High-affinity Agonist Research Articles

One class classification for the detection of β2 adrenergic receptor agonists using single-ligand dynamic interaction data

G protein-coupled receptors are involved in many biological processes, relaying the extracellular signal inside the cell. Signaling is regulated by the interactions between receptors and their ligands, it can be stimulated by agonists, or inhibited by antagonists or inverse agonists. The development of a new drug targeting a member of this family requires to take into account the pharmacological profile of the designed ligands in order to elicit the desired response. The structure-based virtual screening of chemical libraries may prioritize a specific class of ligands by combining docking results and ligand binding information provided by crystallographic structures. The performance of the method depends on the relevance of the structural data, in particular the conformation of the targeted site, the binding mode of the reference ligand, and the approach used to compare the interactions formed by the docked ligand with those formed by the reference ligand in the crystallographic structure. Here, we propose a new method based on the conformational dynamics of a single protein–ligand reference complex to improve the biased selection of ligands with specific pharmacological properties in a structure-based virtual screening exercise. Interactions patterns between a reference agonist and the receptor, here exemplified on the β2 adrenergic receptor, were extracted from molecular dynamics simulations of the agonist/receptor complex and encoded in graphs used to train a one-class machine learning classifier. Different conditions were tested: low to high affinity agonists, varying simulation duration, considering or ignoring hydrophobic contacts, and tuning of the classifier parametrization. The best models applied to post-process raw data from retrospective virtual screening obtained by docking of test libraries effectively filtered out irrelevant poses, discarding inactive and non-agonist ligands while identifying agonists. Taken together, our results suggest that consistency of the binding mode during the simulation is a key to the success of the method.

Transitioning From High-dose Methadone to Buprenorphine Using a Microdosing Approach: Unique Considerations at ASAM Level 3 Facilities.

Transitions from high-dose methadone to buprenorphine for treatment of opioid use disorder (OUD) present risk of precipitated withdrawal related to the introduction of a high-affinity partial agonist at the mu-opioid receptor after occupancy by a lower-affinity full agonist. Various strategies have been explored to maintain patient stability during this process, including microdosing buprenorphine. Current literature lacks consensus on an optimal setting and strategy for initiating a buprenorphine microdosing protocol and gives little detail on patients' conditions after the acute transition period. We report a 6-day microdosing transition from methadone 100 mg directly to sublingual buprenorphine, followed by a 20-day period of monitoring and additional treatment. This patient tolerated a sublingual buprenorphine microdosing protocol while using supportive medications with a peak Clinical Opiate Withdrawal Scale score of 6. The patient's most significant withdrawal symptoms occurred several days after completion of the microdosing process. This case demonstrates the feasibility of using a transmucosal buprenorphine formulation in microdosing transitions from high-dose methadone directly to buprenorphine, and highlights the utility of a medically monitored intensive inpatient setting (American Society of Addiction Medicine level 3.7) in providing appropriate monitoring and treatment during and after a microdosing transition.

Mu-opioid receptor system modulates responses to vocal bonding and distress signals in humans.

Laughter is a contagious prosocial signal that conveys bonding motivation; adult crying conversely communicates desire for social proximity by signalling distress. Endogenous mu-opioid receptors (MORs) modulate sociability in humans and non-human primates. In this combined PET-fMRI study (n = 17), we tested whether central MOR tone is associated with regional brain responses to social signals of laughter and crying. MOR availability was measured with positron emission tomography (PET) using the high-affinity agonist radioligand [11C]carfentanil. Haemodynamic responses to social laughter and crying vocalizations were measured using functional magnetic resonance imaging (fMRI). Social laughter evoked activation in the auditory cortex, insula, cingulate cortex, amygdala, primary and secondary somatosensory cortex, and primary and secondary motor cortex; crying sounds led to more restricted activation in the auditory cortex and nearby areas. MOR availability was negatively correlated with the haemodynamic responses to social laughter in the primary and secondary somatosensory cortex, primary and secondary motor cortex, posterior insula, posterior cingulate cortex, precuneus, cuneus, temporal gyri and lingual gyrus. For crying-evoked activations, MOR availability was negatively correlated with medial and lateral prefrontal haemodynamic responses. Altogether our findings highlight the role of the MOR system in modulating acute brain responses to both positive and negative social signals. This article is part of the theme issue 'Cracking the laugh code: laughter through the lens of biology, psychology and neuroscience'.

Cannabidiol Improves Random-Pattern Skin Flap Survival in Rats: Involvement of Cannabinoid Type-2 Receptors.

One of the major complications associated with random-pattern skin flaps is distal necrosis. Cannabidiol (CBD) has recently gained much attention as a therapeutic anti-inflammatory agent. We aimed to evaluate the efficacy of CBD on the random-pattern skin flap survival (SFS) in rats and to explore the possible involvement of cannabinoid type-2 (CB2) receptors. In this controlled experimental study, we randomly divided male Wistar rats into seven study groups (six rats each). We performed a random-pattern skin flap model in each rat following pretreatment with vehicle (control) or multiple doses of CBD (0.3, 1, 5, or 10 mg/kg). In a separate group, we injected SR144528 (2 mg/kg), a high affinity and selective CB2 inverse agonist, before the most effective dose of CBD (1 mg/kg). A sham nontreated and nonoperated group was also included. Seven days after surgeries, the percentage of necrotic area (PNA) was calculated. Histopathological microscopy, CB2 expression level, and interleukin (IL)-1β and tumor necrosis factor (TNF)-α concentrations were also investigated in the flap tissue samples. A PNA of 72.7 ± 7.5 (95% confidence interval [CI]: 64.8-80.6) was captured in the control group. Following treatment with CBD 0.3, 1, 5, and 10 mg/kg, a dose-dependent effect was observed with PNAs of 51.0 ± 10.0 (95% CI: 40.5-61.5; p <0.05), 15.4 ± 5.8 (95% CI: 9.3-21.5; p <0.001), 37.1 ± 10.2 (95% CI: 26.3-47.8; p <0.001), and 46.4 ± 14.0 (95% CI: 31.7-61.1; p <0.001), respectively. Histopathologically, tissues enhanced significantly. Besides, CB2 expression surged remarkably, IL-1β and TNF-α concentrations decreased considerably after treatment with CBD of 1 mg/kg compared with the control (p <0.05 and <0.001, respectively). Administering SR144528 reversed the favorable effects of CBD of 1 mg/kg, both macroscopically and microscopically. Pretreatment with CBD of 1 mg/kg improved SFS considerably in rats and exerted desirable anti-inflammatory effects which were possibly mediated by CB2 receptors.

Oyster (Crassostrea gigas) Polysaccharide Ameliorates High-Fat-Diet-Induced Oxidative Stress and Inflammation in the Liver via the Bile Acid-FXR-AMPKα Pathway.

Oyster polysaccharides (OPS) have a variety of biological activities. In this study, we aimed to investigate the potential mechanisms of OPS to ameliorate hepatic oxidative stress and inflammation in mice induced by a high-fat diet (HFD). The results showed that OPS reduced the HFD-induced increases in serum transaminase levels and alleviated hepatic oxidative stress and inflammation. Moreover, OPS regulated bile acid metabolism and increased bile acid content in the liver, serum, and feces. Serum bile acid profile results indicated that OPS reduced levels of chenodeoxycholic acid, deoxycholic acid, and lithocholic acid associated with high-affinity agonists of Farnesol X receptor (FXR). Western blot analysis showed that OPS accelerated bile acid metabolism by downregulating hepatic FXR expression and promoting its downstream CYP7A1, CYP27A1, and CYP8B1 protein expression. Meanwhile, OPS ameliorated oxidative stress and inflammation in the liver by modulating FXR-AMPKα-Nrf2/NF-κB signaling to reduce p-IκBα/IκBα, p-NF-κB p65/NF-κB p65, IL-1β, and TNF-α expression and increase p-Nrf2/Nrf2, HO-1, and NQO-1 expression. This study was the first to explore the possible mechanism of OPS in improving liver oxidative stress and inflammation from the perspective of bile acid metabolism, providing a theoretical basis for OPS as a new source of functional food.

Structural insights into the function-modulating effects of nanobody binding to the integrin receptor αMβ2

The integrin receptor αMβ2 mediates phagocytosis of complement-opsonized objects, adhesion to the extracellular matrix, and transendothelial migration of leukocytes. However, the mechanistic aspects of αMβ2 signaling upon ligand binding are unclear. Here, we present the first atomic structure of the human αMβ2 headpiece fragment in complex with the nanobody (Nb) hCD11bNb1 at a resolution of 3.2 Å. We show that the receptor headpiece adopts the closed conformation expected to exhibit low ligand affinity. The crystal structure indicates that in the R77H αM variant, associated with systemic lupus erythematosus, the modified allosteric relationship between ligand binding and integrin outside–inside signaling is due to subtle conformational effects transmitted over a distance of 40 Å. Furthermore, we found the Nb binds to the αI domain of the αM subunit in an Mg2+-independent manner with low nanomolar affinity. Biochemical and biophysical experiments with purified proteins demonstrated that the Nb acts as a competitive inhibitor through steric hindrance exerted on the thioester domain of complement component iC3b attempting to bind the αM subunit. Surprisingly, we show that the Nb stimulates the interaction of cell-bound αMβ2 with iC3b, suggesting that it may represent a novel high-affinity proteinaceous αMβ2-specific agonist. Taken together, our data suggest that the iC3b–αMβ2 complex may be more dynamic than predicted from the crystal structure of the core complex. We propose a model based on the conformational spectrum of the receptor to reconcile these observations regarding the functional consequences of hCD11bNb1 binding to αMβ2.

Diet-Induced Obesity Disrupts Histamine-Dependent Oleoylethanolamide Signaling in the Mouse Liver

Introduction: Previous work suggests the existence of a paracrine signaling mechanism in which histamine released from visceral mast cells into the portal circulation contributes to fasting-induced ketogenesis by stimulating biosynthesis of the endogenous high-affinity PPAR-α agonist oleoylethanolamide (OEA). Methods: Male C57Bl/6J mice were rendered obese by exposure to a high-fat diet (HFD; 60% fat). We measured histamine, OEA, and other fatty-acid ethanolamides by liquid-chromatography/mass spectrometry, gene transcription by RT-PCR, protein expression by ELISA, neutral lipid accumulation in the liver using Red Oil O and BODIPY staining, and collagen levels using picrosirius red staining. Results: Long-term exposure to HFD suppressed both fasting-induced histamine release into portal blood and histamine-dependent OEA production in the liver. Additionally, subchronic OEA administration reduced lipid accumulation, inflammatory responses, and fibrosis in the liver of HFD-exposed mice. Discussion: The results suggest that disruption of histamine-dependent OEA signaling in the liver might contribute to pathology in obesity-associated liver steatosis.

Structure-Functional Selectivity Relationship Studies on A-86929 Analogs and Small Aryl Fragments toward the Discovery of Biased Dopamine D1 Receptor Agonists.

Dopamine regulates normal functions such as movement, reinforcement learning, and cognition, and its dysfunction has been implicated in multiple psychiatric and neurological disorders. Dopamine acts through D1- (D1R and D5R) and D2-class (D2R, D3R, and D4R) receptors and activates both G protein- and β-arrestin-dependent signaling pathways. Current dopamine receptor-based therapies are used to ameliorate motor deficits in Parkinson's disease or as antipsychotic medications for schizophrenia. These drugs show efficacy for ameliorating only some symptoms caused by dopamine dysfunction and are plagued by debilitating side effects. Studies in primates and rodents have shown that shifting the balance of dopamine receptor signaling toward the arrestin pathway can be beneficial for inducing normal movement, while reducing motor side effects such as dyskinesias, and can be efficacious at enhancing cognitive function compared to balanced agonists. Several structure-activity relationship (SAR) studies have embarked on discovering β-arrestin-biased dopamine agonists, focused on D2 partial agonists, noncatechol D1 agonists, and mixed D1/D2R dopamine receptor agonists. Here, we describe an SAR study to identify novel D1R β-arrestin-biased ligands using A-86929, a high-affinity D1R catechol agonist, as a core scaffold to identify chemical motifs responsible for β-arrestin-biased activity at both D1 and D2Rs. Most of the A-86929 analogs screened were G protein-biased, but none of them were exclusively arrestin-biased. Additionally, various small-fragment molecular probes displayed weak bias toward the β-arrestin pathway. Continued in-depth SFSR (structure-functional selectivity relationship) studies informed by structure determination, molecular modeling, and mutagenesis studies will facilitate the discovery of potent and efficacious arrestin-biased dopamine receptor ligands.

Varenicline-induced sucidal behavior: Case report and literature review

IntroductionNowdays there are different strategies for the treatment of smoking cessation. The treatment include drugs such as varenicline, which acts as a high-affinity partial agonist for the alpha-4 beta-2 nicotinic acetylcholine receptor subtype (nACh). We report a case of a suicidal behaviour in a 39 year-old woman with no previous history of mental illness, who was brought to the emergency department after intentional intoxication with benzodiazepines. The patient was on 10th day of treatment with varenicline.ObjectivesTo present a case of sucidal behavior that developed in a 39 year-old woman after starting varenicline. Review of literature and total number of cases reported in the european database of suspected adverse drug reactions (EudraVigilance).MethodsWe carried out a literature review in Pubmed electing those articles focused on mental disorders in those patients that have been taking varenicline. Review number of cases suicidal behavior reported by the European database of suspected adverse drug reactions.ResultsA 39-year-old female was brought to the emergency department after voluntary ingestion of Lorazepam 1mg (40 tablets) in a sucide attempt. The family reported the starting of thoughts of suicide after 1 week of treatment. No previous history of mental disorders. The patient reported low mood and drowsiness in the last 5 days not linked to any cause. After 5 days of discontinuation these mood symptoms and sucidal behavior remited.ConclusionsVarenicline is associated with different neuropsychiatric sypmtoms. In patients with or without history of mental disorders we should warn about the symptoms for discontinuation of the treatment.DisclosureNo significant relationships.

Nociceptor Neurons are Involved in the Host Response to Escherichia coli Urinary Tract Infections.

PurposeUrinary tract infections (UTIs) can evoke a rapid host immune response leading to bladder inflammation and epithelial damage. Neuroimmune interactions are critical for regulating immune function in mucosal tissues. Yet the role of nociceptor neurons in bladder host defense has not been well defined. This study aimed to explore the interaction between nociceptor neurons and bladder immune system during UTIs.MethodsIn this study, whether uropathogenic Escherichia coli (UPEC) and lipopolysaccharide (LPS) can directly stimulate nociceptor neurons was detected. Female C57BL/6J mice were treated with high dose of capsaicin, a high-affinity TRPV1 agonist, to ablate nociceptor neurons. Bladder inflammation, barrier epithelial function and bladder immune cell infiltration were assessed after UPEC infection. The level of neuropeptide calcitonin gene-related peptide (CGRP) in infected bladder was detected. Furthermore, the effects of CGRP on neutrophils and macrophages were evaluated both in vitro and in vivo.ResultsWe found that UPEC and its pathogenic factor LPS could directly excite nociceptor neurons, releasing CGRP into infected bladder, which suppressed the recruitment of neutrophils, the polarization of macrophages and the killing function of UPEC. Both Botulinum neurotoxin A (BoNT/A) and BIBN4096 (CGRP antagonism) blocked neuronal inhibition and prevented against UPEC infection.ConclusionThe present study showed a novel mechanism by which UPEC stimulated the secretion of CGRP from nociceptor neurons to suppress innate immunity.

The Aryl Hydrocarbon Receptor Modulates T Follicular Helper Cell Responses to Influenza Virus Infection in Mice.

T follicular helper (Tfh) cells support Ab responses and are a critical component of adaptive immune responses to respiratory viral infections. Tfh cells are regulated by a network of signaling pathways that are controlled, in part, by transcription factors. The aryl hydrocarbon receptor (AHR) is an environment-sensing transcription factor that modulates many aspects of adaptive immunity by binding a range of small molecules. However, the contribution of AHR signaling to Tfh cell differentiation and function is not known. In this article, we report that AHR activation by three different agonists reduced the frequency of Tfh cells during primary infection of C57BL/6 mice with influenza A virus (IAV). Further, using the high-affinity and AHR-specific agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin, we show that AHR activation reduced Tfh cell differentiation and T cell-dependent B cell responses. Using conditional AHR knockout mice, we demonstrated that alterations of Tfh cells and T cell-dependent B cell responses after AHR activation required the AHR in T cells. AHR activation reduced the number of T follicular regulatory (Tfr) cells; however, the ratio of Tfr to Tfh cells was amplified. These alterations to Tfh and Tfr cells during IAV infection corresponded with differences in expression of BCL6 and FOXP3 in CD4+ T cells and required the AHR to have a functional DNA-binding domain. Overall, these findings support that the AHR modulates Tfh cells during viral infection, which has broad-reaching consequences for understanding how environmental factors contribute to variation in immune defenses against infectious pathogens, such as influenza and severe acute respiratory syndrome coronavirus.

Cannabidiol Enhances Microglial Beta-Amyloid Peptide Phagocytosis and Clearance via Vanilloid Family Type 2 Channel Activation.

Alzheimer’s disease (AD) is associated with the accumulation and aggregation of amyloid in the brain. The cation channel TRPV2 may mediate the pathological changes in mild cognitive impairment. A high-affinity agonist of TRPV2 named cannabidiol is one of the candidate drugs for AD. However, the molecular mechanism of cannabidiol via TRPV2 in AD remains unknown. The present study investigated whether cannabidiol enhances the phagocytosis and clearance of microglial Aβ via the TRPV2 channel. We used a human dataset, mouse primary neuron and microglia cultures, and AD model mice to evaluate TRPV2 expression and the ability of microglial amyloid-β phagocytosis in vivo and in vitro. The results revealed that TRPV2 expression was reduced in the cortex and hippocampus of AD model mice and AD patients. Cannabidiol enhanced microglial amyloid-β phagocytosis through TRPV2 activation, which increased the mRNA expression of the phagocytosis-related receptors, but knockdown of TRPV2 or Trem2 rescued the expression. TRPV2-mediated effects were also dependent on PDK1/Akt signaling, a pathway in which autophagy was indispensable. Furthermore, cannabidiol treatment successfully attenuated neuroinflammation while simultaneously improving mitochondrial function and ATP production via TRPV2 activation. Therefore, TRPV2 is proposed as a potential therapeutic target in AD, while CBD is a promising drug candidate for AD.

Functional Rescue of a Nephrogenic Diabetes Insipidus Causing Mutation in the V2 Vasopressin Receptor by Specific Antagonist and Agonist Pharmacochaperones.

The urine concentrating function of the kidney is essential to maintain the water homeostasis of the human body. It is mainly regulated by the arginine-vasopressin (AVP), which targets the type 2 vasopressin receptor (V2R) in the kidney. The inability of V2R to respond to AVP stimulation leads to decreased urine concentration and congenital nephrogenic diabetes insipidus (NDI). NDI is characterized by polyuria, polydipsia, and hyposthenuria. In this study, we identified a point mutation (S127F) in the AVPR2 gene of an NDI patient, and we characterized the impaired function of the V2R mutant in HEK293 cells. Based on our data, the S127F-V2R mutant is almost exclusively located intracellularly in the endoplasmic reticulum (ER), and very few receptors were detected at the cell surface, where the receptor can bind to AVP. The overexpressed S127F-V2R mutant receptor has negligible cAMP generation capability compared to the wild-type receptor in response to AVP stimulation. Since certain misfolded mutant proteins, that are retained in the ER, can be rescued by pharmacological chaperones, we examined the potential rescue effects of two pharmacochaperones on the S127F-V2R. We found that pretreatment with both tolvaptan (an established V2R inverse agonist) and MCF14 compound (a cell-permeable high-affinity agonist for the V2R) were capable of partially restoring the cAMP generating function of the receptor in response to vasopressin stimulation. According to our data, both cell permeant agonists and antagonists can function as pharmacochaperones, and serve as the starting compounds to develop medicines for patients carrying the S127F mutation.

Structure, Function, and Regulation of the Kainate Receptor.

Neural communication and modulation are complex processes. Ionotropic glutamate receptors (iGluRs) significantly contribute to mediating the fast-excitatory branch of neurotransmission in the mammalian brain. Kainate receptors (KARs), a subfamily of the iGluRs, act as modulators of the neuronal circuitry by playing important roles at both the post- and presynaptic sites of specific neurons. The functional tetrameric receptors are formed by two different gene families, low agonist affinity (GluK1-GluK3) and high agonist affinity (GluK4-GluK5) subunits. These receptors garnered attention in the past three decades, and since then, much work has been done to understand their localization, interactome, physiological functions, and regulation. Cloning of the receptor subunits (GluK1-GluK5) in the early 1990s led to recombinant expression of kainate receptors in heterologous systems. This facilitated understanding of the functional differences between subunit combinations, splice variants, trafficking, and drug discovery. Structural studies of individual domains and recent full-length homomeric and heteromeric kainate receptors have revealed unique functional mechanisms, which have answered several long-standing questions in the field of kainate receptor biology. In this chapter, we review the current understanding of kainate receptors and associated disorders.

Anti-Inflammatory Activity of a CB2 Selective Cannabinoid Receptor Agonist: Signaling and Cytokines Release in Blood Mononuclear Cells.

The endocannabinoid system (ECS) exerts immunosuppressive effects, which are mostly mediated by cannabinoid receptor 2 (CBR2), whose expression on leukocytes is higher than CBR1, mainly localized in the brain. Targeted CBR2 activation could limit inflammation, avoiding CBR1-related psychoactive effects. Herein, we evaluated in vitro the biological activity of a novel, selective and high-affinity CBR2 agonist, called JT11, studying its potential CBR2-mediated anti-inflammatory effect. Trypan Blue and MTT assays were used to test the cytotoxic and anti-proliferative effect of JT11 in Jurkat cells. Its pro-apoptotic activity was investigated analyzing both cell cycle and poly PARP cleavage. Finally, we evaluated its impact on LPS-induced ERK1/2 and NF-kB-p65 activation, TNF-α, IL-1β, IL-6 and IL-8 release in peripheral blood mononuclear cells (PBMCs) from healthy donors. Selective CB2R antagonist SR144528 and CBR2 knockdown were used to further verify the selectivity of JT11. We confirmed selective CBR2 activation by JT11. JT11 regulated cell viability and proliferation through a CBR2-dependent mechanism in Jurkat cells, exhibiting a mild pro-apoptotic activity. Finally, it reduced LPS-induced ERK1/2 and NF-kB-p65 phosphorylation and pro-inflammatory cytokines release in human PBMCs, proving to possess in vitro anti-inflammatory properties. JT11 as CBR2 ligands could enhance ECS immunoregulatory activity and our results support the view that therapeutic strategies targeting CBR2 signaling could be promising for the treatment of chronic inflammatory diseases.

Novel Somatostatin Receptor-4 Agonist SM-I-26 Mitigates Lipopolysaccharide-Induced Inflammatory Gene Expression in Microglia.

Somatostatin receptor subtype 4 (SSTR4) is expressed in BV2 microglia, suggesting that SSTR4 agonists may impact microglia function. This study assessed the high-affinity SSTR4 agonist SM-I-26 (SMI) (0nM, 10nM, 1000nM) against lipopolysaccharide (LPS)-induced inflammation (0, 10 or 100ng/ml) over 6 or 24h in BV2 microglia. Cell viability, nitrite output and mRNA expression changes of genes associated with our target (Sstr4), inflammation (Tnf-α, Il-6, Il-1β, inos), anti-inflammatory and anti-oxidant actions (Il-10, Catalase), and mediators of Aβ binding/phagocytosis (Msr1, Cd33, Trem1, Trem2) were measured. At 6h SMI showed no effect across all conditions. At 24h SMI (10 and 1000nM) upregulated Sstr4 expression under inflammatory and non-inflammatory conditions. At 24h SMI downregulated expression of the inflammatory cytokines Tnf-α (1000nM within all LPS concentrations) and Il-6 (10nM within 0 and 10ng/ml LPS). At 24h 10nM SMI upregulated Il-10, while 1000nM upregulated Catalase under inflammatory and non-inflammatory conditions. At 24h Msr1 and Cd33 were upregulated by 1000nM SMI under non-inflammatory conditions, while Trem1 was downregulated by 10 and 1000nM SMI under mildly inflammatory and non-inflammatory conditions. These results show that SMI had concentration and time-dependent effects on mRNA expression of genes associated with different states of microglial activation. The SMI reduced Tnf-α and Il-6 inflammatory gene expression, and increased Il-10 anti-inflammatory gene expression, identifies anti-inflammatory actions of SSTR4 agonists extend to microglia.

Quantal Ca2+ release mediated by very few IP3 receptors that rapidly inactivate allows graded responses to IP3.

SummaryInositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels that link extracellular stimuli to Ca2+ signals. Ca2+ release from intracellular stores is “quantal”: low IP3 concentrations rapidly release a fraction of the stores. Ca2+ release then slows or terminates without compromising responses to further IP3 additions. The mechanisms are unresolved. Here, we synthesize a high-affinity partial agonist of IP3Rs and use it to demonstrate that quantal responses do not require heterogenous Ca2+ stores. IP3Rs respond incrementally to IP3 and close after the initial response to low IP3 concentrations. Comparing functional responses with IP3 binding shows that only a tiny fraction of a cell’s IP3Rs mediate incremental Ca2+ release; inactivation does not therefore affect most IP3Rs. We conclude, and test by simulations, that Ca2+ signals evoked by IP3 pulses arise from rapid activation and then inactivation of very few IP3Rs. This allows IP3Rs to behave as increment detectors mediating graded Ca2+ release.

Design and discovery of a high affinity, selective and β-arrestin biased 5-HT7 Receptor Agonist.

Compound 1c, 5-chloro-2-(2-(3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-2,3-dihydro-1H-inden-1-one was previously reported from our laboratory showing high affinity binding to the 5-HT7 receptor (Ki = 0.5 nM). However, compound 1c racemizes readily upon enantiomeric separation. To prevent racemization, we have redesigned and synthesized methyl and carboxyethyl analogs, compounds 2 and 3 respectively, whose binding affinities were similar to those of compound 1c. Compounds 2 and 3 cannot undergo racemization since tautomerism was no longer possible and thus, compound 2 was selected for enantiomeric separation and further evaluation. Upon enantiomeric separation, the levorotatory enantiomer, (-)2 or 2a demonstrated a higher affinity (Ki = 1.2 nM) than the (+)2 or 2b enantiomer (Ki = 93 nM) and a β-arrestin biased functional selectivity for the 5-HT7 receptor. Although 2a showed about 8 times less activity than 5-HT in the Gs pathway, it showed over 31 times higher activity than 5-HT in the β-arrestin pathway. This constitutes a significant β-arrestin pathway preference and shows 2a to be more potent and more efficacious than the recently published β-arrestin biased 3-(4-chlorophenyl)-1,4,5,6,7,8-hexahydropyrazolo[3,4-d]azepine, the N-debenzylated analog of JNJ18038683 (Compound 7).

7,8-Dihydroxyflavone improves neuropathological changes in the brain of Tg26 mice, a model for HIV-associated neurocognitive disorder

The combined antiretroviral therapy era has significantly increased the lifespan of people with HIV (PWH), turning a fatal disease to a chronic one. However, this lower but persistent level of HIV infection increases the susceptibility of HIV-associated neurocognitive disorder (HAND). Therefore, research is currently seeking improved treatment for this complication of HIV. In PWH, low levels of brain derived neurotrophic factor (BDNF) has been associated with worse neurocognitive impairment. Hence, BDNF administration has been gaining relevance as a possible adjunct therapy for HAND. However, systemic administration of BDNF is impractical because of poor pharmacological profile. Therefore, we investigated the neuroprotective effects of BDNF-mimicking 7,8 dihydroxyflavone (DHF), a bioactive high-affinity TrkB agonist, in the memory-involved hippocampus and brain cortex of Tg26 mice, a murine model for HAND. In these brain regions, we observed astrogliosis, increased expression of chemokine HIV-1 coreceptors CXCR4 and CCR5, neuroinflammation, and mitochondrial damage. Hippocampi and cortices of DHF treated mice exhibited a reversal of these pathological changes, suggesting the therapeutic potential of DHF in HAND. Moreover, our data indicates that DHF increases the phosphorylation of TrkB, providing new insights about the role of the TrkB–Akt–NFkB signaling pathway in mediating these pathological hallmarks. These findings guide future research as DHF shows promise as a TrkB agonist treatment for HAND patients in adjunction to the current antiviral therapies.

Novel ι-Carrageenan Tetrasaccharide Alleviates Liver Lipid Accumulation via the Bile Acid-FXR-SHP/PXR Pathway to Regulate Cholesterol Conversion and Fatty Acid Metabolism in Insulin-Resistant Mice.

ι-Carrageenan tetrasaccharide (ιCTs), a novel oligosaccharide, was hydrolyzed from ι-carrageenan with targeting marine tool-enzyme Cgi82A. Previously, we have found ιCTs exhibited a hypoglycemic effect, whether it could regulate lipid metabolism remains unknown. In this study, the insulin-resistant mice induced by high-fat-high-sucrose diet were orally administrated with ιCTs (30 mg/kg·bw) for 20 weeks. The results showed that the contents of triglyceride and cholesterol in both serum and liver were reduced by ιCTs, and their excretion in feces were promoted, suggesting lipid accumulation was inhibited. Intriguingly, the overall levels of bile acid in serum, liver, and feces were all raised by ιCTs. Given that bile acids are the essential signal factors for regulating lipid metabolism via the farnesoid-X-receptor (FXR), we conducted serum bile acid profile analysis and found that the levels of high-affinity agonists deoxycholic acid and lithocholic acid were decreased in the ιCTs group, showing that ιCTs failed to activate FXR. Western blot analysis showed that ιCTs downregulated hepatic FXR and small heterodimer partner (SHP) expression and increased downstream CYP7A1 expression via regulating the FXR-SHP signal to accelerate liver cholesterol conversion. Meanwhile, ιCTs decreased the expression of PXR and SREBP1c and elevated the expression of PPARα and CPT1α via regulating the FXR-PXR-SREBP1c/PPARα signal to inhibit fatty acid synthesis and promote fatty acid β-oxidation. To the best of our knowledge, this study for the first time reported that ιCTs alleviated liver lipid accumulation via the bile acid-FXR-SHP/PXR signal to regulate cholesterol conversion and fatty acid metabolism, which highlighted a new idea for ameliorating insulin resistance.