The serotonin 7 receptor (5-HT 7R) regulates various processes in the central nervous system, including mood, learning, and circadian rhythm control, among others. Receptor activation can lead to activation of the Gα s protein and a subsequent increase of intracellular cyclic adenosine monophosphate (cAMP). Receptor interaction with inverse agonists results in a decrease of basal cAMP levels and therefore a downstream effect of reduced neuronal excitability and neurotransmission. Recently, pellotine ( 1a), a Lophophora alkaloid, was unexpectedly shown to be an inverse agonist of the 5-HT 7R. Therefore, we evaluated close analogs of compound 1a, both naturally occurring and synthetic analogs, as inverse agonists of the 5-HT 7R. Functional evaluation in a GloSensor cAMP assay revealed a preference for an 8-hydroxy-6,7-dimethoxy substitution pattern over 6,7,8-trimethoxy analogs or 8-hydroxy-6,7-methylenedioxy analogs. This was supported by molecular dynamics simulations, where the 8-hydroxy substitution allowed more robust interaction with the 5-HT 7R, which correlated with inverse agonism efficacy. Additionally, N-methylation (as in 1a) improved the potency of the evaluated analogs. In this series, the most potent inverse agonist was anhalidine ( 1b) (EC 50 = 219 nM, E max = -95.4%), which lacks the 1-methyl, compared to pellotine ( 1a), and showed a 2-fold higher functional potency. Altogether, these results provide key insights for the further development of potent low molecular weight inverse agonists of the 5-HT 7R.

The serotonin 2A receptor (5-HT2AR) is the most abundant excitatory serotonin receptor in the brain and the primary target for serotonergic psychedelics. Despite its importance, the receptor's mechanism of action and structure-activity relationships are not fully understood. The experimental 5-HT2AR structures have identified a hydrophobic tunnel lateral to the orthosteric site, situated in between the transmembrane helices 4 and 5. Here, we have examined this tunnel and its implications for 5-HT2AR agonist pharmacology. The tunnel characteristics and the key roles of Phe2345x39 and Gly2385x43 were delineated by molecular modeling and the synthesis and functional characterization of ten phenethylamine analogs. Molecular modeling, structure-activity relationship determination, and mutagenesis experiments demonstrate that the agonist potencies at the 5-HT2AR exhibited by analogs with 4-substituents with at least four heavy atoms are rooted in their ability to protrude into and form interactions within this tunnel. Thus, these findings provide new insights into the molecular basis for phenethylamine-induced 5-HT2AR activation and identify this tunnel, for the first time, as a druggable region to employ in designing potent 5-HT2AR agonists.

Biased signaling gives hormones, probes or drugs distinct functional outcomes via the same receptor. The Biased Signaling Atlas ( https://BiasedSignalingAtlas.org ) provides a community hub with data and tools to advance this paradigm, which may yield safer and more potent drugs.

Serotonergic psychedelics are described to have activation of the serotonin 2A receptor (5-HT2A) as their main pharmacological action. Despite their relevance, the molecular mechanisms underlying the psychedelic effects induced by certain 5-HT2A agonists remain elusive. One of the proposed hypotheses is the occurrence of biased agonism, defined as the preferential activation of certain signaling pathways over others. This study comparatively monitored the efficiency of a diverse panel of 4-position-substituted (and N-benzyl-derived) phenylalkylamines to induce recruitment of β-arrestin2 (βarr2) or miniGαq to the 5-HT2A, allowing us to assess structure-activity relationships and biased agonism. All test compounds exhibited agonist properties with a relatively large range of both EC50 and Emax values. Interestingly, the lipophilicity of the 2C-X phenethylamines was correlated with their efficacy in both assays but yielded a stronger correlation in the miniGαq- than in the βarr2-assay. Molecular docking suggested that accommodation of the 4-substituent of the 2C-X analogues in a hydrophobic pocket between transmembrane helices 4 and 5 of 5-HT2A may contribute to this differential effect. Aside from previously used standard conditions (lysergic acid diethylamide (LSD) as a reference agonist and a 2 h activation profile to assess a compound's activity), serotonin was included as a second reference agonist, and the compounds' activities were also assessed using the first 30 min of the activation profile. Under all assessed circumstances, the qualitative structure-activity relationships remained unchanged. Furthermore, the use of two reference agonists allowed for the estimation of both "benchmark bias" (relative to LSD) and "physiology bias" (relative to serotonin).

The N-benzylphenethylamines (NBOMes) are a class of ligands from which compounds with impressive selectivity for the serotonin 2A receptor (5-HT2AR) over the closely related serotonin 2C receptor (5-HT2CR) have emerged. These include 4-(2-((2-hydroxybenzyl)amino)ethyl)-2,5-dimethoxybenzonitrile (25CN-NBOH, 1) and 2-(2,5-dimethoxy-4-bromobenzyl)-6-(2-methoxyphenyl)piperidine (DMPMBB, 2). The present work entails the synthesis and characterization of ligands wherein the structures of these two molecules have been fused. The desired compounds were accessed by a six-step synthetic procedure followed by the chiral resolution of the resulting racemic mixtures, giving one active ((S,S)-3) and three essentially inactive stereoisomers. In silico experiments support that one of the four possible stereoisomers would be active. Further in silico investigations showed that 1, 2, and (S,S)-3 share a common binding mode, further supporting the shared stereochemistry between the active enantiomer ((S,S)-3) and 2.

Serotonin is a neurotransmitter regulating numerous physiological processes also modulated by drugs, for example, schizophrenia, depression, migraine, and obesity. However, these drugs typically have adverse effects caused by promiscuous binding across 12 serotonin and more than 20 homologous receptors. Recently, structures of the entire serotonin receptor family uncovered molecular ligand recognition. Here, we present a map of 19 'selectivity hotspots', that is, nonconserved binding site residues governing selectivity via favorable target interactions or repulsive 'off-target' contacts. Furthermore, we review functional rationale from observed ligand-binding affinities and mutagenesis effects. Unifying knowledge underlying specific probes and drugs is critical toward the functional characterization of different receptors and alleviation of adverse effects.

The orphan G protein-coupled receptor GPR139 is predominantly expressed in the central nervous system and has attracted considerable interest as a therapeutic target. However, the biological role of this receptor remains somewhat elusive, in part due to the lack of quality pharmacological tools to investigate GPR139 function. In an effort to understand GPR139 signaling and to identify improved compounds, in this study we performed virtual screening and analog searches, in combination with multiple pharmacological assays. We characterized GPR139-dependent signaling using previously published reference agonists in Ca2+ mobilization and inositol monophosphate accumulation assays, as well as a novel real-time GPR139 internalization assay. For the four reference agonists tested, the rank order of potency was conserved across signaling and internalization assays: JNJ-63533054 > Compound 1a » Takeda > AC4 > DL43, consistent with previously reported values. We noted an increased efficacy of JNJ-63533054-mediated inositol monophosphate signaling and internalization, relative to Compound 1a. We then performed virtual screening for GPR139 agonist and antagonist compounds that were screened and validated in GPR139 functional assays. We identified four GPR139 agonists that were active in all assays, with similar or reduced potency relative to known compounds. Likewise, compound analogs selected based on GPR139 agonist and antagonist substructure searches behaved similarly to their parent compounds. Thus, we have characterized GPR139 signaling for multiple new ligands using G protein-dependent assays and a new real-time internalization assay. These data add to the GPR139 tool compound repertoire, which could be optimized in future medical chemistry campaigns.

Peptides targeting disease-relevant protein-protein interactions are an attractive class of therapeutics covering the otherwise undruggable space between small molecules and therapeutic proteins. However, peptides generally suffer from poor metabolic stability and low membrane permeability. Hence, peptide cyclization has become a valuable approach to develop linear peptide motifs into metabolically stable and potentially cell-permeable cyclic leads. Furthermore, cyclization of side chains, also known as "stapling", can stabilize particular secondary peptide structures. Here, we demonstrate that a comprehensive examination of cyclization strategies in terms of position, chemistry, and length is a prerequisite for the selection of optimal cyclic peptide scaffolds. Our systematic approach identifies cyclic APP dodecamer peptides targeting the phosphotyrosine binding domain of Mint2 with substantially improved affinity. We show that especially all-hydrocarbon stapling provides improved metabolic stability, a significantly stabilized secondary structure and membrane permeability.

Abstract Biased signaling is a paradigm in signal transduction whereby hormones, probes, or drugs bind the same receptor but engage different intracellular signaling pathways leading to distinct functional outcomes. Whereas there is a wealth of knowledge of bias signaling, it is scattered throughout literature and the vast majority of signaling pathways still lack a biased tool compound and functional annotation. Here, we provide an online Biased Signaling Atlas (https://BiasedSignalingAtlas.org) letting any researcher swiftly navigate 9,000 ligand bias datapoints and 640 functional/therapeutic annotations of signaling pathways, analyze ligand-bias relationships, download machine learning-ready data, select tool compounds, and calculate consistent bias values. We invite the global community to together advance biased signaling by depositing its data into the shared repository. This provides a common hub for the global research community to jointly explore the principals of signal transduction and to translate mapped molecular mechanisms to design drugs with better efficacy and safety.

Serotonin (or 5-hydroxytryptamine, 5-HT) is an important neurotransmitter that activates 12 different G protein-coupled receptors (GPCRs) through selective coupling of G(s), G(i), or G(q) proteins. The structural basis for G protein subtype selectivity by these GPCRs remains elusive. Here, we report the structures of the serotonin receptors 5-HT4, 5-HT6, and 5-HT7 with G(s), and 5-HT4 with G(i1). The structures reveal that transmembrane helices TM5 and TM6 alternate lengths as a macro-switch to determine receptor's selectivity for G(s) and G(i), respectively. We find that the macro-switch by the TM5-TM6 length is shared by class A GPCR-G protein structures. Furthermore, we discover specific residues within TM5 and TM6 that function as micro-switches to form specific interactions with G(s) or G(i). Together, these results present a common mechanism of G(s) versus G(i) protein coupling selectivity or promiscuity by class A GPCRs and extend the basis of ligand recognition at serotonin receptors.

The serotonin 2A receptor (5-HT2AR) is the mediator of the psychedelic effects of serotonergic psychedelics, which have shown promising results in clinical studies for several neuropsychiatric indications. The 5-HT2AR is able to signal through the Gαq and β-arrestin effector proteins, but it is currently not known how the different signaling pathways contribute to the therapeutic effects mediated by serotonergic psychedelics. In the present work, we have evaluated the subtype-selective 5-HT2AR agonist 25CN-NBOH and a series of close analogues for biased signaling at this receptor. These ligands were designed to evaluate the role of interactions with Ser1593×36. The lack of interaction between this hydroxyl moiety and Ser1593×36 resulted in detrimental effects on potency and efficacy in both βarr2 and miniGαq recruitment assays. Remarkably, Gαq-mediated signaling was considerably more affected. This led to the development of the first efficacious βarr2-biased 5-HT2AR agonists 4a–b and 6e–f, βarr2 preferring, relative to lysergic acid diethylamide (LSD).

Dear Editor, The G protein-coupled receptor GPR139 is involved in neuro-modulation,and one of its agonists is in clinical trials for the treatment of cognitive impairment and negative symptoms of schizophrenia.While GPR139 is a understudied 'orphan'receptor,it can be activated by the amino acids L-Trp,L-Phe1 or α-Melanocyte-stimulating hormone (α-MSH) which is an endogenous agonist of melanocortin receptors.2,3 GPR139 activation triggers4-7 several G protein pathways of which Gq/11 is the primary.Of note,the expression of GPR139 correlates with that of the μ-opioid and dopamine D2 receptors in a broad range of the central nervous system (CNS),which acts as a regulator of μ-opioid and dopamine signaling.6-9 For example,GPR139 antagonist JNJ-3792165 increases the sensitivity of the μ-opioid receptor to morphine.6 Hitherto,the structural basis of how ligands interact with and activate GPR139 to transduce diverse signals has remained unknown.Furthermore,the more physiologically relevant inter-mediate states of GPCR-G protein complex structures in the nucleotide-bound forms are also elusive.Here,we report the cryo-electron microscopy (cryo-EM) structures of GPR139 in complex with a key reference ligand JNJ-63533054 which is an analog of agent TAK-04110 in clinical trial,and miniGs/q or Gi in nucleotide-free form,as well as the GPR139-JNJ-63533054-miniGs/q complex in GDP-and GTP-bound states,respectively (Fig.1a-f).

There is an urgent need for novel therapeutic approaches to treat Alzheimer's disease (AD) with the ability to both alleviate the clinical symptoms and halt the progression of the disease. AD is characterized by the accumulation of amyloid-β (Aβ) peptides which are generated through the sequential proteolytic cleavage of the amyloid precursor protein (APP). Previous studies reported that Mint2, a neuronal adaptor protein binding both APP and the γ-secretase complex, affects APP processing and formation of pathogenic Aβ. However, there have been contradicting results concerning whether Mint2 has a facilitative or suppressive effect on Aβ generation. Herein, we deciphered the APP-Mint2 protein-protein interaction (PPI) via extensive probing of both backbone H-bond and side-chain interactions. We also developed a proteolytically stable, high-affinity peptide targeting the APP-Mint2 interaction. We found that both an APP binding-deficient Mint2 variant and a cell-permeable PPI inhibitor significantly reduced Aβ42 levels in a neuronal in vitro model of AD. Together, these findings demonstrate a facilitative role of Mint2 in Aβ formation, and the combination of genetic and pharmacological approaches suggests that targeting Mint2 is a promising therapeutic strategy to reduce pathogenic Aβ levels.

Dopamine (DA) and norepinephrine (NE) are pivotal neuromodulators that regulate a broad range of brain functions, often in concert. Despite their physiological importance, untangling the relationship between DA and NE in the fine control of output function is currently challenging, primarily due to a lack of techniques to allow the observation of spatiotemporal dynamics with sufficiently high selectivity. Although genetically encoded fluorescent biosensors have been developed to detect DA, their poor selectivity prevents distinguishing DA from NE. Here, we report the development of a red fluorescent genetically encoded GPCR (G protein-coupled receptor)-activation reporter for DA termed ‘R-GenGAR-DA’. More specifically, a circular permutated red fluorescent protein (cpmApple) was replaced by the third intracellular loop of human DA receptor D1 (DRD1) followed by the screening of mutants within the linkers between DRD1 and cpmApple. We developed two variants: R-GenGAR-DA1.1, which brightened following DA stimulation, and R-GenGAR-DA1.2, which dimmed. R-GenGAR-DA1.2 demonstrated a reasonable dynamic range ( ΔF/F 0 = − 43%), DA affinity (EC 50 = 0.92 µM) and high selectivity for DA over NE (66-fold) in HeLa cells. Taking advantage of the high selectivity of R-GenGAR-DA1.2, we monitored DA in presence of NE using dual-color fluorescence live imaging, combined with the green-NE biosensor GRAB NE1m , which has high selectivity for NE over DA (> 350-fold) in HeLa cells and hippocampal neurons grown from primary culture. Thus, this is a first step toward the multiplex imaging of these neurotransmitters in, for example, freely moving animals, which will provide new opportunities to advance our understanding of the high spatiotemporal dynamics of DA and NE in normal and abnormal brain function.

Delta selective compound 2 (DS2; 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]benzamide) is one of the most widely used tools to study selective actions mediated by δ-subunit–containing GABAA receptors. DS2 was discovered over 10 years ago, but despite great efforts, the precise molecular site of action has remained elusive. Using a combination of computational modeling, site-directed mutagenesis, and cell-based pharmacological assays, we probed three potential binding sites for DS2 and analogs at α4β1δ receptors: an α4(+)δ(−) interface site in the extracellular domain (ECD), equivalent to the diazepam binding site in αβγ2 receptors, and two sites in the transmembrane domain (TMD) - one in the α4(+)β1(−) and one in the α4(−)β1(+) interface, with the α4(−)β1(+) site corresponding to the binding site for etomidate and a recently disclosed low-affinity binding site for diazepam. We show that mutations in the ECD site did not abrogate DS2 modulation. However, mutations in the TMD α4(+)β1(−) interface, either α4(S303L) of the α4(+) side or β1(I289Q) of the β1(−) side, convincingly disrupted the positive allosteric modulation by DS2. This was consistently demonstrated both in an assay measuring membrane potential changes and by whole-cell patch-clamp electrophysiology and rationalized by docking studies. Importantly, general sensitivity to modulators was not compromised in the mutated receptors. This study sheds important light on the long-sought molecular recognition site for DS2, refutes the misconception that the selectivity of DS2 for δ-containing receptors is caused by a direct interaction with the δ-subunit, and instead points toward a functional selectivity of DS2 and its analogs via a surprisingly well conserved binding pocket in the TMD. SIGNIFICANCE STATEMENT δ-Containing GABAA receptors represent potential drug targets for the treatment of several neurological conditions with aberrant tonic inhibition, yet no drugs are currently in clinical use. With the identification of the molecular determinants responsible for positive modulation by the known compound delta selective compound 2, the ground is laid for design of ligands that selectively target δ-containing GABAA receptor subtypes, for better understanding of tonic inhibition, and ultimately, for rational development of novel drugs.

G-protein-coupled receptors (GPCRs) represent the largest family of drug targets. Upon activation, GPCRs signal primarily via a diverse set of heterotrimeric G proteins. Most GPCRs can couple to several different G protein subtypes. However, how drugs act at GPCRs contributing to the selectivity of G protein recognition is poorly understood. Here, we examined the G protein selectivity profile of the dopamine D2 receptor (D2), a GPCR targeted by antipsychotic drugs. We show that D2 discriminates between six individual members of the Gi/o family, and its profile of functional selectivity is remarkably different across its ligands, which all engaged D2 with a distinct G protein coupling pattern. Using structural modeling, receptor mutagenesis, and pharmacological evaluation, we identified residues in the D2 binding pocket that shape these ligand-directed biases. We further provide pharmacogenomic evidence that natural variants in D2 differentially affect its G protein biases in response to different ligands.

Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter 1 , 2 that activates the largest subtype family of G-protein-coupled receptors 3 . Drugs that target 5-HT 1A , 5-HT 1D , 5-HT 1E and other 5-HT receptors are used to treat numerous disorders 4 . 5-HT receptors have high levels of basal activity and are subject to regulation by lipids, but the structural basis for the lipid regulation and basal activation of these receptors and the pan-agonism of 5-HT remains unclear. Here we report five structures of 5-HT receptor–G-protein complexes: 5-HT 1A in the apo state, bound to 5-HT or bound to the antipsychotic drug aripiprazole; 5-HT 1D bound to 5-HT; and 5-HT 1E in complex with a 5-HT 1E - and 5-HT 1F -selective agonist, BRL-54443. Notably, the phospholipid phosphatidylinositol 4-phosphate is present at the G-protein–5-HT 1A interface, and is able to increase 5-HT 1A -mediated G-protein activity. The receptor transmembrane domain is surrounded by cholesterol molecules—particularly in the case of 5-HT 1A , in which cholesterol molecules are directly involved in shaping the ligand-binding pocket that determines the specificity for aripiprazol. Within the ligand-binding pocket of apo-5-HT 1A are structured water molecules that mimic 5-HT to activate the receptor. Together, our results address a long-standing question of how lipids and water molecules regulate G-protein-coupled receptors, reveal how 5-HT acts as a pan-agonist, and identify the determinants of drug recognition in 5-HT receptors.

Delta selective compound 2 (DS2) is one of the most widely used tools to study selective actions mediated by δ subunit-containing GABA A receptors. DS2 was discovered over 10 years ago, but despite great efforts, the precise molecular site of action has remained elusive. Using a combination of computational modeling, site-directed mutagenesis and cell-based pharmacological assays, we probed three potential binding sites for DS2 and analogs at α 4 β 1 δ receptors: an α 4 (+) δ (-) interface site in the extracellular domain (ECD), equivalent to the diazepam binding site in αβγ 2 receptors, and two sites in the transmembrane domain (TMD); one in the α 4 (+) β 1 (-) and one in the α 4 (-) β 1 (+) interface, with the α 4 (-) β 1 (+) site corresponding to the binding site for etomidate and a recently disclosed low-affinity binding site for diazepam. We show that mutations in the ECD site did not abrogate DS2 modulation. However, mutations in the TMD α 4 (+) β 1 (-) interface, either α 4 (S303L) of the α 4 (+) -side or β 1 (I289Q) of the β 1 (-) -side, convincingly disrupted the positive allosteric modulation by DS2. This was consistently demonstrated both in an assay measuring membrane potential changes and by whole-cell patchclamp electrophysiology and rationalized by docking studies. Importantly, general sensitivity to modulators was not compromised in the mutated receptors. This study sheds important light on the long-sought molecular recognition site for DS2, refutes the misconception that the selectivity of DS2 for δ-containing receptors is caused by a direct interaction with the δ-subunit, and instead points towards a functional selectivity of DS2 and its analogs via a surprisingly well-conserved binding pocket in the TMD. Significance statement δ-Containing GABA A receptors represent potential drug targets for the treatment of several neurological conditions with aberrant tonic inhibition. Yet, no drugs are currently in clinical use. With the identification of the molecular determinants responsible for positive modulation by the know compound DS2, the ground is laid for design of ligands that selectively target δ-containing GABA A receptor subtypes, for better understanding of tonic inhibition, and, ultimately, for rational development of novel drugs.

Despite the therapeutic relevance of δ-containing γ-aminobutyric acid type A receptors (GABAARs) and the need for δ-selective compounds, the structural determinants for the mode and molecular site of action of δ-selective positive allosteric modulator imidazo[1,2-a]pyridine DS2 remain elusive. To guide the quest for insight, we synthesized a series of DS2 analogues guided by a structural receptor model. Using a fluorescence-based fluorometric imaging plate reader membrane potential assay, we found that the δ-selectivity and the pharmacological profile are severely affected by substituents in the 5-position of the imidazopyridine core scaffold. Interestingly, the 5-methyl, 5-bromo, and 5-chloro DS2 analogues, 30, 35, and 36, were shown to be superior to DS2 at α4β1δ as mid-high nanomolar potency δ-selective allosteric modulators, displaying 6-16 times higher potency than DS2. Of these, 30 also displayed at least 60-fold selectivity for α4β1δ over α4β1γ2 receptor subtypes representing a potential tool for the selective characterization of δ-containing GABAARs in general.