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Lysophosphatidic acid (LPA) is a bioactive phospholipid that acts as an agonist of six G protein-coupled receptors named LPA receptors (LPA1-6). LPA elicits diverse intracellular events and modulates several biological functions, including cell proliferation, migration, and invasion. Overactivation of the LPA--LPA receptor system is reported to be involved in several pathologies, including cancer, neuropathic pain, fibrotic diseases, atherosclerosis, and type 2 diabetes. Thus, LPA receptor modulators may be clinically relevant in numerous diseases, making the identification and pharmacodynamic characterization of new LPA receptor ligands of strong interest. In the present work, label-free dynamic mass redistribution (DMR) assay has been used to evaluate the pharmacological activity of some LPA1 and LPA2 standard antagonists at the recombinant human LPA1 and LPA2 receptors. These results are compared to those obtained in parallel experiments with the calcium mobilization assay. Additionally, the same experimental protocol has been used for the pharmacological characterization of the new compound CHI. KI 16425, RO 6842262, and BMS-986020 behaved as LPA1 inverse agonists in DMR experiments and as LPA1 antagonists in calcium mobilization assays. Amgen compound 35 behaved as an LPA2 antagonist, while Merck compound 20 from WO2012028243 was detected as an LPA2 inverse agonist using the DMR test. Of note, for all the compounds, similar potency values were estimated by DMR and calcium assay. The new compound CHI was found to be an LPA1 inverse agonist, but with potency lower than that of the standard compounds. In conclusion, we have demonstrated that DMR assay can be successfully used to characterize LPA1 and LPA2 ligands. Compared to the classical calcium mobilization assay, DMR offers some advantages, in particular allowing the identification of inverse agonists. Finally, in the frame of this study, a new LPA1 inverse agonist has been identified. [ABSTRACT FROM AUTHOR]

Despite the important role of bitter taste for the rejection of potentially harmful food sources, birds have long been suspected to exhibit inferior bitter tasting abilities. Although more recent reports on the bitter recognition spectra of several bird species have cast doubt about the validity of this assumption, the bitter taste of avian species is still an understudied field. Previously, we reported the bitter activation profiles of three zebra finch receptors Tas2r5, -r6, and -r7, which represent orthologs of a single chicken bitter taste receptor, Tas2r1. In order to get a better understanding of the bitter tasting capabilities of zebra finches, we selected another Tas2r gene of this species that is similar to another chicken Tas2r. Using functional calcium mobilization experiments, we screened zebra finch Tas2r1 with 72 bitter compounds and observed responses for 7 substances. Interestingly, all but one of the newly identified bitter agonists were different from those previously identified for Tas2r5, -r6, and -r7 suggesting that the newly investigated receptor fills important gaps in the zebra finch bitter recognition profile. The most potent bitter agonist found in our study is cucurbitacin I, a highly toxic natural bitter substance. We conclude that zebra finch exhibits an exquisitely developed bitter taste with pronounced cucurbitacin I sensitivity suggesting a prominent ecological role of this compound for zebra finch. [ABSTRACT FROM AUTHOR]

Lysophosphatidic acid (LPA) is a bioactive phospholipid that acts as an agonist of six G protein-coupled receptors named LPA receptors (LPA1-6). LPA elicits diverse intracellular events and modulates several biological functions, including cell proliferation, migration, and invasion. Overactivation of the LPA–LPA receptor system is reported to be involved in several pathologies, including cancer, neuropathic pain, fibrotic diseases, atherosclerosis, and type 2 diabetes. Thus, LPA receptor modulators may be clinically relevant in numerous diseases, making the identification and pharmacodynamic characterization of new LPA receptor ligands of strong interest. In the present work, label-free dynamic mass redistribution (DMR) assay has been used to evaluate the pharmacological activity of some LPA1 and LPA2 standard antagonists at the recombinant human LPA1 and LPA2 receptors. These results are compared to those obtained in parallel experiments with the calcium mobilization assay. Additionally, the same experimental protocol has been used for the pharmacological characterization of the new compound CHI. KI 16425, RO 6842262, and BMS-986020 behaved as LPA1 inverse agonists in DMR experiments and as LPA1 antagonists in calcium mobilization assays. Amgen compound 35 behaved as an LPA2 antagonist, while Merck compound 20 from WO2012028243 was detected as an LPA2 inverse agonist using the DMR test. Of note, for all the compounds, similar potency values were estimated by DMR and calcium assay. The new compound CHI was found to be an LPA1 inverse agonist, but with potency lower than that of the standard compounds. In conclusion, we have demonstrated that DMR assay can be successfully used to characterize LPA1 and LPA2 ligands. Compared to the classical calcium mobilization assay, DMR offers some advantages, in particular allowing the identification of inverse agonists. Finally, in the frame of this study, a new LPA1 inverse agonist has been identified.

Despite the important role of bitter taste for the rejection of potentially harmful food sources, birds have long been suspected to exhibit inferior bitter tasting abilities. Although more recent reports on the bitter recognition spectra of several bird species have cast doubt about the validity of this assumption, the bitter taste of avian species is still an understudied field. Previously, we reported the bitter activation profiles of three zebra finch receptors Tas2r5, -r6, and –r7, which represent orthologs of a single chicken bitter taste receptor, Tas2r1. In order to get a better understanding of the bitter tasting capabilities of zebra finches, we selected another Tas2r gene of this species that is similar to another chicken Tas2r. Using functional calcium mobilization experiments, we screened zebra finch Tas2r1 with 72 bitter compounds and observed responses for 7 substances. Interestingly, all but one of the newly identified bitter agonists were different from those previously identified for Tas2r5, -r6, and –r7 suggesting that the newly investigated receptor fills important gaps in the zebra finch bitter recognition profile. The most potent bitter agonist found in our study is cucurbitacin I, a highly toxic natural bitter substance. We conclude that zebra finch exhibits an exquisitely developed bitter taste with pronounced cucurbitacin I sensitivity suggesting a prominent ecological role of this compound for zebra finch.

The mu opioid receptor agonists are the most efficacious pain controlling agents but their use is accompanied by severe side effects. More recent developments indicate that some ligands can differentially activate receptor downstream pathways, possibly allowing for dissociation of analgesia mediated through the G protein from the opioid-related side effects mediated by ß-arrestin pathway. In an effort to identify such biased ligands, here we present a series of thirteen endomorphin-2 (EM-2) analogs with modifications in positions 1, 2, and/or 3. All obtained analogs behaved as mu receptor selective agonists in calcium mobilization assay carried out on cells expressing opioid receptors and chimeric G proteins. A Bioluminescence Resonance Energy Transfer (BRET) approach was employed to determine the ability of analogs to promote the interaction of the mu opioid receptor with G protein or ß-arrestin 2. Nearly half of the developed analogs showed strong bias towards G protein, in addition four compounds were nearly inactive towards ß-arrestin 2 recruitment while blocking the propensity of EM-2 to evoke mu-ß-arrestin 2 interaction. The data presented here contribute to our understanding of EM-2 interaction with the mu opioid receptor and of the transductional propagation of the signal. In addition, the generation of potent and selective mu receptor agonists strongly biased towards G protein provides the scientific community with novel tools to investigate the in vivo consequences of biased agonism at this receptor. [ABSTRACT FROM AUTHOR]

The mu opioid receptor agonists are the most efficacious pain controlling agents but their use is accompanied by severe side effects. More recent developments indicate that some ligands can differentially activate receptor downstream pathways, possibly allowing for dissociation of analgesia mediated through the G protein from the opioid-related side effects mediated by β-arrestin pathway. In an effort to identify such biased ligands, here we present a series of thirteen endomorphin-2 (EM-2) analogs with modifications in positions 1, 2, and/or 3. All obtained analogs behaved as mu receptor selective agonists in calcium mobilization assay carried out on cells expressing opioid receptors and chimeric G proteins. A Bioluminescence Resonance Energy Transfer (BRET) approach was employed to determine the ability of analogs to promote the interaction of the mu opioid receptor with G protein or β-arrestin 2. Nearly half of the developed analogs showed strong bias towards G protein, in addition four compounds were nearly inactive towards β-arrestin 2 recruitment while blocking the propensity of EM-2 to evoke mu-β-arrestin 2 interaction. The data presented here contribute to our understanding of EM-2 interaction with the mu opioid receptor and of the transductional propagation of the signal. In addition, the generation of potent and selective mu receptor agonists strongly biased towards G protein provides the scientific community with novel tools to investigate the in vivo consequences of biased agonism at this receptor.

Recently, mixed opioid/NOP agonists came to the spotlight for their favorable functional profiles and promising outcomes in clinical trials as novel analgesics. This study reports on two novel chimeric peptides incorporating the fragment Tyr-c[D-Lys-Phe-Phe]Asp-NH2 (RP-170), a cyclic peptide with high affinity for µ and κ opioid receptors (or MOP and KOP, respectively), conjugated with the peptide Ac-RYYRIK-NH2, a known ligand of the nociceptin/orphanin FQ receptor (NOP), yielding RP-170-RYYRIK-NH2 (KW-495) and RP-170-Gly3-RYYRIK-NH2 (KW-496). In vitro, the chimeric KW-496 gained affinity for KOP, hence becoming a dual KOP/MOP agonist, while KW-495 behaved as a mixed MOP/NOP agonist with low nM affinity. Hence, KW-495 was selected for further in vivo experiments. Intrathecal administration of this peptide in mice elicited antinociceptive effects in the hot-plate test; this action was sensitive to both the universal opioid receptor antagonist naloxone and the selective NOP antagonist SB-612111. The rotarod test revealed that KW-495 administration did not alter the mice motor coordination performance. Computational studies have been conducted on the two chimeras to investigate the structural determinants at the basis of the experimental activities, including any role of the Gly3 spacer. [ABSTRACT FROM AUTHOR]

The composition of menus and the sequence of foodstuffs consumed during a meal underlies elaborate rules. However, the molecular foundations for the observed taste- and pleasure-raising effects of complex menus are obscure. The molecular identification and characterization of taste receptors can help to gain insight into the complex interrelationships of food items and beverages during meals. In our study, we quantified important bitter compounds in chicory and chicory-based surrogate coffee and used them to identify responsive bitter taste receptors. The two receptors, TAS2R43 and TAS2R46, are exquisitely sensitive to lactucin, lactucopicrin, and 11β,13-dihydrolactucin. Sensory testing demonstrated a profound influence of the sequence of consumption of chicory, surrogate coffee, and roasted coffee on the perceived bitterness by human volunteers. These findings pave the way for a molecular understanding of some of the mixture effects underlying empirical meal compositions.

The careful evaluation of food is important for survival throughout the animal kingdom, and specialized chemoreceptors have evolved to recognize nutrients, minerals, acids, and many toxins. Vertebrate bitter taste, mediated by the taste receptor type 2 (T2R) family, warns against potentially toxic compounds. During evolution T2R receptors appear first in bony fish, but the functional properties of bony fish T2R receptors are mostly unknown. We performed a phylogenetic analysis showing the "living fossil" coelacanth (Latimeria chalumnae) and zebrafish (Danio rerio) to possess T2R repertoires typical for early-diverged species in the lobe-finned and the ray-finned clade, respectively. Receptors from these two species were selected for heterologous expression assays using a diverse panel of bitter substances. Remarkably, the ligand profile of the most basal coelacanth receptor, T2R01, is identical to that of its ortholog in zebrafish, consistent with functional conservation across >400 Myr of separate evolution. The second coelacanth receptor deorphaned, T2R02, is activated by steroid hormones and bile acids, evolutionary old molecules that are potentially endogenously synthesized agonists for extraoral T2Rs. For zebrafish, we report the presence of both specialized and promiscuous T2R receptors. Moreover, we identified an antagonist for one of the zebrafish receptors suggesting that bitter antagonism contributed to shape this receptor family throughout evolution. [ABSTRACT FROM AUTHOR]

In the past, taste interactions between sodium chloride (NaCl) and bitter tastants were investigated in human sensory studies, and the suppression of bitterness by sodium was observed. It is currently not clear if this phenomenon occurs predominantly peripherally or centrally and if the effect is general or only particular bitter compounds are blocked. Therefore, the influence of NaCl at the receptor level was tested by functional expression assays using four out of ∼25 human bitter taste receptors together with prototypical agonists. It was observed that NaCl affected only the responses of particular bitter taste receptor-compound pairs, whereas other bitter responses remained unchanged upon variations of the sodium concentrations. Among the tested receptors, TAS2R16 showed a reduction in signaling in the presence of NaCl. This demonstrates that for some receptor-agonist pairs, NaCl reduces the activation at the receptor level, whereas central effects may dominate the NaCl-induced bitter taste inhibition for other substances.

The potential, multifaceted therapeutic profile of cannabidiol (CBD), a major constituent derived from the Cannabis sativa plant, covers a wide range of neurological and psychiatric disorders, ranging from anxiety to pediatric epilepsy and drug addiction. However, the molecular targets responsible for these effects have been only partially identified. In this view, the involvement of the orexin system, the key regulator in arousal and the sleep/wake cycle, and in motivation and reward processes, including drug addiction, prompted us to explore, using computational and experimental approaches, the possibility that CBD could act as a ligand of orexin receptors, orexin 1 receptor of type 1 (OX1R) and type 2 (OX2R). Ligand-binding assays showed that CBD is a selective ligand of OX1R in the low micromolar range (Ki 1.58 ± 0.2 μM) while in vitro functional assays, carried out by intracellular calcium imaging and mobilization assays, showed that CBD acts as an antagonist at this receptor. Finally, the putative binding mode of CBD has been inferred by molecular docking and molecular dynamics simulations and its selectivity toward the OX1R subtype rationalized at the molecular level. This study provides the first evidence that CBD acts as an OX1R antagonist, supporting its potential use in addictive disorders and/or body weight regulation.

Abstract GPR139 is a Gq‐coupled receptor activated by the essential amino acids L‐tryptophan (L‐Trp) and L‐phenylalanine (L‐Phe). We carried out mutagenesis studies of the human GPR139 receptor to identify the critical structural motifs required for GPR139 activation. We applied site‐directed and high throughput random mutagenesis approaches using a double addition normalization strategy to identify novel GPR139 sequences coding receptors that have altered sensitivity to endogenous ligands. This approach resulted in GPR139 clones with gain‐of‐function, reduction‐of‐function or loss‐of‐function mutations. The agonist pharmacology of these mutant receptors was characterized and compared to wild‐type receptor using calcium mobilization, radioligand binding, and protein expression assays. The structure‐activity data were incorporated into a homology model which highlights that many of the gain‐of‐function mutations are either in or immediately adjacent to the purported orthosteric ligand binding site, whereas the loss‐of‐function mutations were largely in the intracellular G‐protein binding area or were disrupters of the helix integrity. There were also some reduction‐of‐function mutations in the orthosteric ligand binding site. These findings may not only facilitate the rational design of novel agonists and antagonists of GPR139, but also may guide the design of transgenic animal models to study the physiological function of GPR139.

GPR139 is a Gq‐coupled receptor activated by the essential amino acids L‐tryptophan (L‐Trp) and L‐phenylalanine (L‐Phe). We carried out mutagenesis studies of the human GPR139 receptor to identify the critical structural motifs required for GPR139 activation. We applied site‐directed and high throughput random mutagenesis approaches using a double addition normalization strategy to identify novel GPR139 sequences coding receptors that have altered sensitivity to endogenous ligands. This approach resulted in GPR139 clones with gain‐of‐function, reduction‐of‐function or loss‐of‐function mutations. The agonist pharmacology of these mutant receptors was characterized and compared to wild‐type receptor using calcium mobilization, radioligand binding, and protein expression assays. The structure‐activity data were incorporated into a homology model which highlights that many of the gain‐of‐function mutations are either in or immediately adjacent to the purported orthosteric ligand binding site, whereas the loss‐of‐function mutations were largely in the intracellular G‐protein binding area or were disrupters of the helix integrity. There were also some reduction‐of‐function mutations in the orthosteric ligand binding site. These findings may not only facilitate the rational design of novel agonists and antagonists of GPR139, but also may guide the design of transgenic animal models to study the physiological function of GPR139. [ABSTRACT FROM AUTHOR]

Morphiceptin (Tyr-Pro-Phe-Pro-NH 2 ) is a selective ligand of the mu opioid receptor, an important target in pain regulation. In this study, morphiceptin was modified at positions 2 or 3 by introduction of β 2 - or β 3 -amino acids and additionally in position 1 by replacing Tyr by Dmt (2′,6′-dimethyltyrosine), which resulted in obtaining enzymatically stable analogs with mixed opioid receptor affinity profiles. An analog of the sequence Dmt- d -Ala-( R )-β 2 -1-Nal-Pro-NH 2 [Nal = 3-(1-naphthyl)-alanine] showed very high activity at the mu and delta receptors in the calcium mobilization functional test but did not cross the artificial membrane imitating the blood-brain barrier. In the in vivo test this analog induced strong antinociceptive effect in the writhing test in mice after intraperitioneal but also oral administration and inhibited diarrhea similarly to loperamide. Therefore, it may become an interesting lead compound in the development of peripherally restricted drugs for the treatment of gastrointestinal disorders. [ABSTRACT FROM AUTHOR]

Cyclic pentapeptide Tyr-c[D-Lys-Phe-Phe-Asp]NH 2, based on the structure of endomorphin-2 (EM-2), which shows high affinity to the μ-opioid receptor (MOR) and a very strong antinociceptive activity in mice was used as a parent compound for the structure–activity relationship studies. In this report we synthesized analogs of a general sequence Dmt-c[D-Lys-Xaa-Yaa-Asp]NH 2 , with D-1- or D-2-naphthyl-3-alanine (D-1-Nal or D-2-Nal) in positions 3 or 4. In our earlier papers we have indicated that replacing a phenylalanine residue by the more extended aromatic system of naphthylalanines may result in increased bioactivities of linear analogs. The data obtained here showed that only cyclopeptides modified in position 4 retained the sub-nanomolar MOR and nanomolar κ-opioid receptor (KOR) affinity, similar but not better than that of a parent cyclopeptide. In the in vivo mouse hot-plate test, the most potent analog, Dmt-c[D-Lys-Phe-D-1-Nal-Asp]NH 2 , exhibited higher than EM-2 but slightly lower than the cyclic parent peptide antinociceptive activity after peripheral (ip) and also central administration (icv). Conformational analyses in a biomimetic environment and molecular docking studies disclosed the structural determinants responsible for the different pharmacological profiles of position 3- versus position 4-modified analogs. [ABSTRACT FROM AUTHOR]

Nociceptin/orphanin FQ (N/OFQ) is a heptadecapeptide acting as the endogenous ligand of the N/OFQ peptide receptor (NOP). N/OFQ(1–13)-NH 2 is the shortest N/OFQ sequence maintaining the same potency and efficacy as the natural peptide. Thus N/OFQ(1–13)-NH 2 was used as chemical template for investigating the structure activity relationship of threonine in position 5. 28 [X 5 ]N/OFQ(1–13)-NH 2 derivatives, in which Thr was substituted with natural and unnatural residues, were synthesized and characterized pharmacologically for their effects at the human NOP receptor. Two different functional assays were used: agonist stimulated [ 35 S]GTPγS binding in cell membranes and calcium mobilization in whole cells co-expressing chimeric G proteins. All [X 5 ]N/OFQ(1–13)-NH 2 derivatives behaved as full NOP agonists showing large differences in their potency. There was an excellent correlation between the results obtained in the two assays. The results of this study suggest that: position 5 does not play a pivotal role in receptor activation; the secondary alcoholic function of Thr is not important for receptor binding; side chain size, lipo/hydrophilic balance as well as hydrogen bond capability are also not crucial for receptor binding; an aliphatic amino function positively charged with at least 3 carbon atom distance from the peptide backbone has a huge disrupting effect on receptor binding. In conclusion this study demonstrates that a simple ethyl side chain as in compound 23 is sufficient in N/OFQ position 5 for maintaining bioactivity. [ABSTRACT FROM AUTHOR]

The composition of menus and the sequence of foodstuffs consumed during a meal underlies elaborate rules. However, the molecular foundations for the observed taste- and pleasure-raising effects of complex menus are obscure. The molecular identification and characterization of taste receptors can help to gain insight into the complex interrelationships of food items and beverages during meals. In our study, we quantified important bitter compounds in chicory and chicory-based surrogate coffee and used them to identify responsive bitter taste receptors. The two receptors, TAS2R43 and TAS2R46, are exquisitely sensitive to lactucin, lactucopicrin, and 11β,13-dihydrolactucin. Sensory testing demonstrated a profound influence of the sequence of consumption of chicory, surrogate coffee, and roasted coffee on the perceived bitterness by human volunteers. These findings pave the way for a molecular understanding of some of the mixture effects underlying empirical meal compositions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lang, Lang, Dunkel, Ziegler and Behrens.)

In this study, the Gαqi5 protein was used to force the human nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor to signal through the Ca2+ pathway in CHO cells. [Ca2+]i levels were monitored using the fluorometer FlexStation II and the Ca2+ dye Fluo 4 AM. Concentration response curves were generated with a panel of full and partial agonists, while NOP antagonists were assessed in inhibition-response curves. The following rank order of potency of antagonists was measured: $${\text{SB}} - {\text{612111}} >{\text{J}} - 113397 = {\text{Trap}} - 101 \geqslant {\text{UFP}} - 101 >\left[ {{\text{Nphe}}^{\text{1}} } \right]{{\text{N}} \mathord{\left/ {\vphantom {{\text{N}} {{\text{OFQ}}}}} \right. \kern-\nulldelimiterspace} {{\text{OFQ}}}}\left( {1 - 13} \right){\text{NH}}_{\text{2}} >>$$ naloxone, which is superimposable to literature findings. The rank order of potency of full and partial agonists is also similar to that obtained in previous studies with the exception of a panel of ligands (UFP-112, Ro 64-6198, ZP120, UFP-113) whose potency was relatively low in the Gαqi5–NOP receptor calcium assay. Interestingly, these NOP ligands are characterized by slow kinetic of interaction with the NOP receptor, as demonstrated by bioassay experiments. These results demonstrated that the FlexStation II–Gαqi5–NOP receptor calcium assay represents an adequate and useful screening for NOP receptor ligands, particularly for antagonists. [ABSTRACT FROM AUTHOR]

Aim: To develop homogeneous calcium mobilization assay for high-throughput screening (HTS) of mas-related gene (Mrg) receptor agonists. Methods: CHO-K1 cells stably expressing the full-length MrgD receptor and a calcium-sensitive dye were used to develop an HTS assay based on intracellular calcium influx. This method was applied to large-scale screening of a library containing 8000 synthetic compounds and natural product extracts. cAMP measurements were carried out to verify the bioactivities of the hits found by the calcium mobilization assay. Similar approaches were also employed in the identification of the MrgA1 receptor agonists following HTS of 16 000 samples. Results: EC50 values of the positive control compounds (β-alanine for MrgD receptor and dynorphin A for MrgA1 receptor) determined by the calcium mobilization assay were consistent with those reported in the literature, and the Z′ factors were 0.65 and 0.50 for MrgD and MrgA1 receptor assay, respectively. About 31 compounds for the MrgD receptor and 48 compounds for the MrgA1 receptor showing ≥20% of the maximal agonist activities found in the controls were initially identified as hits. Secondary screening confirmed that 2 compounds for each receptor possessed specific agonist activities. Intracellular cAMP level measurements indicated that the 2 confirmed hits displayed the functionality of the MrgD receptor agonists. Conclusion: A series of validation studies demonstrated that the homogeneous calcium mobilization assay developed was highly efficient, amenable to automation and a robust tool to screen potential MrgD and MrgA1 receptor agonists. Its application may be expanded to other G-protein coupled receptors that mobilize calcium influx upon activation. [ABSTRACT FROM AUTHOR]

Abstract: We found that zaprinast, a well-known cyclic guanosine monophosphate-specific phosphodiesterase inhibitor, acted as an agonist for a G protein-coupled receptor, GPR35. In our intracellular calcium mobilization assay, zaprinast activated rat GPR35 strongly (geometric mean EC50 value of 16nM), whereas it activated human GPR35 moderately (geometric mean EC50 value of 840nM). We also demonstrated that GPR35 acted as a Gαi/o- and Gα16-coupled receptor for zaprinast when heterologously expressed in human embryonic kidney 293 (HEK 293) cells. These findings will facilitate the research on GPR35 and the drug discovery of the GPR35 modulators. [Copyright &y& Elsevier]

The human P2Y1 receptor was expressed in the yeast Saccharomyces cerevisiae strain MPY578q5, which is engineered to couple to mammalian G protein-coupled receptors (GPCRs) and requires agonist-induced activation for growth. A range of known P2Y1 receptor agonists were examined with the yeast growth assay system, and the results were validated by comparing with potencies in the transfected 1321N1 astrocytoma cell line, in which calcium mobilization was measured with a FLIPR (fluorometric-imaging plate reader). The data were also compared with those from phospholipase C activation and radioligand binding with the use of a newly available radioligand [3H]MRS2279 (2-chloro- N 6-methyl-( N)-methanocarba-2’-deoxyadenosine-3’,5’bisphosphate). In the yeast growth assay, the rank order of potency of 2-MeSADP (2-methylthioadenosine 5’-diphosphate), ADP (adenosine 5’-diphosphate), and ATP (adenosine 5’-triphosphate) is the same as those in other assay systems, i.e., 2-MeSADP>ADP>ATP. The P2Y1-selective antagonist MRS2179 ( N 6-methyl-2-deoxyadenosine-3’,5’-bisphosphate) was shown to act as an antagonist with similar potency in all systems. The results suggest that the yeast expression system is suitable for screening P2Y1 receptor ligands, both agonists and antagonists. The yeast system should be useful for random mutagenesis of GPCRs to identify mutants with certain properties, such as selective potency enhancement for small synthetic molecules and constitutive activity. [ABSTRACT FROM AUTHOR]

The potential, multifaceted therapeutic profile of cannabidiol (CBD), a major constituent derived from the Cannabis sativa plant, covers a wide range of neurological and psychiatric disorders, ranging from anxiety to pediatric epilepsy and drug addiction. However, the molecular targets responsible for these effects have been only partially identified. In this view, the involvement of the orexin system, the key regulator in arousal and the sleep/wake cycle, and in motivation and reward processes, including drug addiction, prompted us to explore, using computational and experimental approaches, the possibility that CBD could act as a ligand of orexin receptors, orexin 1 receptor of type 1 (OX1R) and type 2 (OX2R). Ligand-binding assays showed that CBD is a selective ligand of OX1R in the low micromolar range (Ki 1.58 ± 0.2 μM) while in vitro functional assays, carried out by intracellular calcium imaging and mobilization assays, showed that CBD acts as an antagonist at this receptor. Finally, the putative binding mode of CBD has been inferred by molecular docking and molecular dynamics simulations and its selectivity toward the OX1R subtype rationalized at the molecular level. This study provides the first evidence that CBD acts as an OX1R antagonist, supporting its potential use in addictive disorders and/or body weight regulation. [ABSTRACT FROM AUTHOR]

Opioid-induced constipation is the most prevalent adverse effect of opioid drugs. Peripherally acting mu opioid receptor antagonists (PAMORAs), including naloxegol, are indicated for the treatment of opioid-induced constipation. The aim of this study was the in vitro and in vivo pharmacological characterization of naloxegol in comparison with naloxone. In vitro experiments were performed to measure calcium mobilization in cells coexpressing opioid receptors and chimeric G proteins and mu receptor interaction with G protein and β-arrestin 2 using bioluminescence resonance energy transfer. In vivo experiments were performed in mice to measure pain threshold using the tail withdrawal assay and colonic transit using the bead expulsion assay. In vitro, naloxegol behaved as a selective and competitive mu receptor antagonist similarly to naloxone, being 3-10-fold less potent. In vivo, naloxone was effective in blocking fentanyl actions when given subcutaneously (sc), but not per os (po). In contrast, naloxegol elicited very similar effects with sc or po administration counteracting in a dose dependent manner the constipating effects of fentanyl without interfering with the fentanyl mediated analgesia. Thus, a useful PAMORA action could be obtained with naloxegol both after po and sc administration. • PAMORAs are peripherally acting mu opioid receptor antagonists. • Naloxegol is a PAMORA clinically used for opioid-induced constipation. • In cells naloxegol behaved as a selective and competitive mu antagonist slightly less potent than naloxone. • In mice naloxegol blocked the constipating effects of fentanyl. • In mice naloxegol did not block the analgesic effects of fentanyl. [ABSTRACT FROM AUTHOR]

Summary Non-caloric sweeteners are widely used for the formulation of calorie-reduced beverages for health-conscious consumers. However, disadvantages such as undesired off-tastes limit the use of non-nutritive sweeteners. Therefore, the food industry is constantly searching for novel sweeteners and frequently resorts to using blends combining non-caloric sweeteners in a single formulation. The earliest blend allowing higher sweetness levels with reduced bitter off-taste combined saccharin with cyclamate. However, the mechanism by which sweetener blends become superior to single compounds remained obscure. By functional expression of human bitter taste receptors, we found the explanation for the phenomenon observed ∼60 years ago. We demonstrate that cyclamate potently blocks the receptors responsible for saccharin's bitter off-taste. This effect occurs at concentrations where cyclamate itself does not elicit a side taste. Intriguingly, also saccharin inhibits cyclamate-activated bitter receptors. Our experiments demonstrate that heterologous assays are useful for understanding perceptual phenomena and the development of novel tastant formulations. [ABSTRACT FROM AUTHOR]

Morphine and related drugs, which are the most effective analgesics for the relief of severe pain, act through activating opioid receptors. The endogenous ligands of these receptors are opioid peptides which cannot be used as antinociceptive agents due to their low bioactivity and stability in biological fluids. The major goal of opioid research is to understand the mechanism of action of opioid receptor agonists in order to improve therapeutic utility of opioids. Analgesic effects of morphine are mediated mostly through activation of the mu opioid receptor. However, in the search for safer and more effective drug candidates, analogs with mixed opioid receptor profile gained a lot of interest. Recently, the concept of biased agonists able to differentially activate GPCR downstream pathways, became a new approach in the design of novel drug candidates. It is hypothesized that compounds promoting G-protein signaling may produce analgesia while β-arrestin recruitment may be responsible for opioid side effects. In this report we showed that replacement of the tyrosine residue in the mu-selective ligand Tyr-c[d-Lys-Phe-Asp]NH 2 with 2',6'-dimethyltyrosine (Dmt) produced a cyclopeptide Dmt-c[d-Lys-Phe-Asp]NH 2 with mu/delta opioid receptor agonist profile. This analog showed improved antinociception in the hot-plate test, probably due to the simultaneous activation of mu and delta receptors but also significantly inhibited the gastrointestinal transit. Using the bioluminescence resonance energy transfer (BRET) assay it was shown that this analog was a mu receptor agonist biased toward β-arrestin. β-Arrestin-dependent signaling is most likely responsible for the observed inhibition of gastrointestinal motility exerted by the novel cyclopeptide., (Copyright © 2018. Published by Elsevier Inc.)

As part of our continuing studies on the structure-activity relationships of cyclic pentapeptides based on the structure of endomorphin-2 (EM-2), we report here the synthesis and biological activities of a new series of analogues of a general sequence Tyr/Dmt-c[d-Lys-Phe-Phe-Asp]NH2 (where Dmt = 2',6'-dimethyltyrosine), incorporating fluorinated amino acids: 4-fluorophenylalanine (4-F-Phe), 2,4-difluorophenylalanine (2,4-F-Phe), or 4-trifluoromethylphenylalanine (4-CF3-Phe) instead of the Phe residue in position 3 or 4. Depending on the fluorinated amino acid residue and its position in the sequence, analogues were mixed, high affinity MOP/KOP receptor agonists, MOP/DOP/KOP agonists, or selective KOP agonists. The in vitro potencies and efficacies of all novel analogues were assessed in calcium mobilization assay. The most potent analogues, Dmt-c[d-Lys-Phe-4-F-Phe-Asp]NH2 and Dmt-c[d-Lys-Phe-2,4-F-Phe-Asp]NH2, were tested in vivo in the mouse hot-plate test. They produced strong antinociceptive effect not only after intracerebroventricular but also after intraperitoneal injection, indicating that they were able to cross the blood-brain barrier.