Discovery and structure - activity relationships of 2,4,5-trimethoxyphenyl pyrimidine derivatives as selective D5 receptor partial agonists.

Dopamine receptors are therapeutic targets for the treatment of various neurological and psychiatric disorders, including Parkinson's and Alzheimer's. Previously, PF-06649751 (tavapadon), PF-2562 and PW0464 have been discovered as potent and selective G protein-biased D1/D5 receptor agonists with optimal pharmacokinetic properties. However, no selective D5R agonist has been reported yet. In this context, we designed and synthesized forty non-catecholamines-based pyrimidine derivatives and identified four pyrimidine derivatives as selective D5R partial agonists. Using cAMP-based GloSensor assay in transiently transfected HEK293T cells with human D1 or D5 receptors, we discovered that compound 5c (4-(4-bromophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine) exhibited modest D5R agonist activity. This leads us to explore various modifications of this scaffold to improve the D5 agonist potency and efficacy. Using molecular docking, and rational design followed by their evaluation at D1 and D5 receptors for agonist activity, we identified three new derivatives, 5j, 5h, and 5e. The most potent compound of this series 5j (4-(4-iodophenyl)-6-(2,4,5-trimethoxyphenyl)pyrimidin-2-amine), exhibited EC50 of 269.7 ± 6.6 nM. Mice microsomal stability studies revealed that 5j is quite stable (>70 % at 1 hr). Furthermore, pharmacokinetic analysis of 5j (20 mg/kg, p.o) in C57BL/6j mice showed that 5j is readily absorbed via oral route of dosing and also enters into the brain (plasma Tmax: 1 h, Cmax: 51.10 ± 13.51 ng/ml; Brain Tmax: 0.5 h, Cmax: 22.54 ± 4.08 ng/ml). We further determined the in-vivo effect of 5j on cognition in scopolamine-induced amnesia in C57BL/6j mice. We observed that 5j (10 mg/kg, p.o) alleviated scopolamine-induced impairment in short-term memory and social recognition, which were blocked by D1/D5 antagonist SCH23390 (0.1 mg/kg, i.p.). Furthermore, 5j did not exhibit any cytotoxicity (up to 10 µM) or in vivo acute toxicity up to 200 mg/kg (p.o). These results strongly suggest that 5j could be further developed for treating neurological disorders wherein the D5 receptors play pivotal roles.

Kappa Opioid Receptor Activation Induces Epigenetic Silencing of Brain-Derived Neurotropic Factor via HDAC5 in Depression.

Treatment-resistant depression (TRD) occurs in almost 50% of the depressed patients. Central kappa opioid receptor (KOR) agonism has been demonstrated to induce depression and anxiety, while KOR antagonism alleviates depression-like symptoms in rodent models and TRD in clinical studies. Previously, we have shown that sustained KOR activation leads to a TRD-like phenotype in mice, and modulation of brain-derived neurotrophic factor (BDNF) expression in the prefrontal cortex (PFC) appears to be one of the molecular determinants of the antidepressant response. In the present study, we observed that sustained KOR activation by a selective agonist, U50488, selectively reduced the levels of Bdnf transcripts II, IV, and Bdnf CDS (protein-coding Exon IX) in the PFC and cultured primary cortical neurons, which was blocked by selective KOR antagonist, norbinaltorphimine. Considering the crucial role of epigenetic pathways in BDNF expression, we further investigated the role of various epigenetic markers in KOR-induced BDNF downregulation in mice. We observed that treatment with U50488 resulted in selective and specific downregulation of acetylation at the ninth lysine residue of the histone H3 protein (H3K9ac) and upregulation of histone deacetylase 5 (HDAC5) expression in the PFC. Further, using anti-H3K9ac and anti-HDAC5 antibodies in the chromatin immune precipitation assay, we detected decreased enrichment of H3K9ac and increased HDAC5 binding at Bdnf II and IV transcripts after U50488 treatment, which were blocked by a selective KOR antagonist, norbinaltorphimine. Further mechanistic studies using HDAC5 selective inhibitor, LMK235, in primary cortical neurons and adeno-associated viral shRNA-mediated HDAC5-knockdown in the PFC of mice demonstrated an essential role of HDAC5 in KOR-mediated reduction of Bdnf expression in the PFC and in depression-like symptoms in mice. These results suggest that KOR engages multiple pathways to induce depression-like symptoms in mice and provide novel insights into the mechanisms by which activation of KOR regulates major depressive disorders.

Molecular mechanism of distinct chemokine engagement and functional divergence of the human Duffy antigen receptor.

The Duffy antigen receptor is a seven-transmembrane (7TM) protein expressed primarily at the surface of red blood cells and displays strikingly promiscuous binding to multiple inflammatory and homeostatic chemokines. It serves as the basis of the Duffy blood group system in humans and also acts as the primary attachment site for malarial parasite Plasmodium vivax and pore-forming toxins secreted by Staphylococcus aureus. Here, we comprehensively profile transducer coupling of this receptor, discover potential non-canonical signaling pathways, and determine the cryoelectron microscopy (cryo-EM) structure in complex with the chemokine CCL7. The structure reveals a distinct binding mode of chemokines, as reflected by relatively superficial binding and a partially formed orthosteric binding pocket. We also observe a dramatic shortening of TM5 and 6 on the intracellular side, which precludes the formation of the docking site for canonical signal transducers, thereby providing a possible explanation for the distinct pharmacological and functional phenotype of this receptor.

Molecular insights into intrinsic transducer-coupling bias in the CXCR4-CXCR7 system.

Chemokine receptors constitute an important subfamily of G protein-coupled receptors (GPCRs), and they are critically involved in a broad range of immune response mechanisms. Ligand promiscuity among these receptors makes them an interesting target to explore multiple aspects of biased agonism. Here, we comprehensively characterize two chemokine receptors namely, CXCR4 and CXCR7, in terms of their transducer-coupling and downstream signaling upon their stimulation by a common chemokine agonist, CXCL12, and a small molecule agonist, VUF11207. We observe that CXCR7 lacks G-protein-coupling while maintaining robust ßarr recruitment with a major contribution of GRK5/6. On the other hand, CXCR4 displays robust G-protein activation as expected but exhibits significantly reduced ßarr-coupling compared to CXCR7. These two receptors induce distinct ßarr conformations even when activated by the same agonist, and CXCR7, unlike CXCR4, fails to activate ERK1/2 MAP kinase. We also identify a key contribution of a single phosphorylation site in CXCR7 for ßarr recruitment and endosomal localization. Our study provides molecular insights into intrinsic-bias encoded in the CXCR4-CXCR7 system with broad implications for drug discovery.

Recent Advancements in Nanocarrier-assisted Brain Delivery of Phytochemicals Against Neurological Diseases.

Despite ongoing advancements in research, the inability of therapeutics to cross the blood-brain barrier (BBB) makes the treatment of neurological disorders (NDs) a challenging task, offering only partial symptomatic relief. Various adverse effects associated with existing approaches are another significant barrier that prompts the usage of structurally diverse phytochemicals as preventive/therapeutic lead against NDs in preclinical and clinical settings. Despite numerous beneficial properties, phytochemicals suffer from poor pharmacokinetic profile which limits their pharmacological activity and necessitates the utility of nanotechnology for efficient drug delivery. Nanocarriers have been shown to be proficient carriers that can enhance drug delivery, bioavailability, biocompatibility, and stability of phytochemicals. We, thus, conducted a meticulous literature survey using several electronic databases to gather relevant studies in order to provide a comprehensive summary about the use of nanocarriers in delivering phytochemicals as a treatment approach for NDs. Additionally, the review highlights the mechanisms of drug transport of nanocarriers across the BBB and explores their potential future applications in this emerging field.

Molecular determinants associated with temporal succession of SARS-CoV-2 variants in Uttar Pradesh, India.

The emergence and rapid evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused a global crisis that required a detailed characterization of the dynamics of mutational pattern of the viral genome for comprehending its epidemiology, pathogenesis and containment. We investigated the molecular evolution of the SASR-CoV-2 genome during the first, second and third waves of COVID-19 in Uttar Pradesh, India. Nanopore sequencing of the SARS-CoV-2 genome was undertaken in 544 confirmed cases of COVID-19, which included vaccinated and unvaccinated individuals. In the first wave (unvaccinated population), the 20A clade (56.32%) was superior that was replaced by 21A Delta in the second wave, which was more often seen in vaccinated individuals in comparison to unvaccinated (75.84% versus 16.17%, respectively). Subsequently, 21A delta got outcompeted by Omicron (71.8%), especially the 21L variant, in the third wave. We noticed that Q677H appeared in 20A Alpha and stayed up to Delta, D614G appeared in 20A Alpha and stayed in Delta and Omicron variants (got fixed), and several other mutations appeared in Delta and stayed in Omicron. A cross-sectional analysis of the vaccinated and unvaccinated individuals during the second wave revealed signature combinations of E156G, F157Del, L452R, T478K, D614G mutations in the Spike protein that might have facilitated vaccination breach in India. Interestingly, some of these mutation combinations were carried forward from Delta to Omicron. In silico protein docking showed that Omicron had a higher binding affinity with the host ACE2 receptor, resulting in enhanced infectivity of Omicron over the Delta variant. This work has identified the combinations of key mutations causing vaccination breach in India and provided insights into the change of [virus's] binding affinity with evolution, resulting in more virulence in Delta and more infectivity in Omicron variants of SARS-CoV-2. Our findings will help in understanding the COVID-19 disease biology and guide further surveillance of the SARS-CoV-2 genome to facilitate the development of vaccines with better efficacies.

Targeting G protein coupled receptors for alleviating neuropathic pain.

Pain sensation is a normal physiological response to alert and prevent further tissue damage. It involves the perception of external stimuli by somatosensory neurons, then transmission of the message to various other types of neurons present in the spinal cord and brain to generate an appropriate response. Currently available analgesics exhibit very modest efficacy, and that too in only a subset of patients with chronic pain conditions, particularly neuropathic pain. The G protein-coupled receptors (GPCRs) are expressed on presynaptic, postsynaptic terminals, and soma of somatosensory neurons, which binds to various types of ligands to modulate neuronal activity and thus pain sensation in both directions. Fundamentally, neuropathic pain arises due to aberrant neuronal plasticity, which includes the sensitization of peripheral primary afferents (dorsal root ganglia and trigeminal ganglia) and the sensitization of central nociceptive neurons in the spinal cord or trigeminal nucleus or brain stem and cortex. Owing to the expression profiles of GPCRs in somatosensory neurons and other neuroanatomical regions involved in pain processing and transmission, this article shall focus only on four families of GPCRs: 1- Opioid receptors, 2-Cannabinoid receptors, 3-Adenosine receptors, and 4-Chemokine receptors.

Sonochemical synthesis and biological evaluation of isoquinolin-1(2H)-one/isoindolin-1-one derivatives: Discovery of a positive ago-allosteric modulator (PAAM) of 5HT2CR.

Efforts have been devoted for the discovery and development of positive allosteric modulators (PAMs) of 5-HT2CR because of their potential advantages over the orthosteric agonist like Lorcaserin that was withdrawn from the market. On the other hand, pursuing a positive ago-allosteric modulator (PAAM) is considered as beneficial particularly when an agonist is not capable of affecting the potency of the endogenous agonist sufficiently. In search of a suitable PAAM of 5-HT2CR we adopted an in silico based approach that indicated the potential of the 3-(1-hydroxycycloalkyl) substituted isoquinolin-1-one derivatives against the 5-HT2CR as majority of these molecules interacted with the site other than that of Lorcaserin with superior docking scores. These compounds along with the regioisomeric 3-methyleneisoindolin-1-one derivatives were prepared via the Cu(OAc)2 catalyzed coupling of 2-iodobenzamide with 1-ethynylcycloalkanol under ultrasound irradiation. According to the in vitro studies, most of these compounds were not only found to be potent and selective agonists but also emerged as PAAM of 5-HT2CR whereas Lorcaserin did not show PAAM activities. According to the SAR study the isoquinolin-1(2H)-ones appeared as better PAAM than isoindolin-1-ones whereas the presence of hydroxyl group appeared to be crucial for the activity. With the potent PAAM activity for 5-HT2CR (EC50 = 1 nM) and 107 and 86-fold selectivity towards 5-HT2C over 5-HT2A and 5-HT2B the compound 4i was identified as a hit molecule. The compound showed good stability in male BALB/c mice brain homogenate (∼85 % remaining after 2 h), moderate stability in the presence of rat liver microsomes (42 % remaining after 1 h) and acceptable PK properties with fast reaching in the brain maintaining âˆ¼ 1:1 brain/plasma concentration ratio. The compound at a dose of 50 mg/kg exhibited decreased trend in the food intake starting from day 3 in S.D. rats, which reached significant by 5th day, and the effect was comparable to Lorcaserin (10 mg/kg) on day 5. Thus, being the first example of PAAM of 5-HT2CR the compound 4i is of further medicinal interest.

Synthesis and Assessment of Fused ß-Carboline Derivatives as Kappa Opioid Receptor Agonists.

The synthesis of 5-formyl-6-aryl-6H-indolo[3,2,1-de][1,5] naphthyridine-2-carboxylates by reaction between 1-formyl-9H-ß-carbolines and cinnamaldehydes in the presence of pyrrolidine in water with microwave irradiation is described. Pharmacophoric modification of the formyl group offered several new fused ß-carboline derivatives, which were investigated for their κ-opioid receptor (KOR) agonistic activity. Two compounds 4 a and 4 c produced appreciable agonist activity on KOR with EC50 values of 46±19 and 134±9 nM, respectively. Moreover, compound-induced KOR signaling studies suggested both compounds to be extremely G-protein-biased agonists. The analgesic effect of 4 a was validated by the increase in tail flick latency in mice in a time-dependent manner, which was completely blocked by the KOR-selective antagonist norBNI. Moreover, unlike U50488, an unbiased full KOR agonist, 4 a did not induce sedation. The docking of 4 a with the human KOR was studied to rationalize the result.

Novel Tetrahydroquinazolinamines as Selective Histamine 3 Receptor Antagonists for the Treatment of Obesity.

The histamine 3 receptor (H3R) is a presynaptic receptor, which modulates several neurotransmitters including histamine and various essential physiological processes, such as feeding, arousal, cognition, and pain. The H3R is considered as a drug target for the treatment of several central nervous system disorders. We have synthesized and identified a novel series of 4-aryl-6-methyl-5,6,7,8-tetrahydroquinazolinamines that act as selective H3R antagonists. Among all the synthesized compounds, in vitro and docking studies suggested that the 4-methoxy-phenyl-substituted tetrahydroquinazolinamine compound 4c has potent and selective H3R antagonist activity (IC50 < 0.04 µM). Compound 4c did not exhibit any activity on the hERG ion channel and pan-assay interference compounds liability. Pharmacokinetic studies showed that 4c crosses the blood brain barrier, and in vivo studies demonstrated that 4c induces anorexia and weight loss in obese, but not in lean mice. These data reveal the therapeutic potential of 4c as an anti-obesity candidate drug via antagonizing the H3R.

Identification of dual role of piperazine-linked phenyl cyclopropyl methanone as positive allosteric modulator of 5-HT2C and negative allosteric modulator of 5-HT2B receptors.

Allosteric modulators of G-protein-coupled receptors have lately gained significant traction in drug discovery. Recent studies have shown that allosteric modulation of serotonin 2C receptor (5-HT2C) as a viable strategy for the treatment of various central nervous system (CNS) disorders. Considering the critical role of 5-HT2C in the modulation of appetite, a selective positive allosteric modulator (PAM) of 5-HT2C offers a new opportunity for anti-obesity therapeutic development. In this study, phenyl cyclopropyl-linked N-heterocycles were synthesized and evaluated at 5-HT2C for agonist and PAM activity. Our study shows that imidazole linked phenyl cyclopropyl methanones has PAM activity on both 5-HT2C and serotonin 2B receptor (5-HT2B). Interestingly, piperazine linked phenyl cyclopropyl methanones (58) was active as PAM of 5-HT2C (increased the Emax of 5-HT to 139%), and as negative allosteric modulator (NAM) of 5-HT2B (decreases EC50 of 5-HT 10 times without affecting Emax). Similar effect of compound 58 was observed with synthetic orthosteric agonist lorcaserin on 5-HT2B. Molecular docking study revealed that all active compounds were binding to the predicted allosteric site on 5-HT2C and shared a common interacting residues. Finally, compound 58 suppressed food intake in Sprague Dawley (SD) rats similar to lorcaserin after i.c.v. administration. Therefore, these results suggest that piperazine moiety is essential for dual activity (PAM & NAM) of compounds 58, and supports the hypothesis of 5-HT2C PAM for the treatment of obesity similar to the full agonist.

Chronic histamine 3 receptor antagonism alleviates depression like conditions in mice via modulation of brain-derived neurotrophic factor and hypothalamus-pituitary adrenal axis.

The last two decades of research has established histamine (HA) as a neurotransmitter. Since H3R antagonists are known to modulate several neurotransmitters besides HA, H3R antagonists have shown potential for the treatment of different central nervous system disorders, including depression. However, molecular mechanisms underlying the beneficial effects of H3R antagonism in depression are not clear, yet. In the present study, we investigated the antidepressant potential of ciproxifan, a selective H3R antagonist, in chronic unpredictable stress (CUS) model of depression in C57BL/6 J mice. We observed that chronic treatment of CUS mice with ciproxifan (3 mg/kg i.p.; for three weeks) alleviates depression-like symptoms such as helplessness measured by forced swim and tail suspension test (FST and TST), anhedonia measured by sucrose preference test (SPT) and social deficit measured in social behavior test. Chronic ciproxifan treatment restored CUS induced BDNF expression in the prefrontal cortex (PFC) and hippocampus. We also observed that ciproxifan modulates CUS induced NUCB2/nesfatin-1 and CRH expression in the hypothalamus and plasma corticosterone. We also determined the direct effect of HA on BDNF expression in neurons by western blotting and immunocytochemistry, and found that HA significantly induced BDNF expression, which was blocked by the H4R selective antagonist, but not by other HA receptor selective antagonists. Furthermore, ciproxifan significantly modulated NMDA glutamate receptor subunits NR2B and NR2A. Thus, these results suggest that increased HA signaling in the brain produces antidepressant-like effects in mice and modulates BDNF expression and HPA-axis.

Docosahexaenoic acid modulates brain-derived neurotrophic factor via GPR40 in the brain and alleviates diabesity-associated learning and memory deficits in mice.

GPR40 (Free fatty acid receptor 1) has emerged as an important therapeutic target for diabetes. Several studies have demonstrated the association of comorbid psychiatric conditions with decreased n-3 polyunsaturated fatty acids, which may act as an agonist for GPR40. In this study, we for the first time provide evidence of reduced GPR40 signaling in the hippocampus and cortex which may be a critical underlying mechanism mediating cognitive deficits in diabesity (diabetes and obesity together). Specifically, we showed decreased GPR40 and brain-derived neurotrophic factor (BDNF) expression in the brain regions of high-fat-diet-induced obese and db/db mice. Next, we demonstrated that chronic treatment with docosahexaenoic acid (DHA) or the synthetic GPR40 agonist, GW9508, significantly alleviates cognitive functions in mice, which correlates with increased BDNF expression in the hippocampus. This supports the hypothesis that DHA improves cognitive function in diabesity via GPR40 agonism. We also showed that DHA specifically activates GPR40 and modulates BDNF expression in primary cortical neurons mediated by the extracellular receptor kinase (ERK) and P38-mitogen-activated protein kinase (MAPK) pathways. Finally, the central nervous system (CNS)-specific blockade of GPR40 signaling abrogated the memory potentiating effects of DHA, and induction of BDNF expression in the hippocampus. Thus, we provided evidence that DHA stimulation of GPR40 mediate some of DHA's beneficial effects in metabolic syndrome and identify GPR40 as a viable therapeutic target for the treatment of CNS-related comorbidities associated with diabesity.

Discovery of coumarin-dihydroquinazolinone analogs as niacin receptor 1 agonist with in-vivo anti-obesity efficacy.

In this study, we presented rational designing and synthesis of coumarin-dihydroquinazolinone conjugates to evaluate their agonist activity at GPR109a receptor. Among the synthesized small molecule library, compound 10c displayed robust agonist action at GPR109a with EC50 < 11 nM. Homology model of human GPR109a protein was generated to realize the binding interaction of the active molecule with the active site of GPR109a. Further, the efficacy of active compound 10c was supported by in-vivo experiments which showed reduced body weight in diet induced obese mice model. Interestingly, compound 10c reduced leptin in blood plasma and total serum cholesterol. These results suggest that the coumarin-dihydroquinazolinone conjugate is a suitable scaffold to further expand the chemical diversity and make them potential niacin receptor 1 agonist.

Identification of novel ß-lactams and pyrrolidinone derivatives as selective Histamine-3 receptor (H3R) modulators as possible anti-obesity agents.

Four series of structurally related ß-lactams, 2,5-pyrrolidinediones, azaspirodecatrienediones (ASDT) and dihydropyrroloquinoxalinetriones (DPQT) were synthesized by utilizing post-Ugi modifications in one-pot, and their activity towards human histamine-3 receptor (H3R) was evaluated. Out of 94 compounds, screened against histamine-3 receptor (H3R), 21 compounds showed high H3R selective agonist property with EC50 values ranging from 187 nM to 0.1 nM, whereas none of the compound was found to have the affinity towards other receptors of histamine family such as histamine H1, H2, and H4 receptor. All active compounds have no assay interference activity as determined by in-silico analysis and receptor independent luciferase assay and cell cytotoxicity assay. Given the important role of H3R in hypophagia, we also evaluated the in vivo effect of the representative compound 6k on the cumulative food intake in diet induce obese C57BL6/J mice. Interestingly, we observed that single dose administration (20 mg/kg, intraperitoneal injection) of 6k significantly suppressed cumulative food intake, while no significant effect was observed at 10 mg/kg. These results suggest that ß-lactams, 2,5-pyrrolidinediones, azaspirodecatrienediones (ASDT) and dihydropyrroloquinoxalinetriones (DPQT) could be useful for the development of anti-obesity candidate drugs.

A synthetic intrabody-based selective and generic inhibitor of GPCR endocytosis.

Beta-arrestins (ßarrs) critically mediate desensitization, endocytosis and signalling of G protein-coupled receptors (GPCRs), and they scaffold a large number of interaction partners. However, allosteric modulation of their scaffolding abilities and direct targeting of their interaction interfaces to modulate GPCR functions selectively have not been fully explored yet. Here we identified a series of synthetic antibody fragments (Fabs) against different conformations of ßarrs from phage display libraries. Several of these Fabs allosterically and selectively modulated the interaction of ßarrs with clathrin and ERK MAP kinase. Interestingly, one of these Fabs selectively disrupted ßarr-clathrin interaction, and when expressed as an intrabody, it robustly inhibited agonist-induced endocytosis of a broad set of GPCRs without affecting ERK MAP kinase activation. Our data therefore demonstrate the feasibility of selectively targeting ßarr interactions using intrabodies and provide a novel framework for fine-tuning GPCR functions with potential therapeutic implications.

GloSensor assay for discovery of GPCR-selective ligands.

G protein-coupled receptors (GPCRs) are modulators of almost every physiological process, and therefore, are most favorite therapeutic target for wide spectrum of diseases. Ideally, high-throughput functional assays should be implemented that allow the screening of large compound libraries in cost-effective manner to identify agonist, antagonist, and allosteric modulators in the same assay. Taking advantage of the increased understanding of the GPCR structure and signaling, several commercially available functional assays based on fluorescence or chemiluminescence detection are being used in both academia and industry. In this chapter, we provide step-by-step method and guidelines to perform cAMP measurement using GloSensor assay. Finally, we have also discussed the analysis and interpretation of results obtained using this assay by providing several examples of Gs- and Gi-coupled GPCRs.

Opioid Receptor Modulators with a Cinnamyl Group.

To obtain selective and potent opioid receptor ligands, we synthesized dehydro derivatives of alvimopan and found compound (28f), a selective but modest affinity MOR antagonist weaker than alvimopan (1). We replaced the arylpiperidine unit by an arylpiperazine to obtain the 1-(α-carboxycinnamyl)-4-arylpiperazines like 13h, which to our surprise had no MOR or DOR activity but was a KOR agonist with moderate affinity. In contrast, literature examples of arylpiperazines 4 and 5 were reported to be pan opioid receptor antagonists, while 6 was a MOR agonist. Two compounds (13l and 11b) showed analgesic response in tail flick test which was blocked by pretreatment with norbinaltorphimine (norBNI). Among 10 1-(α-carboxycinnamyl)-4-arylpiperidines, compound 28g and five others were specific MOR antagonists. Interestingly, compound 26b of this series was found to be more potent than naloxone but weaker than 1. Docking studies have explained differential activities of the above piperazines and piperidines.

Core engagement with ß-arrestin is dispensable for agonist-induced vasopressin receptor endocytosis and ERK activation.

G protein-coupled receptors (GPCRs) exhibit highly conserved activation and signaling mechanisms by which agonist stimulation leads to coupling of heterotrimeric G proteins and generation of second messenger response. This is followed by receptor phosphorylation, primarily in the carboxyl terminus but also in the cytoplasmic loops, and subsequent binding of arrestins. GPCRs typically recruit arrestins through two different sets of interactions, one involving phosphorylated receptor tail and the other mediated by the receptor core. The engagement of both set of interactions (tail and core) is generally believed to be necessary for arrestin-dependent functional outcomes such as receptor desensitization, endocytosis, and G protein-independent signaling. Here we demonstrate that a vasopressin receptor (V2R) mutant with truncated third intracellular loop (V2RΔICL3) can interact with ß-arrestin 1 (ßarr1) only through the phosphorylated tail without engaging the core interaction. Of interest, such a partially engaged V2RΔICL3-ßarr1 complex can efficiently interact with clathrin terminal domain and ERK2 MAPK in vitro. Furthermore, this core interaction-deficient V2R mutant exhibits efficient endocytosis and ERK activation upon agonist stimulation. Our data suggest that core interaction with ßarr is dispensable for V2R endocytosis and ERK activation and therefore provide novel insights into refining the current understanding of functional requirements in biphasic GPCR-ßarr interaction.