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.

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.

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.

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.

Intranasal Eutectic Powder of Zolmitriptan with Enhanced Bioavailability in the Rat Brain.

Intranasal administration can potentially deliver drugs to the brain because of the proximity of the delivery site to the olfactory lobe. We prepared triturates of micronized or crystalline zolmitriptan with a GRAS substance, nicotinamide, to form a eutectic. We characterized the formulation using differential scanning calorimetry, powder X-ray diffraction, and FTIR spectroscopy to confirm its eutectic nature and generated a phase diagram. The eutectic formulation was aerosolized using an in-house insufflator into the nares of rats. Groups of rats received zolmitriptan intravenously or intranasally, or intranasal eutectic formulation. Zolmitriptan was estimated in the olfactory lobe, cerebral cortex, cerebellum, and blood plasma at different time-points by LC-MS. Pharmacokinetics in these tissues indicated the superiority of the intranasal eutectic formulation for brain targeting when compared with results of IV solution and intranasal pure zolmitriptan powder. Enhancement of nose-to-brain transport is likely to have resulted from more rapid dissolution of the eutectic as compared to pure drug.

Intestinal epithelial cell-derived µ-opioid signaling protects against ischemia reperfusion injury through PI3K signaling.

Intestinal ischemia has a wide variety of causes, including, but not limited to, atherosclerosis, thrombosis, hypotension, and chronic inflammation. In severe cases, ischemic injury can result in death. µ-Opioid receptor (MOR) signaling has previously been shown to protect against chemically induced colitis, but the cellular origin of this effect remains unknown. Herein, we evaluated the role of intestinal epithelial cell (IEC)-derived MOR signaling in host responses to ischemia/reperfusion-induced injury. Ileal ischemia was accomplished through obstruction of the distal branches of the superior mesenteric artery (60 minutes) and reperfusion for 90 minutes (ischemia-reperfusion). Floxed-MOR mice were crossed to Villin-cre transgenic mice to selectively delete the MOR gene in IECs (MOR(IEC-/-)). Radio-ligand binding assays demonstrated selective loss of MOR signaling in IECs of MOR(IEC-/-) mice. The s.c. administration of the MOR agonist, [D-Arg2, Lys4] dermorphin (1-4) amide (DALDA), 10 minutes before surgery protected against both ischemic and reperfusion phases of intestinal injury, an effect abolished in MOR(IEC-/-) mice. This cytoprotective effect was associated with enterocyte-mediated phosphoinositide 3-kinase (PI3K)/glycogen synthase kinase 3ß signaling and decreased apoptosis, as determined by IHC and caspase-3 processing. PI3K blockade with Ly294002 resulted in loss of MOR-mediated cytoprotective function. Together, these data show that IEC-derived µ-opioid signaling uses the PI3K pathway to protect cells against the damaging effect of ischemia-reperfusion. Targeting MOR signaling may represent a novel mean to alleviate intestinal injury and promote the wound-healing response.

Discovery of ß-arrestin-biased dopamine D2 ligands for probing signal transduction pathways essential for antipsychotic efficacy.

Elucidating the key signal transduction pathways essential for both antipsychotic efficacy and side-effect profiles is essential for developing safer and more effective therapies. Recent work has highlighted noncanonical modes of dopamine D(2) receptor (D(2)R) signaling via ß-arrestins as being important for the therapeutic actions of both antipsychotic and antimanic agents. We thus sought to create unique D(2)R agonists that display signaling bias via ß-arrestin-ergic signaling. Through a robust diversity-oriented modification of the scaffold represented by aripiprazole (1), we discovered UNC9975 (2), UNC0006 (3), and UNC9994 (4) as unprecedented ß-arrestin-biased D(2)R ligands. These compounds also represent unprecedented ß-arrestin-biased ligands for a G(i)-coupled G protein-coupled receptor (GPCR). Significantly, UNC9975, UNC0006, and UNC9994 are simultaneously antagonists of G(i)-regulated cAMP production and partial agonists for D(2)R/ß-arrestin-2 interactions. Importantly, UNC9975 displayed potent antipsychotic-like activity without inducing motoric side effects in inbred C57BL/6 mice in vivo. Genetic deletion of ß-arrestin-2 simultaneously attenuated the antipsychotic actions of UNC9975 and transformed it into a typical antipsychotic drug with a high propensity to induce catalepsy. Similarly, the antipsychotic-like activity displayed by UNC9994, an extremely ß-arrestin-biased D(2)R agonist, in wild-type mice was completely abolished in ß-arrestin-2 knockout mice. Taken together, our results suggest that ß-arrestin signaling and recruitment can be simultaneously a significant contributor to antipsychotic efficacy and protective against motoric side effects. These functionally selective, ß-arrestin-biased D(2)R ligands represent valuable chemical probes for further investigations of D(2)R signaling in health and disease.

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 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.

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.

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.

Antagonist functional selectivity: 5-HT2A serotonin receptor antagonists differentially regulate 5-HT2A receptor protein level in vivo.

Dysregulation of the 5-HT(2A) receptor is implicated in both the etiology and treatment of schizophrenia. Although the essential role of 5-HT(2A) receptors in atypical antipsychotic drug actions is widely accepted, the contribution of 5-HT(2A) down-regulation to their efficacy is not known. We hypothesized that down-regulation of cortical 5-HT(2A) receptors contributes to the therapeutic action of atypical antipsychotic drugs. To test this hypothesis, we assessed the effect of chronically administered antipsychotics (clozapine, olanzapine, and haloperidol) and several 5-HT(2A) antagonists [ketanserin, altanserin, α-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol (M100907), α-phenyl-1-(2-phenylethyl)-4-piperidinemethano (M11939), 4-[(2Z)-3-{[2-(dimethylamino)ethoxy]amino}-3-(2-fluorophenyl)prop-2-en-1-ylidene]cyclohexa-2,5-dien-1-one (SR46349B), and pimavanserin], on the phencyclidine (PCP)-induced hyperlocomotor response and cortical 5-HT(2A) receptor levels in C57BL/6J mice. Clozapine and olanzapine, but not haloperidol, induced receptor down-regulation and attenuated PCP-induced locomotor responses. Of the selective 5-HT(2A) antagonists tested, only ketanserin caused significant receptor protein down-regulation, whereas SR46349B up-regulated 5-HT(2A) receptors and potentiated PCP-hyperlocomotion; the other 5-HT(2A) receptor antagonists were without effect. The significance of these findings with respect to atypical antipsychotic drug action is discussed.

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.

PSD-95 is essential for hallucinogen and atypical antipsychotic drug actions at serotonin receptors.

Here, we report that postsynaptic density protein of 95 kDa (PSD-95), a postsynaptic density scaffolding protein, classically conceptualized as being essential for the regulation of ionotropic glutamatergic signaling at the postsynaptic membrane, plays an unanticipated and essential role in mediating the actions of hallucinogens and atypical antipsychotic drugs at 5-HT(2A) and 5-HT(2C) serotonergic G-protein-coupled receptors. We show that PSD-95 is crucial for normal 5-HT(2A) and 5-HT(2C) expression in vivo and that PSD-95 maintains normal receptor expression by promoting apical dendritic targeting and stabilizing receptor turnover in vivo. Significantly, 5-HT(2A)- and 5-HT(2C)-mediated downstream signaling is impaired in PSD-95(null) mice, and the 5-HT(2A)-mediated head-twitch response is abnormal. Furthermore, the ability of 5-HT(2A) inverse agonists to normalize behavioral changes induced by glutamate receptor antagonists is abolished in the absence of PSD-95 in vivo. These results demonstrate that PSD-95, in addition to the well known role it plays in scaffolding macromolecular glutamatergic signaling complexes, profoundly modulates metabotropic 5-HT(2A) and 5-HT(2C) receptor function.

Parallel functional activity profiling reveals valvulopathogens are potent 5-hydroxytryptamine(2B) receptor agonists: implications for drug safety assessment.

Drug-induced valvular heart disease (VHD) is a serious side effect of a few medications, including some that are on the market. Pharmacological studies of VHD-associated medications (e.g., fenfluramine, pergolide, methysergide, and cabergoline) have revealed that they and/or their metabolites are potent 5-hydroxytryptamine(2B) (5-HT(2B)) receptor agonists. We have shown that activation of 5-HT(2B) receptors on human heart valve interstitial cells in vitro induces a proliferative response reminiscent of the fibrosis that typifies VHD. To identify current or future drugs that might induce VHD, we screened approximately 2200 U.S. Food and Drug Administration (FDA)-approved or investigational medications to identify 5-HT(2B) receptor agonists, using calcium-based high-throughput screening. Of these 2200 compounds, 27 were 5-HT(2B) receptor agonists (hits); 14 of these had previously been identified as 5-HT(2B) receptor agonists, including seven bona fide valvulopathogens. Six of the hits (guanfacine, quinidine, xylometazoline, oxymetazoline, fenoldopam, and ropinirole) are approved medications. Twenty-three of the hits were then "functionally profiled" (i.e., assayed in parallel for 5-HT(2B) receptor agonism using multiple readouts to test for functional selectivity). In these assays, the known valvulopathogens were efficacious at concentrations as low as 30 nM, whereas the other compounds were less so. Hierarchical clustering analysis of the pEC(50) data revealed that ropinirole (which is not associated with valvulopathy) was clearly segregated from known valvulopathogens. Taken together, our data demonstrate that patterns of 5-HT(2B) receptor functional selectivity might be useful for identifying compounds likely to induce valvular heart disease.

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.

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.

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.

Insights into the regulation of 5-HT2A serotonin receptors by scaffolding proteins and kinases.

5-HT(2A) serotonin receptors are essential molecular targets for the actions of LSD-like hallucinogens and atypical antipsychotic drugs. 5-HT(2A) serotonin receptors also mediate a variety of physiological processes in peripheral and central nervous systems including platelet aggregation, smooth muscle contraction, and the modulation of mood and perception. Scaffolding proteins have emerged as important regulators of 5-HT(2A) receptors and our recent studies suggest multiple scaffolds exist for 5-HT(2A) receptors including PSD95, arrestin, and caveolin. In addition, a novel interaction has emerged between p90 ribosomal S6 kinase and 5-HT(2A) receptors which attenuates receptor signaling. This article reviews our recent studies and emphasizes the role of scaffolding proteins and kinases in the regulation of 5-HT(2A) trafficking, targeting and signaling.

Purification and mass spectrometric analysis of the kappa opioid receptor.

A clonal human embryonic kidney (HEK) 293 cell line was established that stably expressed the rat kappa-opioid receptor (rKOR) with a FLAG epitope at the amino terminus. The Kd for [3H]diprenorphine was 1.1+/-0.2 nM, and the Bmax was 2.6+/-0.4 pmol/mg. Dynorphin A (1-13), U69,593 and naloxone competitively inhibited [3H]diprenorphine binding with Ki values of 2.0, 18 and 18 nM, respectively, in good agreement with previously reported affinities for the unmodified receptor. U69,593 stimulated [35S]GTPgammaS binding in a concentration-dependent manner and caused phosphorylation of mitogen-activated protein (MAP) kinase, indicating that the activated epitope-tagged receptor triggered appropriate signaling pathways. Immunoblot analysis demonstrated that two immunoreactive receptor species with apparent molecular masses of 42 and 52 kDa were expressed. Previous studies indicated that the 42 kDa protein was localized intracellularly and was a precursor of the 52 kDa receptor, which was present at the cell surface. rKOR was extracted from transfected HEK 293 cell membranes with n-dodecyl-beta-D-maltopyranoside. Sequential use of wheat germ agglutinin chromatography, Sephacryl S300 gel filtration chromatography, anti-FLAG immunoaffinity chromatography and SDS/PAGE permitted purification of the 52 kDa receptor. MALDI-TOF mass spectrometry was used to identify peptides derived from rKOR following sequential in-gel digestion with trypsin and cyanogen bromide. Eighteen rKOR peptides were detected, corresponding to 27.1% coverage of the receptor. Precursor-selective MS/MS confirmed the identity of most of these peptides. In addition, we have identified heat shock protein 70 (HSP70) as a rKOR-interacting protein.