting antinociception, and domains within the C-terminus of the NK1R are necessary for the full pronociceptive actions of SP. The results support the hypothesis that endosomal signaling of GPCRs mediates nociception and provides insight into strategies for antagonizing GPCRs in intracellular locations for the treatment of diverse diseases.

The small molecule activator S3969 stimulates the epithelial sodium channel by interacting with a specific binding pocket in the channel's ß-subunit.

The epithelial sodium channel (ENaC) is essential for mediating sodium absorption in several epithelia. Its impaired function leads to severe disorders, including pseudohypoaldosteronism type 1 and respiratory distress. Therefore, pharmacological ENaC activators have potential therapeutic implications. Previously, a small molecule ENaC activator (S3969) was developed. So far, little is known about molecular mechanisms involved in S3969-mediated ENaC stimulation. Here, we identified an S3969-binding site in human ENaC by combining structure-based simulations with molecular biological methods and electrophysiological measurements of ENaC heterologously expressed in Xenopus laevis oocytes. We confirmed a previous observation that the extracellular loop of ß-ENaC is essential for ENaC stimulation by S3969. Molecular dynamics simulations predicted critical residues in the thumb domain of ß-ENaC (Arg388, Phe391, and Tyr406) that coordinate S3969 within a binding site localized at the ß-γ-subunit interface. Importantly, mutating each of these residues reduced (R388H; R388A) or nearly abolished (F391G; Y406A) the S3969-mediated ENaC activation. Molecular dynamics simulations also suggested that S3969-mediated ENaC stimulation involved a movement of the α5 helix of the thumb domain of ß-ENaC away from the palm domain of γ-ENaC. Consistent with this, the introduction of two cysteine residues (ßR437C - γS298C) to form a disulfide bridge connecting these two domains prevented ENaC stimulation by S3969 unless the disulfide bond was reduced by DTT. Finally, we demonstrated that S3969 stimulated ENaC endogenously expressed in cultured human airway epithelial cells (H441). These new findings may lead to novel (patho-)physiological and therapeutic concepts for disorders associated with altered ENaC function.

"Photo-Adrenalines": Photoswitchable ß2 -Adrenergic Receptor Agonists as Molecular Probes for the Study of Spatiotemporal Adrenergic Signaling.

ß2 -adrenergic receptor (ß2 -AR) agonists are used for the treatment of asthma and chronic obstructive pulmonary disease, but also play a role in other complex disorders including cancer, diabetes and heart diseases. As the cellular and molecular mechanisms in various cells and tissues of the ß2 -AR remain vastly elusive, we developed tools for this investigation with high temporal and spatial resolution. Several photoswitchable ß2 -AR agonists with nanomolar activity were synthesized. The most potent agonist for ß2 -AR with reasonable switching is a one-digit nanomolar active, trans-on arylazopyrazole-based adrenaline derivative and comprises valuable photopharmacological properties for further biological studies with high structural accordance to the native ligand adrenaline.

Structure-guided discovery of orexin receptor-binding PET ligands.

Molecular imaging using positron emission tomography (PET) can serve as a promising tool for visualizing biological targets in the brain. Insights into the expression pattern and the in vivo imaging of the G protein-coupled orexin receptors OX1R and OX2R will further our understanding of the orexin system and its role in various physiological and pathophysiological processes. Guided by crystal structures of our lead compound JH112 and the approved hypnotic drug suvorexant bound to OX1R and OX2R, respectively, we herein describe the design and synthesis of two novel radioligands, [18F]KD23 and [18F]KD10. Key to the success of our structural modifications was a bioisosteric replacement of the triazole moiety with a fluorophenyl group. The 19F-substituted analog KD23 showed high affinity for the OX1R and selectivity over OX2R, while the high affinity ligand KD10 displayed similar Ki values for both subtypes. Radiolabeling starting from the respective pinacol ester precursors resulted in excellent radiochemical yields of 93% and 88% for [18F]KD23 and [18F]KD10, respectively, within 20 min. The new compounds will be useful in PET studies aimed at subtype-selective imaging of orexin receptors in brain tissue.

Inhibiting a promiscuous GPCR: iterative discovery of bitter taste receptor ligands.

The human GPCR family comprises circa 800 members, activated by hundreds of thousands of compounds. Bitter taste receptors, TAS2Rs, constitute a large and distinct subfamily, expressed orally and extra-orally and involved in physiological and pathological conditions. TAS2R14 is the most promiscuous member, with over 150 agonists and 3 antagonists known prior to this study. Due to the scarcity of inhibitors and to the importance of chemical probes for exploring TAS2R14 functions, we aimed to discover new ligands for this receptor, with emphasis on antagonists. To cope with the lack of experimental structure of the receptor, we used a mixed experimental/computational methodology which iteratively improved the performance of the predicted structure. The increasing number of active compounds, obtained here through experimental screening of FDA-approved drug library, and through chemically synthesized flufenamic acid derivatives, enabled the refinement of the binding pocket, which in turn improved the structure-based virtual screening reliability. This mixed approach led to the identification of 10 new antagonists and 200 new agonists of TAS2R14, illustrating the untapped potential of rigorous medicinal chemistry for TAS2Rs. 9% of the ~ 1800 pharmaceutical drugs here tested activate TAS2R14, nine of them at sub-micromolar concentrations. The iterative framework suggested residues involved in the activation process, is suitable for expanding bitter and bitter-masking chemical space, and is applicable to other promiscuous GPCRs lacking experimental structures.

Visualization of ß-adrenergic receptor dynamics and differential localization in cardiomyocytes.

A key question in receptor signaling is how specificity is realized, particularly when different receptors trigger the same biochemical pathway(s). A notable case is the two ß-adrenergic receptor (ß-AR) subtypes, ß1 and ß2, in cardiomyocytes. They are both coupled to stimulatory Gs proteins, mediate an increase in cyclic adenosine monophosphate (cAMP), and stimulate cardiac contractility; however, other effects, such as changes in gene transcription leading to cardiac hypertrophy, are prominent only for ß1-AR but not for ß2-AR. Here, we employ highly sensitive fluorescence spectroscopy approaches, in combination with a fluorescent ß-AR antagonist, to determine the presence and dynamics of the endogenous receptors on the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. These techniques allow us to visualize that the ß2-AR is confined to and diffuses within the T-tubular network, as opposed to the ß1-AR, which is found to diffuse both on the outer plasma membrane as well as on the T-tubules. Upon overexpression of the ß2-AR, this compartmentalization is lost, and the receptors are also seen on the cell surface. Such receptor segregation depends on the development of the T-tubular network in adult cardiomyocytes since both the cardiomyoblast cell line H9c2 and the cardiomyocyte-differentiated human-induced pluripotent stem cells express the ß2-AR on the outer plasma membrane. These data support the notion that specific cell surface targeting of receptor subtypes can be the basis for distinct signaling and functional effects.

Phosphoproteomic Analysis of Dopamine D2 Receptor Signaling Reveals Interplay of G Protein- and ß-Arrestin-Mediated Effects.

Leveraging biased signaling of G protein-coupled receptors has been proposed as a promising strategy for the development of drugs with higher specificity. However, the consequences of selectively targeting G protein- or ß-arrestin-mediated signaling on cellular functions are not comprehensively understood. In this study, we utilized phosphoproteomics to gain a systematic overview of signaling induced by the four biased and balanced dopamine D2 receptor (D2R) ligands MS308, BM138, quinpirole, and sulpiride in an in vitro D2R transfection model. Quantification of 14,160 phosphosites revealed a low impact of the partial G protein agonist MS308 on cellular protein phosphorylation, as well as surprising similarities between the balanced agonist quinpirole and the inverse agonist sulpiride. Analysis of the temporal profiles of ligand-induced phosphorylation events showed a transient impact of the G protein-selective agonist MS308, whereas the ß-arrestin-preferring agonist BM138 elicited a delayed, but more pronounced response. Functional enrichment analysis of ligand-impacted phosphoproteins and treatment-linked kinases confirmed multiple known functions of D2R signaling while also revealing novel effects, for example of MS308 on sterol regulatory element-binding protein-related gene expression. All raw data were deposited in MassIVE (MSV000089457).

The beer component hordenine inhibits alcohol addiction-associated behaviours in mice.

Alcohol consumption is a widespread behaviour that may eventually result in the development of alcohol use disorder (AUD). Alcohol, however, is rarely consumed in pure form but in fruit- or corn-derived preparations, like beer. These preparations add other compounds to the consumption, which may critically modify alcohol intake and AUD risk. We investigated the effects of hordenine, a barley-derived beer compound on alcohol use-related behaviours. We found that the dopamine D2 receptor agonist hordenine (50 mg/kg) limited ongoing alcohol consumption and prophylactically diminished relapse drinking after withdrawal in mice. Although not having reinforcing effects on its own, hordenine blocked the establishment of alcohol-induced conditioned place preference (CPP). However, it independently enhanced alcohol CPP retrieval. Hordenine had a dose-dependent inhibitory effect on locomotor activity. Chronic hordenine exposure enhanced monoamine tissue levels in many brain regions. Further characterization revealed monoaminergic binding sites of hordenine and found a strong binding on the serotonin and dopamine transporters, and dopamine D3 , and adrenergic α1A and α2A receptor activation but no effects on GABAA receptor or glycinergic signalling. These findings suggest that natural ingredients of beer, like hordenine, may work as an inhibitory and use-regulating factor by their modulation of monoaminergic signalling in the brain.

Structure-based development of a subtype-selective orexin 1 receptor antagonist.

Orexins are neuropeptides that activate the rhodopsin-like G protein-coupled receptors OX1R and OX2R. The orexin system plays an important role in the regulation of the sleep-wake cycle and the regulation of feeding and emotions. The nonselective orexin receptor antagonist suvorexant has been the first drug on the market targeting the orexin system and is prescribed for the treatment of insomnia. Subtype-selective OX1R antagonists are valuable tools to further investigate the functions and physiological role of the OX1R in vivo and promising lead compounds for the treatment of drug addiction, anxiety, pain or obesity. Starting from the OX1R and OX2R crystal structures bound to suvorexant, we exploited a single amino acid difference in the orthosteric binding site by using molecular docking and structure-based drug design to optimize ligand interactions with the OX1R while introducing repulsive interactions with the OX2R. A newly established enantiospecific synthesis provided ligands showing up to 75-fold selectivity for the OX1R over the OX2R subtype. The structure of a new OX1R antagonist with subnanomolar affinity (JH112) was determined by crystallography in complex with the OX1R and corresponded closely to the docking-predicted geometry. JH112 exhibits high selectivity over a panel of different GPCRs, is able to cross the blood-brain barrier and acts as slowly diffusing and insurmountable antagonist for Gq protein activation and in particular ß-arrestin-2 recruitment at OX1R. This study demonstrates the potential of structure-based drug design to develop more subtype-selective GPCR ligands with potentially reduced side effects and provides an attractive probe molecule and lead compound.

Design of Drug Efficacy Guided by Free Energy Simulations of the ß2 -Adrenoceptor.

G-protein-coupled receptors (GPCRs) play important roles in physiological processes and are modulated by drugs that either activate or block signaling. Rational design of the pharmacological efficacy profiles of GPCR ligands could enable the development of more efficient drugs, but is challenging even if high-resolution receptor structures are available. We performed molecular dynamics simulations of the ß2 adrenergic receptor in active and inactive conformations to assess if binding free energy calculations can predict differences in ligand efficacy for closely related compounds. Previously identified ligands were successfully classified into groups with comparable efficacy profiles based on the calculated shift in ligand affinity upon activation. A series of ligands were then predicted and synthesized, leading to the discovery of partial agonists with nanomolar potencies and novel scaffolds. Our results demonstrate that free energy simulations enable design of ligand efficacy and the same approach can be applied to other GPCR drug targets.

Photochromic Fentanyl Derivatives for Controlled µ-Opioid Receptor Activation.

Photoswitchable ligands as biological tools provide an opportunity to explore the kinetics and dynamics of the clinically relevant µ-opioid receptor. These ligands can potentially activate or deactivate the receptor when desired by using light. Spatial and temporal control of biological activity allows for application in a diverse range of biological investigations. Photoswitchable ligands have been developed in this work, modelled on the known agonist fentanyl, with the aim of expanding the current "toolbox" of fentanyl photoswitchable ligands. In doing so, ligands have been developed that change geometry (isomerize) upon exposure to light, with varying photophysical and biochemical properties. This variation in properties could be valuable in further studying the functional significance of the µ-opioid receptor.

Activation of the α2B adrenoceptor by the sedative sympatholytic dexmedetomidine.

The α2 adrenergic receptors (α2ARs) are G protein-coupled receptors (GPCRs) that respond to adrenaline and noradrenaline and couple to the Gi/o family of G proteins. α2ARs play important roles in regulating the sympathetic nervous system. Dexmedetomidine is a highly selective α2AR agonist used in post-operative patients as an anxiety-reducing, sedative medicine that decreases the requirement for opioids. As is typical for selective αAR agonists, dexmedetomidine consists of an imidazole ring and a substituted benzene moiety lacking polar groups, which is in contrast to ßAR-selective agonists, which share an ethanolamine group and an aromatic system with polar, hydrogen-bonding substituents. To better understand the structural basis for the selectivity and efficacy of adrenergic agonists, we determined the structure of the α2BAR in complex with dexmedetomidine and Go at a resolution of 2.9 Å by single-particle cryo-EM. The structure reveals the mechanism of α2AR-selective activation and provides insights into Gi/o coupling specificity.

An allosteric modulator binds to a conformational hub in the ß2 adrenergic receptor.

Most drugs acting on G-protein-coupled receptors target the orthosteric binding pocket where the native hormone or neurotransmitter binds. There is much interest in finding allosteric ligands for these targets because they modulate physiologic signaling and promise to be more selective than orthosteric ligands. Here we describe a newly developed allosteric modulator of the ß2-adrenergic receptor (ß2AR), AS408, that binds to the membrane-facing surface of transmembrane segments 3 and 5, as revealed by X-ray crystallography. AS408 disrupts a water-mediated polar network involving E1223.41 and the backbone carbonyls of V2065.45 and S2075.46. The AS408 binding site is adjacent to a previously identified molecular switch for ß2AR activation formed by I3.40, P5.50 and F6.44. The structure reveals how AS408 stabilizes the inactive conformation of this switch, thereby acting as a negative allosteric modulator for agonists and positive allosteric modulator for inverse agonists.

Development of disulfide-functionalized peptides covalently binding G protein-coupled receptors.

A broadly applicable synthesis of peptides incorporating mixed disulfides between cysteine and homocysteine and cysteamine was developed. The method was established using pharmacologically relevant G protein-coupled receptor (GPCR) ligands including the µ-receptor agonist Dmt-DALDA and extended to the orexin derivative Oxa(17-33) and NT(8-13), the C-terminal hexapeptide of neurotensin. The newly developed NT(8-13) analog 6b incorporating an S-functionalized homocysteine revealed covalent binding of the neurotensin receptor 1 (NTSR1) in a radioligand depletion study.

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.

The Bitter Taste Receptor TAS2R14 as a Drug Target.

G protein-coupled receptors (GPCRs) mediate most of our physiological responses to hormones, neurotransmitters and environmental stimulants. Besides human senses like vision and olfaction, taste perception is mostly mediated by GPCRs. Hence, the bitter taste receptor family TAS2R comprises 25 distinct receptors and plays a key role in food acceptance and drug compliance. The TAS2R14 subtype is the most broadly tuned bitter taste receptor, recognizing a range of chemically highly diverse agonists. Besides other tissues, it is expressed in human airway smooth muscle and may represent a novel drug target for airway diseases. Several natural products as well as marketed drugs including flufenamic acid have been identified to activate TAS2R14, but higher potency ligands are needed to investigate the ligand-controlled physiological function and to facilitate the targeted modulate for potential future clinical applications. A combination of structure-based molecular modeling with chemical synthesis and in vitro profiling recently resulted in new flufenamic acid agonists with improved TAS2R14 potency and provided a validated and refined structural model of ligand-TAS2R14 interactions, which can be applied for future drug design projects.