How GPCR Phosphorylation Patterns Orchestrate Arrestin-Mediated Signaling.

Binding of arrestin to phosphorylated G-protein-coupled receptors (GPCRs) controls many aspects of cell signaling. The number and arrangement of phosphates may vary substantially for a given GPCR, and different phosphorylation patterns trigger different arrestin-mediated effects. Here, we determine how GPCR phosphorylation influences arrestin behavior by using atomic-level simulations and site-directed spectroscopy to reveal the effects of phosphorylation patterns on arrestin binding and conformation. We find that patterns favoring binding differ from those favoring activation-associated conformational change. Both binding and conformation depend more on arrangement of phosphates than on their total number, with phosphorylation at different positions sometimes exerting opposite effects. Phosphorylation patterns selectively favor a wide variety of arrestin conformations, differently affecting arrestin sites implicated in scaffolding distinct signaling proteins. We also reveal molecular mechanisms of these phenomena. Our work reveals the structural basis for the long-standing "barcode" hypothesis and has important implications for design of functionally selective GPCR-targeted drugs.

GPCR-G Protein-ß-Arrestin Super-Complex Mediates Sustained G Protein Signaling.

Classically, G protein-coupled receptor (GPCR) stimulation promotes G protein signaling at the plasma membrane, followed by rapid ß-arrestin-mediated desensitization and receptor internalization into endosomes. However, it has been demonstrated that some GPCRs activate G proteins from within internalized cellular compartments, resulting in sustained signaling. We have used a variety of biochemical, biophysical, and cell-based methods to demonstrate the existence, functionality, and architecture of internalized receptor complexes composed of a single GPCR, ß-arrestin, and G protein. These super-complexes or "megaplexes" more readily form at receptors that interact strongly with ß-arrestins via a C-terminal tail containing clusters of serine/threonine phosphorylation sites. Single-particle electron microscopy analysis of negative-stained purified megaplexes reveals that a single receptor simultaneously binds through its core region with G protein and through its phosphorylated C-terminal tail with ß-arrestin. The formation of such megaplexes provides a potential physical basis for the newly appreciated sustained G protein signaling from internalized GPCRs.

Restructuring G-protein- coupled receptor activation.

G-protein-coupled receptors serve as key signal transduction conduits, linking extracellular inputs with diverse cellular responses. These receptors eluded structural characterization for decades following their identification. A landmark structure of rhodopsin provided a basis for structure-function studies and homology modeling, but advances in receptor biology suffered from a lack of receptor-specific structural insights. The recent explosion in GPCR structures confirms some features predicted by rhodopsin-based models, and more importantly, it reveals unexpected ligand-binding modes and critical aspects of the receptor activation process. The new structures also promise to foster studies testing emerging models for GPCR function such as receptor dimerization and ligand-biased signaling.

Pharmacological fingerprint of antipsychotic drugs at the serotonin 5-HT2A receptor.

The intricate involvement of the serotonin 5-HT2A receptor (5-HT2AR) both in schizophrenia and in the activity of antipsychotic drugs is widely acknowledged. The currently marketed antipsychotic drugs, although effective in managing the symptoms of schizophrenia to a certain extent, are not without their repertoire of serious side effects. There is a need for better therapeutics to treat schizophrenia for which understanding the mechanism of action of the current antipsychotic drugs is imperative. With bioluminescence resonance energy transfer (BRET) assays, we trace the signaling signature of six antipsychotic drugs belonging to three generations at the 5-HT2AR for the entire spectrum of signaling pathways activated by serotonin (5-HT). The antipsychotic drugs display previously unidentified pathway preference at the level of the individual Gα subunits and ß-arrestins. In particular, risperidone, clozapine, olanzapine and haloperidol showed G protein-selective inverse agonist activity. In addition, G protein-selective partial agonism was found for aripiprazole and cariprazine. Pathway-specific apparent dissociation constants determined from functional analyses revealed distinct coupling-modulating capacities of the tested antipsychotics at the different 5-HT-activated pathways. Computational analyses of the pharmacological and structural fingerprints support a mechanistically based clustering that recapitulate the clinical classification (typical/first generation, atypical/second generation, third generation) of the antipsychotic drugs. The study provides a new framework to functionally classify antipsychotics that should represent a useful tool for the identification of better and safer neuropsychiatric drugs and allows formulating hypotheses on the links between specific signaling cascades and in the clinical outcomes of the existing drugs.

The AF-2 cofactor binding region is key for the selective SUMOylation of estrogen receptor alpha by antiestrogens.

Antiestrogens (AEs) are used to treat all stages of estrogen receptor (ER)-positive breast cancer. Selective estrogen receptor modulators such as tamoxifen have tissue-specific partial agonist activity, while selective estrogen receptor downregulators such as fulvestrant (ICI182,780) display a more complete antiestrogenic profile. We have previously observed that fulvestrant-induced ERα SUMOylation contributes to transcriptional suppression, but whether this effect is seen with other AEs and is specific to ERα is unclear. Here we show that several AEs induce SUMOylation of ERα, but not ERß, at different levels. Swapping domains between ERα and ERß indicates that the ERα identity of the ligand-binding domain helices 3 and 4 (H3-H4 region), which contribute to the static part of the activation function-2 (AF-2) cofactor binding groove, is sufficient to confer fulvestrant-induced SUMOylation to ERß. This region does not contain lysine residues unique to ERα, suggesting that ERα-specific residues in H3-H4 determine the capacity of the AE-bound ERα ligand-binding domain to recruit the SUMOylation machinery. We also show that the SUMO E3 ligase protein inhibitor of activated STAT 1 increases SUMOylation of ERα and of ERß containing the H3-H4 region of ERα, but not of ERß. Together, these results shed new light on the molecular basis for the differential capacity of selective estrogen receptor modulators and selective estrogen receptor downregulators to suppress transcription by ERα.

The ubiquitination status of the glucagon receptor determines signal bias.

The pancreatic hormone glucagon activates the glucagon receptor (GCGR), a class B seven-transmembrane G protein-coupled receptor that couples to the stimulatory heterotrimeric G protein and provokes PKA-dependent signaling cascades vital to hepatic glucose metabolism and islet insulin secretion. Glucagon-stimulation also initiates recruitment of the endocytic adaptors, ßarrestin1 and ßarrestin2, which regulate desensitization and internalization of the GCGR. Unlike many other G protein-coupled receptors, the GCGR expressed at the plasma membrane is constitutively ubiquitinated and upon agonist-activation, internalized GCGRs are deubiquitinated at early endosomes and recycled via Rab4-containing vesicles. Herein we report a novel link between the ubiquitination status and signal transduction mechanism of the GCGR. In the deubiquitinated state, coupling of the GCGR to Gs is diminished, while binding to ßarrestin is enhanced with signaling biased to a ßarrestin1-dependent p38 mitogen activated protein kinase (MAPK) pathway. This ubiquitin-dependent signaling bias arises through the modification of lysine333 (K333) on the cytoplasmic face of transmembrane helix V. Compared with the GCGR-WT, the mutant GCGR-K333R has impaired ubiquitination, diminished G protein coupling, and PKA signaling but unimpaired potentiation of glucose-stimulated-insulin secretion in response to agonist-stimulation, which involves p38 MAPK signaling. Both WT and GCGR-K333R promote the formation of glucagon-induced ßarrestin1-dependent p38 signaling scaffold that requires canonical upstream MAPK-Kinase3, but is independent of Gs, Gi, and ßarrestin2. Thus, ubiquitination/deubiquitination at K333 in the GCGR defines the activation of distinct transducers with the potential to influence various facets of glucagon signaling in health and disease.

Gαs is dispensable for ß-arrestin coupling but dictates GRK selectivity and is predominant for gene expression regulation by ß2-adrenergic receptor.

ß-arrestins play a key role in G protein-coupled receptor (GPCR) internalization, trafficking, and signaling. Whether ß-arrestins act independently of G protein-mediated signaling has not been fully elucidated. Studies using genome-editing approaches revealed that whereas G proteins are essential for mitogen-activated protein kinase activation by GPCRs., ß-arrestins play a more prominent role in signal compartmentalization. However, in the absence of G proteins, GPCRs may not activate ß-arrestins, thereby limiting the ability to distinguish G protein from ß-arrestin-mediated signaling events. We used ß2-adrenergic receptor (ß2AR) and its ß2AR-C tail mutant expressed in human embryonic kidney 293 cells wildtype or CRISPR-Cas9 gene edited for Gαs, ß-arrestin1/2, or GPCR kinases 2/3/5/6 in combination with arrestin conformational sensors to elucidate the interplay between Gαs and ß-arrestins in controlling gene expression. We found that Gαs is not required for ß2AR and ß-arrestin conformational changes, ß-arrestin recruitment, and receptor internalization, but that Gαs dictates the GPCR kinase isoforms involved in ß-arrestin recruitment. By RNA-Seq analysis, we found that protein kinase A and mitogen-activated protein kinase gene signatures were activated by stimulation of ß2AR in wildtype and ß-arrestin1/2-KO cells but absent in Gαs-KO cells. These results were validated by re-expressing Gαs in the corresponding KO cells and silencing ß-arrestins in wildtype cells. These findings were extended to cellular systems expressing endogenous levels of ß2AR. Overall, our results support that Gs is essential for ß2AR-promoted protein kinase A and mitogen-activated protein kinase gene expression signatures, whereas ß-arrestins initiate signaling events modulating Gαs-driven nuclear transcriptional activity.

Feedback control of the Gpr161-Gαs-PKA axis contributes to basal Hedgehog repression in zebrafish.

Hedgehog (Hh) ligands act as morphogens to direct patterning and proliferation during embryonic development. Protein kinase A (PKA) is a central negative regulator of Hh signalling, and in the absence of Hh ligands, PKA activity prevents inappropriate expression of Hh target genes. The orphan G-protein-coupled receptor Gpr161 contributes to the basal Hh repression machinery by activating PKA. Gpr161 acts as an A-kinase-anchoring protein, and is itself phosphorylated by PKA, but the functional significance of PKA phosphorylation of Gpr161 in the context of Hh signalling remains unknown. Here, we show that loss of Gpr161 in zebrafish leads to constitutive activation of medium and low, but not maximal, levels of Hh target gene expression. Furthermore, we find that PKA phosphorylation-deficient forms of Gpr161, which we show directly couple to Gαs, display an increased sensitivity to Shh, resulting in excess high-level Hh signalling. Our results suggest that PKA feedback-mediated phosphorylation of Gpr161 may provide a mechanism for fine-tuning Gpr161 ciliary localisation and PKA activity.

BRET-based effector membrane translocation assay monitors GPCR-promoted and endocytosis-mediated Gq activation at early endosomes.

G protein-coupled receptors (GPCRs) are gatekeepers of cellular homeostasis and the targets of a large proportion of drugs. In addition to their signaling activity at the plasma membrane, it has been proposed that their actions may result from translocation and activation of G proteins at endomembranes-namely endosomes. This could have a significant impact on our understanding of how signals from GPCR-targeting drugs are propagated within the cell. However, little is known about the mechanisms that drive G protein movement and activation in subcellular compartments. Using bioluminescence resonance energy transfer (BRET)-based effector membrane translocation assays, we dissected the mechanisms underlying endosomal Gq trafficking and activity following activation of Gq-coupled receptors, including the angiotensin II type 1, bradykinin B2, oxytocin, thromboxane A2 alpha isoform, and muscarinic acetylcholine M3 receptors. Our data reveal that GPCR-promoted activation of Gq at the plasma membrane induces its translocation to endosomes independently of ß-arrestin engagement and receptor endocytosis. In contrast, Gq activity at endosomes was found to rely on both receptor endocytosis-dependent and -independent mechanisms. In addition to shedding light on the molecular processes controlling subcellular Gq signaling, our study provides a set of tools that will be generally applicable to the study of G protein translocation and activation at endosomes and other subcellular organelles, as well as the contribution of signal propagation to drug action.

Development of conformational BRET biosensors that monitor ezrin, radixin and moesin activation in real time.

Ezrin, radixin and moesin compose the family of ERM proteins. They link actin filaments and microtubules to the plasma membrane to control signaling and cell morphogenesis. Importantly, their activity promotes invasive properties of metastatic cells from different cancer origins. Therefore, a precise understanding of how these proteins are regulated is important for the understanding of the mechanism controlling cell shape, as well as providing new opportunities for the development of innovative cancer therapies. Here, we developed and characterized novel bioluminescence resonance energy transfer (BRET)-based conformational biosensors, compatible with high-throughput screening, that monitor individual ezrin, radixin or moesin activation in living cells. We showed that these biosensors faithfully monitor ERM activation and can be used to quantify the impact of small molecules, mutation of regulatory amino acids or depletion of upstream regulators on their activity. The use of these biosensors allowed us to characterize the activation process of ERMs that involves a pool of closed-inactive ERMs stably associated with the plasma membrane. Upon stimulation, we discovered that this pool serves as a cortical reserve that is rapidly activated before the recruitment of cytoplasmic ERMs.

Agonist-induced formation of unproductive receptor-G12 complexes.

G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V2 receptors (V2R) associate with both Gs and G12 heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V2R-Gs complexes, V2R-G12 complexes are not destabilized by guanine nucleotides and do not promote G12 activation. Activating V2R does not lead to signaling responses downstream of G12 activation, but instead inhibits basal G12-mediated signaling, presumably by sequestering G12 heterotrimers. Overexpressing G12 inhibits G protein receptor kinase (GRK) and arrestin recruitment to V2R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G12 that are insensitive to nucleotides, suggesting that unproductive GPCR-G12 complexes are not unique to V2R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.

Emerging Paradigm of Intracellular Targeting of G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) recognize a diverse array of extracellular stimuli, and they mediate a broad repertoire of signaling events involved in human physiology. Although the major effort on targeting GPCRs has typically been focused on their extracellular surface, a series of recent developments now unfold the possibility of targeting them from the intracellular side as well. Allosteric modulators binding to the cytoplasmic surface of GPCRs have now been described, and their structural mechanisms are elucidated by high-resolution crystal structures. Furthermore, pepducins, aptamers, and intrabodies targeting the intracellular face of GPCRs have also been successfully utilized to modulate receptor signaling. Moreover, small molecule compounds, aptamers, and synthetic intrabodies targeting ß-arrestins have also been discovered to modulate GPCR endocytosis and signaling. Here, we discuss the emerging paradigm of intracellular targeting of GPCRs, and outline the current challenges, potential opportunities, and future outlook in this particular area of GPCR biology.

Michaelis-Menten Quantification of Ligand Signaling Bias Applied to the Promiscuous Vasopressin V2 Receptor.

Activation of G protein-coupled receptors by agonists may result in the activation of one or more G proteins and recruitment of arrestins. The extent of the activation of each of these pathways depends on the intrinsic efficacy of the ligand. Quantification of intrinsic efficacy relative to a reference compound is essential for the development of novel compounds. In the operational model, changes in efficacy can be compensated by changes in the "functional" affinity, resulting in poorly defined values. To separate the effects of ligand affinity from the intrinsic activity of the receptor, we developed a Michaelis-Menten based quantification of G protein activation bias that uses experimentally measured ligand affinities and provides a single measure of ligand efficacy. We used it to evaluate the signaling of a promiscuous model receptor, the Vasopressin V2 receptor (V2R). Using BRET-based biosensors, we show that the V2R engages many different G proteins across all G protein subfamilies in response to its primary endogenous agonist, arginine vasopressin, including Gs and members of the Gi/o and G12/13 families. These signaling pathways are also activated by the synthetic peptide desmopressin, oxytocin, and the nonmammalian hormone vasotocin. We compared bias quantification using the operational model with Michaelis-Menten based quantification; the latter accurately quantified ligand efficacies despite large difference in ligand affinities. Together, these results showed that the V2R is promiscuous in its ability to engage several G proteins and that its' signaling profile is biased by small structural changes in the ligand. SIGNIFICANCE STATEMENT: By modelling the G protein activation as Michaelis-Menten reaction, we developed a novel way of quantifying signalling bias. V2R activates, or at least engages, G proteins from all G protein subfamilies, including Gi2, Gz, Gq, G12, and G13. Their relative activation may explain its Gs-independent signalling.

Diverse activation pathways in class A GPCRs converge near the G-protein-coupling region.

Class A G-protein-coupled receptors (GPCRs) are a large family of membrane proteins that mediate a wide variety of physiological functions, including vision, neurotransmission and immune responses. They are the targets of nearly one-third of all prescribed medicinal drugs such as beta blockers and antipsychotics. GPCR activation is facilitated by extracellular ligands and leads to the recruitment of intracellular G proteins. Structural rearrangements of residue contacts in the transmembrane domain serve as 'activation pathways' that connect the ligand-binding pocket to the G-protein-coupling region within the receptor. In order to investigate the similarities in activation pathways across class A GPCRs, we analysed 27 GPCRs from diverse subgroups for which structures of active, inactive or both states were available. Here we show that, despite the diversity in activation pathways between receptors, the pathways converge near the G-protein-coupling region. This convergence is mediated by a highly conserved structural rearrangement of residue contacts between transmembrane helices 3, 6 and 7 that releases G-protein-contacting residues. The convergence of activation pathways may explain how the activation steps initiated by diverse ligands enable GPCRs to bind a common repertoire of G proteins.

New oral protease-activated receptor 4 antagonist BMS-986120: tolerability, pharmacokinetics, pharmacodynamics, and gene variant effects in humans.

BMS-986120 is a novel first-in-class oral protease-activated receptor 4 (PAR4) antagonist exhibiting robust antithrombotic activity that has shown low bleeding risk in monkeys. We sought to assess pharmacokinetics, pharmacodynamics, and tolerability of BMS-986120 in healthy participants and platelet responses to BMS-986120 in participants carrying PAR4 A120T variants. Phase I, randomized, double-blind, placebo-controlled single-ascending-dose (SAD; N = 56) and multiple-ascending-dose (MAD; N = 32) studies were conducted. Exposure was approximately dose-proportional: maximum concentrations 27.3 and 1536 ng/mL, areas under the curve (AUC) to infinity of 164 and 15,603 h*ng/mL, and half-lives of 44.7 and 84.1 hours for 3.0 and 180 mg, respectively. The accumulation index suggested an ~2-fold AUC increase at steady state. Single doses of 75 and 180 mg BMS-986120 produced ≥80% inhibition of 12.5 µM PAR4 agonist peptide (AP)-induced platelet aggregation through at least 24 hours postdose, and doses ≥10 mg for ~7 days inhibited aggregation completely through 24 hours. No differences in PAR4-mediated platelet response were seen between AA120 versus TT120 PAR4 variants. In cells expressing A120 or T120 PAR4 proteins, no differences in half-maximal effective concentration in receptor activation by PAR4-AP were observed. BMS-986120 was well tolerated with dose-proportional pharmacokinetics and concentration-dependent pharmacodynamics in healthy participants over a wide dose range.ClinicalTrials.gov ID: NCT02208882.

Apelin protects against abdominal aortic aneurysm and the therapeutic role of neutral endopeptidase resistant apelin analogs.

Abdominal aortic aneurysm (AAA) remains the second most frequent vascular disease with high mortality but has no approved medical therapy. We investigated the direct role of apelin (APLN) in AAA and identified a unique approach to enhance APLN action as a therapeutic intervention for this disease. Loss of APLN potentiated angiotensin II (Ang II)-induced AAA formation, aortic rupture, and reduced survival. Formation of AAA was driven by increased smooth muscle cell (SMC) apoptosis and oxidative stress in Apln-/y aorta and in APLN-deficient cultured murine and human aortic SMCs. Ang II-induced myogenic response and hypertension were greater in Apln-/y mice, however, an equivalent hypertension induced by phenylephrine, an α-adrenergic agonist, did not cause AAA or rupture in Apln-/y mice. We further identified Ang converting enzyme 2 (ACE2), the major negative regulator of the renin-Ang system (RAS), as an important target of APLN action in the vasculature. Using a combination of genetic, pharmacological, and modeling approaches, we identified neutral endopeptidase (NEP) that is up-regulated in human AAA tissue as a major enzyme that metabolizes and inactivates APLN-17 peptide. We designed and synthesized a potent APLN-17 analog, APLN-NMeLeu9-A2, that is resistant to NEP cleavage. This stable APLN analog ameliorated Ang II-mediated adverse aortic remodeling and AAA formation in an established model of AAA, high-fat diet (HFD) in Ldlr-/- mice. Our findings define a critical role of APLN in AAA formation through induction of ACE2 and protection of vascular SMCs, whereas stable APLN analogs provide an effective therapy for vascular diseases.

Genetically encoded intrabody sensors report the interaction and trafficking of ß-arrestin 1 upon activation of G-protein-coupled receptors.

Agonist stimulation of G-protein-coupled receptors (GPCRs) typically leads to phosphorylation of GPCRs and binding to multifunctional proteins called ß-arrestins (ßarrs). The GPCR-ßarr interaction critically contributes to GPCR desensitization, endocytosis, and downstream signaling, and GPCR-ßarr complex formation can be used as a generic readout of GPCR and ßarr activation. Although several methods are currently available to monitor GPCR-ßarr interactions, additional sensors to visualize them may expand the toolbox and complement existing methods. We have previously described antibody fragments (FABs) that recognize activated ßarr1 upon its interaction with the vasopressin V2 receptor C-terminal phosphopeptide (V2Rpp). Here, we demonstrate that these FABs efficiently report the formation of a GPCR-ßarr1 complex for a broad set of chimeric GPCRs harboring the V2R C terminus. We adapted these FABs to an intrabody format by converting them to single-chain variable fragments and used them to monitor the localization and trafficking of ßarr1 in live cells. We observed that upon agonist simulation of cells expressing chimeric GPCRs, these intrabodies first translocate to the cell surface, followed by trafficking into intracellular vesicles. The translocation pattern of intrabodies mirrored that of ßarr1, and the intrabodies co-localized with ßarr1 at the cell surface and in intracellular vesicles. Interestingly, we discovered that intrabody sensors can also report ßarr1 recruitment and trafficking for several unmodified GPCRs. Our characterization of intrabody sensors for ßarr1 recruitment and trafficking expands currently available approaches to visualize GPCR-ßarr1 binding, which may help decipher additional aspects of GPCR signaling and regulation.

Chemogenetics defines receptor-mediated functions of short chain free fatty acids.

Differentiating actions of short chain fatty acids (SCFAs) at free fatty acid receptor 2 (FFA2) from other free fatty acid-responsive receptors and from non-receptor-mediated effects has been challenging. Using a novel chemogenetic and knock-in strategy, whereby an engineered variant of FFA2 (FFA2-DREADD) that is unresponsive to natural SCFAs but is instead activated by sorbic acid replaced the wild-type receptor, we determined that activation of FFA2 in differentiated adipocytes and colonic crypt enteroendocrine cells of mouse accounts fully for SCFA-regulated lipolysis and release of the incretin glucagon-like peptide-1 (GLP-1), respectively. In vivo studies confirmed the specific role of FFA2 in GLP-1 release and also demonstrated a direct role for FFA2 in accelerating gut transit. Thereby, we establish the general principle that such a chemogenetic knock-in strategy can successfully define novel G-protein-coupled receptor (GPCR) biology and provide both target validation and establish therapeutic potential of a 'hard to target' GPCR.

Publisher Correction: Structural insights into binding specificity, efficacy and bias of a ß2AR partial agonist.

In the version of this paper originally published, the structure for epinephrine shown in Figure 1a was redrawn with an extra carbon. The structure has been replaced in the HTML and PDF versions of the article. The original and corrected versions of the structure are shown below.

Novel potent (dihydro)benzofuranyl piperazines as human histamine receptor ligands - Functional characterization and modeling studies on H3 and H4 receptors.

Histamine acts through four different receptors (H1R-H4R), the H3R and H4R being the most explored in the last years as drug targets. The H3R is a potential target to treat narcolepsy, Parkinson's disease, epilepsy, schizophrenia and several other CNS-related conditions, while H4R blockade leads to anti-inflammatory and immunomodulatory effects. Our group has been exploring the dihydrobenzofuranyl-piperazines (LINS01 series) as human H3R/H4R ligands as potential drug candidates. In the present study, a set of 12 compounds were synthesized from adequate (dihydro)benzofuran synthons through simple reactions with corresponding piperazines, giving moderate to high yields. Four compounds (1b, 1f, 1g and 1h) showed high hH3R affinity (pKi > 7), compound 1h being the most potent (pKi 8.4), and compound 1f showed the best efficiency (pKi 8.2, LE 0.53, LLE 5.85). BRET-based assays monitoring Gαi activity indicated that the compounds are potent antagonists. Only one compound (2c, pKi 7.1) presented high affinity for hH4R. In contrast to what was observed for hH3R, it showed partial agonist activity. Docking experiments indicated that bulky substituents occupy a hydrophobic pocket in hH3R, while the N-allyl group forms favorable interactions with hydrophobic residues in the TM2, 3 and 7, increasing the selectivity towards hH3R. Additionally, the importance of the indole NH in the interaction with Glu5.46 from hH4R was confirmed by the modeling results, explaining the affinity and agonistic activity of compound 2c. The data reported in this work represent important findings for the rational design of future compounds for hH3R and hH4R.