Weak Partial Agonist Research Articles

Shining light on the histamine H2 receptor: Synthesis of carbamoylguanidine-type agonists as a pharmacological tool to study internalization

So far, only little is known about the internalization process of the histamine H2 receptor (H2R). One promising approach to study such dynamic processes is the use of agonistic fluorescent ligands. Therefore, a series of carbamoylguanidine-type H2R agonists containing various fluorophores, heterocycles, and linkers (28–40) was synthesized. The ligands were pharmacologically characterized in several binding and functional assays. These studies revealed a significantly biased efficacy (Emax) for some of the compounds, e.g. 32: whereas 32 acted as strong partial (Emax: 0.77, mini-Gs recruitment) or full agonist (Emax: 1.04, [35S]GTPγS binding) with respect to G protein activation, it was only a weak partial agonist regarding β-arrestin1/2 recruitment (Emax: 0.09–0.12) and failed to promote H2R internalization (confocal microscopy). On the other hand, H2R internalization was observed for compounds that exhibited moderate agonistic activity in the β-arrestin1/2 pathways (Emax ≥ 0.22). The presented differently-biased fluorescent ligands are versatile molecular tools for future H2R studies on receptor trafficking and internalization e.g. using fluorescence microscopy.

N-linoleyltyrosine exerts neuroprotective effects in APP/PS1 transgenic mice via cannabinoid receptor-mediated autophagy

Anandamide (AEA) analogs show fair effects in counteracting the deterioration of Alzheimer's disease (AD). Our previous studies demonstrated that AEA analog-N-linoleyltyrosine (NITyr) exerted significant activities. In our current research, the role and mechanisms of NITyr were assessed in APP/PS1 mice mimicking the AD model. NITyr improved motor coordination in the rotarod test (RRT) and ameliorated spatial memory in the Morris water maze (MWM) but did not increase spontaneous locomotor activity in the open field test (OFT). In addition, NITyr protected neurons against β-amyloid (Aβ) injury via hematoxylin-eosin (HE) and Nissl staining. Moreover, the related biochemical indexes showed that NITyr reduced the levels of Aβ40 and Aβ42 in the hippocampus but did not affect the expression of p-APP and β-secretase 1 (BACE1). Furthermore, the autophagy inhibitor 3-methyladenine (3 MA) attenuated the effect of NITyr on animal behaviors and neurons. Meanwhile, NITyr upregulated the expression levels of LC3-II and Beclin-1, which were weakened by AM630 (an antagonist of CB2 receptor and a weak partial agonist of CB1 receptors). AM630 also weakened the role of NITyr in animal behaviors. Thus, NITyr improved behavioral impairment and neural loss by inducing autophagy mainly mediated by the CB2 receptor, and weakly mediated by the CB1 receptor.

Development of a Protocol for Virtual Screening of PPARγ Weak Partial Agonists and Their Metabolites: Case Study on Naturally-derived Oleanane Triterpenoids

Triterpenoids are well known metabolic syndrome (MetS) modulators. One of the suggested molecular mechanisms of action involves peroxisome proliferator-activated receptor gamma (PPARγ) activation. In this study we aimed to: (i) develop a virtual screening (VS) protocol for PPARγ weak partial agonists, (ii) predict potential metabolic transformations of naturally-derived triterpenoids, and (iii) perform VS of the triterpenoids and their metabolites. The NIH PubMed system was searched for publications about naturally-derived oleanane triterpenoids which are agonists or up-regulators of PPARγ. Structure- and ligand-based methods were combined in the development of the VS protocol. Metabolites were predicted using Meteor Nexus expert system (Lhasa Limited). Two in-house virtual libraries of PPARγ weak partial agonists and naturally-derived triterpenoids with their predicted metabolites were compiled. The pharmacophore-based docking protocol was applied for VS of the collected triterpenoids. Most of the docking poses reproduced the binding mode of caulophyllogenin (a weak partial agonist) in a complex with PPARγ (PDB ID 5F9B). Our results contribute to the mechanistic explanation of the effects of triterpenoids suggesting possible weak partial agonistic activity toward PPARγ. This research can direct further studies on triterpenoids’ role in MetS modulation. The developed protocol can be applied for VS of any PPARγ weak partial agonists.

A Functional Comparison of Homopentameric Nicotinic Acetylcholine Receptors (ACR-16) Receptors From Necator americanus and Ancylostoma ceylanicum.

Effective control of hookworm infections in humans and animals relies on using a small group of anthelmintics. Many of these drugs target cholinergic ligand-gated ion channels, yet the direct activity of anthelmintics has only been studied in a subset of these receptors, primarily in the non-parasitic nematode, Caenorhabditis elegans. Here we report the characterization of a homopentameric ionotropic acetylcholine receptor (AChR), ACR-16, from Necator americanus and Ancylostoma ceylanicum, the first known characterization of human hookworm ion channels. We used two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes to determine the pharmacodynamics of cholinergics and anthelmintics on ACR-16 from both species of hookworm. The A. ceylanicum receptor (Ace-ACR-16) was more sensitive to acetylcholine (EC50 = 20.64 ± 0.32 μM) and nicotine (EC50 = 24.37 ± 2.89 μM) than the N. americanus receptor (Nam-ACR-16) (acetylcholine EC50 = 170.1 ± 19.23 μM; nicotine EC50 = 597.9 ± 59.12 μM), at which nicotine was a weak partial agonist (% maximal acetylcholine response = 30.4 ± 7.4%). Both receptors were inhibited by 500 μM levamisole (Ace-ACR-16 = 65.1 ± 14.3% inhibition, Nam-ACR-16 = 79.5 ± 7.7% inhibition), and responded to pyrantel, but only Ace-ACR-16 responded to oxantel. We used in silico homology modeling to investigate potential structural differences that account for the differences in agonist binding and identified a loop E isoleucine 130 of Nam-ACR-16 as possibly playing a role in oxantel insensitivity. These data indicate that key functional differences exist among ACR-16 receptors from closely related species and suggest mechanisms for differential drug sensitivity.

Synthesis, Radiosynthesis and Biological Evaluation of Buprenorphine-Derived Phenylazocarboxamides as Novel μ-Opioid Receptor Ligands.

Targeted structural modifications have led to a novel type of buprenorphine‐derived opioid receptor ligand displaying an improved selectivity profile for the μ‐OR subtype. On this basis, it is shown that phenylazocarboxamides may serve as useful bioisosteric replacements for the widely occurring cinnamide units, without loss of OR binding affinity or subtype selectivity. This study further includes functional experiments pointing to weak partial agonist properties of the novel μ‐OR ligands, as well as docking and metabolism experiments. Finally, the unique bifunctional character of phenylazocarboxylates, herein serving as precursors for the azocarboxamide subunit, was exploited to demonstrate the accessibility of an 18F‐fluorinated analogue.

Discovery of δ opioid receptor full agonists lacking a basic nitrogen atom and their antidepressant-like effects

We have recently reported that the elaboration of the N-substituent in the δ opioid receptor (DOR) antagonist naltrindole (NTI) enabled the regulation of the DOR activities from full inverse agonists to weak partial agonists. The investigations of amide-type NTI derivatives revealed that N-phenylacetyl and N-dihydrocinnamoyl derivatives 3a and 3b were DOR full agonists. The same transformations were applied to a DOR agonist KNT-127 to provide the more potent DOR agonists 6a and 6b. Among the tested compounds, the most efficacious compound 6a showed dose-dependent antidepressant-like effects in the mouse forced swim test. The antidepressant-like effects by 6a seemed to be more potent than those of KNT-127, which is a more potent DOR agonist in in vitro assays. The amide-type compound like 6a may more fully penetrate into the central nervous system.

In vitro and in vivo pharmacological evaluation of the synthetic cannabinoid receptor agonist EG-018

Synthetic cannabinoid receptor agonists (SCRAs) possess high abuse liability and complex toxicological profiles, making them serious threats to public health. EG-018 is a SCRA that has been detected in both illicit products and human samples, but it has received little attention to date. The current studies investigated EG-018 at human CB1 and CB2 receptors expressed in HEK293 cells in [3H]CP55,940 competition binding, [35S]GTPγS binding and forskolin-stimulated cAMP production. EG-018 was also tested in vivo for its ability to produce cannabimimetic and abuse-related effects in the cannabinoid tetrad and THC drug discrimination, respectively. EG-018 exhibited high affinity at CB1 (21 nM) and at CB2 (7 nM), but in contrast to typical SCRAs, behaved as a weak partial agonist in [35S]GTPγS binding, exhibiting lower efficacy but greater potency, than that of THC at CB1 and similar potency and efficacy at CB2. EG-018 inhibited forskolin-stimulated cAMP with similar efficacy but lower potency, compared to THC, which was likely due to high receptor density facilitating saturation of this signaling pathway. In mice, EG-018 (100 mg/kg, 30 min) administered intraperitoneally (i.p.) did not produce effects in the tetrad or drug discrimination nor did it shift THC's ED50 value in drug discrimination when administered before THC, suggesting EG-018 has negligible occupancy of brain CB1 receptors following i.p. administration. Following intravenous (i.v.) administration, EG-018 (56 mg/kg) produced hypomotility, catalepsy, and hypothermia, but only catalepsy was blocked by the selective CB1 antagonist rimonabant (3 mg/kg, i.v.). Additional studies of EG-018 and its structural analogues could provide further insight into how cannabinoids exert efficacy through the cannabinoid receptors.

Buprenorphine Analogs to Treat Relapse

Substance use disorders (SUDs) – the overuse and dependence on at least one substance – have a lifetime prevalence of ~10% in the U.S. While buprenorphine (BUP) treatment reduces relapse rates, it is only marginally effective, as 70% of SUD patients relapse within a year. Due to BUP’s marginal effectiveness and abuse potential, there is a need to improve SUD therapies that prevent relapse to multiple drugs of abuse after exposure to stress, drug‐associated context and drug priming. Here we evaluate BUP and two analogs for their ability to inhibit cocaine‐ and stress‐induced relapse using a conditioned place preference (CPP) paradigm. Pharmacologically, BUP is a potent (sub nM) partial agonist at the m‐opioid receptor (MOR) with potent antagonist activity at the k‐(KOR), and d‐opioid receptors (DOR). In vitro BUP can act as a weak (mM) partial agonist at the nociceptin/OrphaninFQ (NOP) receptor with uncertain in vivo consequences. We measured in vitro potency and efficacy using [35S]GTPgS functional assays in CHO cells separately expressing DOR, KOR, MOR or NOP. Since NOP receptor agonists reduce relapse in animal models, we compared BU10119 with NOP agonist activity (EC50 = 120 nM ± 20; EMAX = 44% ± 5%) or BU12004 with NOP antagonist activity (KB = 190 nM ±30) to BUP (EC50 = 1300 nM ± 320; EMAX = 39% ± 7%). Furthermore, BU12004 and BU10119 were MOR antagonists (KB = 0.42 nM ± 0.08 and KB = 1.1 nM ± 0.2, respectively) with less abuse liability than the MOR partial agonist BUP (EC50 = 0.79 nM ± 0.31; EMAX = 34% ± 6%). BUP, BU10119, and BU12004 were all potent antagonists at DOR and KOR. BU10119 (1.0 mg/kg) attenuated cocaine‐primed reinstatement in CPP, but 10 mg/kg BU12004 did not. BUP trended toward reduced drug‐primed reinstatement. The in vivo rank order of BU10119 >BUP >BU12004 matches the rank order for NOP agonism in vitro. Additionally, 1.0 mg/kg of either BU10119 or BU12004 blocked swim stress‐primed reinstatement, ostensibly due to their KOR antagonist activity. Taken together, BU10119 provides a promising lead to treat SUDs with better effectiveness and reduced abuse potential compared to BUP.Support or Funding InformationSupported by NIDA R01 DA07315 and 035316.

SNAP23 depletion enables more SNAP25/calcium channel excitosome formation to increase insulin exocytosis in type 2 diabetes.

SNAP23 is the ubiquitous SNAP25 isoform that mediates secretion in non-neuronal cells, similar to SNAP25 in neurons. However, some secretory cells like pancreatic islet β cells contain an abundance of both SNAP25 and SNAP23, where SNAP23 is believed to play a redundant role to SNAP25. We show that SNAP23, when depleted in mouse β cells in vivo and human β cells (normal and type 2 diabetes [T2D] patients) in vitro, paradoxically increased biphasic glucose-stimulated insulin secretion corresponding to increased exocytosis of predocked and newcomer insulin granules. Such effects on T2D Goto-Kakizaki rats improved glucose homeostasis that was superior to conventional treatment with sulfonylurea glybenclamide. SNAP23, although fusion competent in slower secretory cells, in the context of β cells acts as a weak partial fusion agonist or inhibitory SNARE. Here, SNAP23 depletion promotes SNAP25 to bind calcium channels more quickly and longer where granule fusion occurs to increase exocytosis efficiency. β Cell SNAP23 antagonism is a strategy to treat diabetes.

A Pharmacological Comparison of Two Isomeric Nicotinic Receptor Agonists: The Marine Toxin Isoanatabine and the Tobacco Alkaloid Anatabine.

Many organisms possess “secondary” compounds to avoid consumption or to immobilize prey. While the most abundant or active compounds are initially investigated, more extensive analyses reveal other “minor” compounds with distinctive properties that may also be of biomedical and pharmaceutical significance. Here, we present an initial in vitro investigation of the actions of two isomeric tetrahydropyridyl ring-containing anabasine analogs: isoanatabine, an alkaloid isolated from a marine worm, and anatabine, a relatively abundant minor alkaloid in commercial tobacco plants. Both compounds have a double bond that is distal to the piperidine ring nitrogen of anabasine. Racemic isoanatabine and anatabine were synthesized and their S- and R-enantiomers were isolated by chiral high pressure liquid chromatography (HPLC). Both isoanatabines displayed higher efficacies at α4β2 nicotinic acetylcholine receptors (nAChRs) relative to the anatabines; R-isoanatabine was most potent. Radioligand binding experiments revealed similar α4β2 nAChR binding affinities for the isoanatabines, but R-anatabine affinity was twice that of S-anatabine. While the two anatabines and S-isoanatabine were highly efficacious agonists at α7 nAChRs, R-isoanatabine was only a weak partial agonist. The four compounds share an ability to stimulate both α4β2 and α7 nAChRs, a property that may be useful in developing more efficacious drugs to treat neurodegenerative and other medical disorders.

Effect of 6-Benzoyl-benzothiazol-2-one scaffold on the pharmacological profile of α-alkoxyphenylpropionic acid derived PPAR agonists

A series of nitrogen heterocycles containing α–ethoxyphenylpropionic acid derivatives were designed as dual PPARα/γ agonist ligands for the treatment of type 2 diabetes (T2D) and its complications. 6-Benzoyl-benzothiazol-2-one was the most tolerant of the tested heterocycles in which incorporation of O-methyl oxime ether and trifluoroethoxy group followed by enantiomeric resolution led to the (S)-stereoisomer 44 b displaying the best in vitro pharmacological profile. Compound 44 b acted as a very potent full PPARγ agonist and a weak partial agonist on the PPARα receptor subtype. Compound 44 b showed high efficacy in an ob/ob mice model with significant decreases in serum triglyceride, glucose and insulin levels but mostly with limited body-weight gain and could be considered as a selective PPARγ modulator (SPPARγM).

Dual inhibitory action of trazodone on dorsal raphe serotonergic neurons through 5-HT1A receptor partial agonism and α1-adrenoceptor antagonism

Trazodone is an antidepressant drug with considerable affinity for 5-HT1A receptors and α1-adrenoceptors for which the drug is competitive agonist and antagonist, respectively. In this study, we used cell-attached or whole-cell patch-clamp recordings to characterize the effects of trazodone at somatodendritic 5-HT1A receptors (5-HT1AARs) and α1-adrenoceptors of serotonergic neurons in rodent dorsal raphe slices. To reveal the effects of trazodone at α1-adrenoceptors, the baseline firing of 5-HT neurons was facilitated by applying the selective α1-adrenoceptor agonist phenylephrine at various concentrations. In the absence of phenylephrine, trazodone (1–10 μM) concentration-dependently silenced neurons through activation of 5-HT1AARs. The effect was fully antagonized by the selective 5-HT1A receptor antagonist Way-100635. With 5-HT1A receptors blocked by Way-100635, trazodone (1–10 μM) concentration-dependently inhibited neuron firing facilitated by 1 μM phenylephrine. Parallel rightward shift of dose-response curves for trazodone recorded in higher phenylephrine concentrations (10–100 μM) indicated competitive antagonism at α1-adrenoceptors. Both effects of trazodone were also observed in slices from Tph2-/- mice that lack synthesis of brain serotonin, showing that the activation of 5-HT1AARs was not mediated by endogenous serotonin. In whole-cell recordings, trazodone activated 5-HT1AAR-coupled G protein-activated inwardly-rectifying (GIRK) channel conductance with weak partial agonist efficacy (~35%) compared to that of the full agonist 5-CT. Collectively our data show that trazodone, at concentrations relevant to its clinical effects, exerts weak partial agonism at 5-HT1AARs and disfacilitation of firing through α1-adrenoceptor antagonism. These two actions converge in inhibiting dorsal raphe serotonergic neuron activity, albeit with varying contribution depending on the intensity of α1-adrenoceptor stimulation.

A GPCR seen in the active state

Structural Biology G protein–coupled receptors (GPCRs) are exceptionally good targets for drug development. Warne et al. describe four crystal structures of complexes of a GPCR—the β1-adrenergic receptor—in its active state. They used nanobodies (recombinant variable domains of heavy-chain antibodies) and engineered G protein to stabilize the β1-adrenergic receptor bound to a full agonist, two partial agonists, and a weak partial agonist. Comparison of these structures to the inactive state elucidates how agonist binding is altered in the active conformation. Science , this issue p. [775][1] [1]: /lookup/doi/10.1126/science.aau5595

In silico and in vivo studies of Astragalus glycyphylloides saponin(s) with relevance to metabolic syndrome modulation

Triterpenoids are well known modulators of metabolic syndrome. One of the suggested modes of action (MoAs) involves peroxisome proliferator-activated receptor gamma (PPARγ) binding.In this study we aimed to: (i) evaluate in silico potential metabolites and PPARγ-mediated MoA of the sapogenin of the main saponin present in a purified saponins' mixture (PSM) from Astragalus glycyphylloides; (ii) estimate in silico and in vivo PSM's toxicity; and (iii) investigate in vivo antihyperglycaemic, hypolipidaemic, antioxidant and hepatoprotective effects of PSM.Metabolites and toxicity were predicted using Meteor and Derek Nexus expert systems (Lhasa Limited) and PPARγ binding was investigated using the software MOE (CCG Inc.). PSM's acute oral toxicity was evaluated in mice and the pharmacological effects were assessed in streptozotocin-induced diabetic spontaneously hypertensive rats (SHRs). Liver histopathology was studied as well.PPARγ weak partial agonism was predicted in silico for 24 probable/plausible Phase I metabolites which docking poses were clustered in 12 different binding modes with characteristic protein-ligand interactions. PSM's beneficial effects on the levels of blood glucose, triglycerides, and total cholesterol, on oxidative stress markers and liver histology in diabetic SHRs were comparable to those of the PPARγ ligand pioglitazone. PSM's safety profile was confirmed in silico and in vivo.

Molecular basis for high-affinity agonist binding in GPCRs.

G protein-coupled receptors (GPCRs) in the G protein-coupled active state have higher affinity for agonists as compared with when they are in the inactive state, but the molecular basis for this is unclear. We have determined four active-state structures of the β1-adrenoceptor (β1AR) bound to conformation-specific nanobodies in the presence of agonists of varying efficacy. Comparison with inactive-state structures of β1AR bound to the identical ligands showed a 24 to 42% reduction in the volume of the orthosteric binding site. Potential hydrogen bonds were also shorter, and there was up to a 30% increase in the number of atomic contacts between the receptor and ligand. This explains the increase in agonist affinity of GPCRs in the active state for a wide range of structurally distinct agonists.

Functional characterization and optimization of a bacterial cyclic nucleotide–gated channel

Cyclic nucleotide-gated (CNG) channels produce the initial electrical signal in mammalian vision and olfaction. They open in response to direct binding of cyclic nucleotide (cAMP or cGMP) to a cytoplasmic region of the channel. However, the conformational rearrangements occurring upon binding to produce pore opening (i.e. gating) are not well understood. SthK is a bacterial CNG channel that has the potential to serve as an ideal model for structure-function studies of gating but is currently limited by its toxicity, native cysteines, and low open probability (Po). Here, we expressed SthK in giant Escherichia coli spheroplasts and performed patch-clamp recordings to characterize SthK gating in a bacterial membrane. We demonstrated that the Po in cAMP is higher than has been previously published and that cGMP acts as a weak partial SthK agonist. Additionally, we determined that SthK expression is toxic to E. coli because of gating by cytoplasmic cAMP. We overcame this toxicity by developing an adenylate cyclase-knockout E. coli cell line. Finally, we generated a cysteine-free SthK construct and introduced mutations that further increase the Po in cAMP. We propose that this SthK model will help elucidate the gating mechanism of CNG channels.

Allosteric Agonism of α7 Nicotinic Acetylcholine Receptors: Receptor Modulation Outside the Orthosteric Site.

Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of ligand-gated ion channels. Typically, channel activation follows the binding of agonists to the orthosteric binding sites of the receptor. α7 nAChRs have a very low probability of channel activation, which can be reversed by the binding of α7 selective positive allosteric modulators (PAMs) to putative sites within the transmembrane domains. Although typical PAMs, like PNU-120596, require coapplication of an orthosteric agonist to produce large channel activations, some, like GAT107 and B-973B [(S)-3-(3,4-difluorophenyl)-N-(1-(6-(4-(pyridin-2-yl)piperazin-1-yl)pyrazin-2-yl)ethyl)propanamide], are characterized as allosteric activating PAMs, which also bind to an allosteric activation (AA) site in the extracellular domain and activate the α7 ion channel by themselves. We had previously characterized N,N-diethyl-N'-phenylpiperazine analogs with various functions. In this work, we docked members of this family to a homology model of the α7 receptor extracellular domain. The compound 1,1-diethyl-4(naphthalene-2-yl)piperazin-1-ium (2NDEP) a weak partial agonist, showed particularly favorable docking and binding energies at the putative AA site of the receptor. We hypothesized that 2NDEP could couple with PAMs through the AA site. This hypothesis was tested with the α7 mutant C190A, which is not activated by orthosteric agonists but is effectively activated by GAT107. The results showed that 2NDEP acts as an allosteric agonist of α7C190A when coapplied with the PAM PNU-120596. Also, the allosteric activity was nearly abolished upon coapplication with the AA site-selective antagonist 2,3,5,6MP-TQS (cis-trans-4-(2,3,5,6-tetramethylphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide), consistent with AA site involvement. Overall, our findings show a novel mode of agonism through an allosteric site in the extracellular domain of α7 nAChR.

Buprenorphine C7‐Esters with Improved Nociceptin Receptor Agonist Potency as Analgesics

There is an urgent need for analgesics with reduced abuse liability, withdrawal, respiratory depression and constipation to treat chronic pain patients. While clinical opiates produce analgesia and associated side effects through Mu Opioid Receptor (MOR) agonist activity, preclinical evidence indicates activity at the Kappa‐ (KOR), Delta‐ (DOR), and Nociception Receptors (NOP) subtypes can minimize side effects. Compounds displaying NOP agonist and weak MOR partial agonist activity produce antinociception with minimal abuse liability in primates and therefore are desirable therapeutic target. In the present study we evaluated buprenorphine analogues with C7 ester substituents for opioid receptor affinity by competition binding assay and for function using the [35S]‐GTPgS assay at MOR, NOP, DOR and KOR. Compounds showed a nearly 1000‐fold range in binding and [35S]‐GTPgS activity at NOP, ranging from low nanomolar to micromolar. One compound, containing a phenyl ester at C7, and lacking a keto group at C6, showed the highest potency NOP activity (EC50= 0.97 +/− 0.22 nM, EMAX= 47% +/− 11) with weak partial agonist potency at MOR (EC50= 3.9 +/− 1.3 nM, EMAX= 23% +/− 7). Since this compound has less MOR agonist efficacy than buprenorphine, it would be predicted to have minimal abuse liability. The series also showed a synergistic relationship in NOP activity between the C7 functional group and the C6 oxidation state. For example, the presence of a keto group at C6 decreased NOP potency significantly (EC50= 164 +/− 51 nM, EMAX= 47% +/− 9). This pattern was observed with numerous C7 functional groups. All compounds in the series were putative antagonists at DOR and KOR as they bound with high affinity but did stimulate significant activity in the [35S]‐GTPgS assay. This series of compounds provides different degrees of partial agonism at MOR and NOP and should help to determine the minimal MOR activity necessary for effective antinociception while minimizing potential abuse liability.Support or Funding InformationSupported by R01 DA07315 and 035316.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Binding and Signaling Properties of the Leu8 and Pro8 Isoforms of Oxytocin at Vasopressin V1a Receptors from Primate Species Expressing Leu8 or Pro8 Oxytocin

Among New World monkeys, there is a remarkable correlation between multiple aspects of social behavior and their expression of an oxytocin (OT) variant with proline at the eighth position (Pro8OT) vs. the common mammalian OT ligand that has leucine at position 8 (Leu8OT). In light of evidence for OT involvement in normative and dysfunctional social behavior, a better understanding of the impact of OT ligand variation on receptor binding and signaling could provide insights into the pharmacological modulation of sociality. We previously showed that binding and calcium signaling at oxytocin receptors (OTRs) from four primate species were not different for Leu8 vs. Pro8 (Taylor JH et al., J Pharmacol Exp Ther, 367:101–107, 2018). OT also binds and activates vasopressin V1a receptors (V1aRs), whose preferred ligand is the closely related nonapeptide hormone arginine vasopressin (AVP). Thus the binding and signaling of Leu8OT and Pro8OT at V1aRs from Leu8OT‐ vs. Pro8OT‐expressing species was examined. V1aRs from marmoset (a Pro8OT species), macaque (a Leu8OT species), and human (a Leu8OT species) were expressed in CHO cells. Competition binding assays with intact cells on ice were performed using 125I‐OVTA. IC50 values in nM for Leu8OT, Pro8OT, and arginine vasopressin (AVP), respectively, were 220, 150, and 0.78 for marmoset; 28, 21, and 1.1 for macaque; and 16, 8, and 0.63 for human. Pro8OT thus has a slightly higher affinity than Leu8OT at all three receptors. Both OT isoforms exhibited about 20‐fold lower affinity than AVP for the macaque and human V1aRs but a nearly 200‐fold lower affinity for the marmoset V1aR. Calcium signaling downstream of V1aRs was assessed with FlexStation‐II assays. EC50 values in nM for Leu8OT, Pro8OT, and AVP, respectively, were 8.4, 6.7, and 0.06 for marmoset; 21, 10, and 0.03 for macaque; and 145, 110, and 2.1 for human. Thus the potency for Pro8OT is slightly higher than for Leu8OT, and the potencies for both OT isoforms are 50–100 times lower than for AVP, at all three V1aRs. These signaling potency differences are similar to the binding affinity differences for the OT isoforms at each receptor. The maximal calcium response to Leu8OT was about 80% of that for Pro8OT and AVP at the marmoset and macaque receptors; in contrast, the maximal Leu8OT and Pro8OT responses were only about 20% of the response to AVP for the human receptor, indicating that both OT isoforms are relatively weak partial agonists for the human V1aR. This difference in partial agonism at human V1aRs compared to marmoset and macaque V1aRs for both OT isoforms is intriguing. However, neither differences in V1aR ligand binding or calcium signaling provide an obvious explanation for the different behaviors of Pro8OT‐ versus Leu8OT‐expressing species, similar to our previous data showing minimal differences between the OT isoforms for binding and signaling at OTRs. Differential signaling to other downstream responses merits further investigation.Support or Funding InformationSupported by NIH grant 1‐R01‐HD089147 to JAF.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cannabinoid Receptor 2 Signalling Bias Elicited by 2,4,6-Trisubstituted 1,3,5-Triazines.

Cannabinoid receptor 2 (CB2) is predominantly distributed in immune tissues and cells and is a promising therapeutic target for modulating inflammation. In this study we designed and synthesised a series of 2,4,6-trisubstituted 1,3,5-triazines with piperazinylalkyl or 1,2-diethoxyethane (PEG2) chains as CB2 agonists, all of which were predicted to be considerably more polar than typical cannabinoid ligands. In this series, we found that triazines containing an adamantanyl group were conducive to CB2 binding whereas those with a cyclopentyl group were not. Although the covalent attachment of a PEG2 linker to the adamantyl triazines resulted in a decrease in binding affinity, some of the ligands produced very interesting hCB2 signalling profiles. Six compounds with notable hCB2 orthosteric binding were functionally characterised in three pathways; internalisation, cyclic adenosine monophosphate (cAMP) and ERK phosphorylation (pERK). These were predominantly confirmed to be hCB2 agonists, and upon comparison to a reference ligand (CP 55,940), four compounds exhibited signalling bias. Triazines 14 (UOSD017) and 15 were biased towards internalisation over cAMP and pERK, and 7 was biased away from pERK activation relative to cAMP and internalisation. Intriguingly, the triazine with an amino-PEG2-piperazinyl linker (13 [UOSD008]) was identified to be a mixed agonist/inverse agonist, exhibiting apparent neutral antagonism in the internalisation pathway, transient inverse agonism in the cAMP pathway and weak partial agonism in the pERK pathway. Both the cAMP and pERK signalling were pertussis toxin (PTX) sensitive, implying that 13 is acting as both a weak agonist and inverse agonist at CB2 via Gαi/o. Compound 10 (UOSD015) acted as a balanced high intrinsic efficacy agonist with the potential to produce greater hCB2-mediated efficacy than reference ligand CP 55,940. As 10 includes a Boc-protected PEG2 moiety it is also a promising candidate for further modification, for example with a secondary reporter or fluorophore. The highest affinity compound in this set of relatively polar hCB2 ligands was compound 16, which acted as a slightly partial balanced agonist in comparison with CP 55,940. The ligands characterised here may therefore exhibit unique functional properties in vivo and have the potential to be valuable in the future development of CB2-directed therapeutics.