Allosteric Modulation

The pentameric ligand-gated ion channel (pLGIC) from Gloeobacter violaceus (GLIC) has provided insightful structure–function views on the permeation process and the allosteric regulation of the pLGICs family. However, GLIC is activated by pH instead of a neurotransmitter and a clear picture for the gating transition driven by protons is still lacking. We used an electrostatics-based (finite difference Poisson–Boltzmann/Debye–Hückel) method to predict the acidities of all aspartic and glutamic residues in GLIC, both in its active and closed-channel states. Those residues with a predicted pK a close to the experimental pH 50 were individually replaced by alanine and the resulting variant receptors were titrated by ATR/FTIR spectroscopy. E35, located in front of loop F far away from the orthosteric site, appears as the key proton sensor with a measured individual pK a at 5.8. In the GLIC open conformation, E35 is connected through a water-mediated hydrogen-bond network first to the hig...

En haut : Vue en microscopie électronique des synapses neuromusculaires du muscle triangulaire de sternum de souris (grossissement de 13 333 fois). Les pelotes noires sont générées par des molécules d'alpha-neurotoxine « à trois doigts », isolée d'un venin de cobra et radioiodée, fixées aux récepteurs nicotiniques de l'acétylcholine associés aux membranes post-synaptiques. (Copyright Pascale Marchot et Jean-Louis Boudier) Sommaire Pages

The neurotensin receptor 1 (NTS1) is a G protein-coupled receptor (GPCR) with promise as a drug target for the treatment of pain, schizophrenia, obesity, addiction, and various cancers. A detailed picture of the NTS1 structural landscape has been established by X-ray crystallography and cryo-EM and yet, the molecular determinants for why a receptor couples to G protein versus arrestin transducers remain poorly defined. We used 13 C ε H3methionine NMR spectroscopy to show that phosphatidylinositol-4,5-bisphosphate (PIP2) promotes transducer complexation not by dramatically altering the receptor structure but by strengthening long-range allosteric connections, in the form of correlated conformational kinetics, between the orthosteric pocket and highlyconserved activation motifs. β-arrestin-1 further remodels the receptor ensemble by reducing conformational exchange kinetics for a subset of resonances, whereas G protein coupling has little to no effect on the rate. A βarrestin biased allosteric modulator transforms the NTS1:G protein complex into a concatenation of substates, without triggering transducer dissociation, suggesting that it may function by stabilizing signaling incompetent G protein conformations such as the non-canonical state. Together, our work demonstrates the importance of kinetic information to a complete picture of the GPCR activation landscape.

Light-operated drugs constitute a major target in drug discovery, since they may provide spatiotemporal resolution for the treatment of complex diseases (i.e. chronic pain). JF-NP-26 is an inactive photocaged derivative of the metabotropic glutamate type 5 (mGlu5) receptor negative allosteric modulator raseglurant. Violet light illumination of JF-NP-26 induces a photochemical reaction prompting the active-drug's release, which effectively controls mGlu5 receptor activity both in ectopic expressing systems and in striatal primary neurons. Systemic administration in mice followed by local light-emitting diode (LED)-based illumination, either of the thalamus or the peripheral tissues, induced JF-NP-26-mediated light-dependent analgesia both in neuropathic and in acute/tonic inflammatory pain models. These data offer the first example of optical control of analgesia in vivo using a photocaged mGlu5 receptor negative allosteric modulator. This approach shows potential for precisely t...

Metabotropic glutamate receptors (mGluRs) are important drug targets because of their involvement in several neurological diseases. Among mGluRs, mGlu5 is a particularly high-profile target because its positive or negative allosteric modulation can potentially treat schizophrenia or anxiety and chronic pain, respectively. Here, we computationally and experimentally probe the functional binding of a novel photoswitchable mGlu5 NAM, termed alloswitch-1, which loses its NAM functionality under violet light. We show alloswitch-1 binds deep in the allosteric pocket in a similar fashion to mavoglurant, the co-crystallized NAM in the mGlu5 transmembrane domain crystal structure. Alloswitch-1, like NAM2-Methyl-6-(phenylethynyl)pyridine(MPEP), is significantly affected by P655M mutation deep in the allosteric pocket, eradicating its functionality. In MD simulations, we show alloswitch-1 and MPEP stabilize the co-crystallized water molecule located at the bottom of the allosteric site that is seemingly characteristic of the inactive receptor state. Furthermore, both NAMs form H-bonds with S809 on helix 7, which may constitute an important stabilizing interaction for NAM-induced mGlu5 inactivation. Alloswitch-1, through isomerization of its amide group from trans to cis is able to form an additional interaction with N747 on helix 5. This may be an important interaction for amidecontaining mGlu5 NAMs, helping to stabilize their binding in a potentially unusual cis-amide state. Simulated conformational switching of alloswitch-1 in silico suggests photoisomerization of its azo group from trans to cis may be possible within the allosteric pocket. However, photoexcited alloswitch-1 binds in an unstable fashion, breaking H-bonds with the protein and destabilizing the co-crystallized water molecule. This suggests photoswitching may have destabilizing effects on mGlu5 binding and functionality.

NMDA receptors are glutamate-activated ion-channels involved in many essential brain functions including learning, memory, cognition, and behavior. Given this broad range of function it is not surprising that the initial attempts to correct NMDA receptor-mediated pathologies with en-mass receptor blockade were derailed by unacceptable side effects. Recent successes with milder or more targeted pharmaceuticals and increasing knowledge of how these receptors operate offer new incentives for rational development of effective NMDA receptor-targeted therapies. In this article we review evidence that L-alanine, a glycine-site partial agonist and pregnanolone sulfate, a usedependent allosteric inhibitor, while attenuating NMDA receptor activity to similar levels elicit remarkably dissimilar functional outcomes. We suggest that detailed understanding of NMDA receptor activation mechanisms and of structural correlates of function will help better match modulator with function and neurological condition and may unleash the yet untapped potential of NMDA receptor pharmaceutics.

Light-operated drugs constitute a major target in drug discovery, since they may provide spatiotemporal resolution for the treatment of complex diseases (i.e. chronic pain). JF-NP-26 is an inactive photocaged derivative of the metabotropic glutamate type 5 (mGlu5) receptor negative allosteric modulator raseglurant. Violet light illumination of JF-NP-26 induces a photochemical reaction prompting the active-drug's release, which effectively controls mGlu5 receptor activity both in ectopic expressing systems and in striatal primary neurons. Systemic administration in mice followed by local light-emitting diode (LED)-based illumination, either of the thalamus or the peripheral tissues, induced JF-NP-26-mediated light-dependent analgesia both in neuropathic and in acute/tonic inflammatory pain models. These data offer the first example of optical control of analgesia in vivo using a photocaged mGlu5 receptor negative allosteric modulator. This approach shows potential for precisely t...

Metabotropic glutamate receptors (mGluRs) are important drug targets because of their involvement in several neurological diseases. Among mGluRs, mGlu5 is a particularly high-profile target because its positive or negative allosteric modulation can potentially treat schizophrenia or anxiety and chronic pain, respectively. Here, we computationally and experimentally probe the functional binding of a novel photoswitchable mGlu5 NAM, termed alloswitch-1, which loses its NAM functionality under violet light. We show alloswitch-1 binds deep in the allosteric pocket in a similar fashion to mavoglurant, the co-crystallized NAM in the mGlu5 transmembrane domain crystal structure. Alloswitch-1, like NAM2-Methyl-6-(phenylethynyl)pyridine(MPEP), is significantly affected by P655M mutation deep in the allosteric pocket, eradicating its functionality. In MD simulations, we show alloswitch-1 and MPEP stabilize the co-crystallized water molecule located at the bottom of the allosteric site that is seemingly characteristic of the inactive receptor state. Furthermore, both NAMs form H-bonds with S809 on helix 7, which may constitute an important stabilizing interaction for NAM-induced mGlu5 inactivation. Alloswitch-1, through isomerization of its amide group from trans to cis is able to form an additional interaction with N747 on helix 5. This may be an important interaction for amidecontaining mGlu5 NAMs, helping to stabilize their binding in a potentially unusual cis-amide state. Simulated conformational switching of alloswitch-1 in silico suggests photoisomerization of its azo group from trans to cis may be possible within the allosteric pocket. However, photoexcited alloswitch-1 binds in an unstable fashion, breaking H-bonds with the protein and destabilizing the co-crystallized water molecule. This suggests photoswitching may have destabilizing effects on mGlu5 binding and functionality.

The A adenosine (AR) and D dopamine (DR) receptors form oligomers in the cell membrane and allosteric interactions across the AR-DR heteromer represent a target for development of drugs against central nervous system disorders. However, understanding of the molecular determinants of AR-DR heteromerization and the allosteric antagonistic interactions between the receptor protomers is still limited. In this work, a structural model of the AR-DR heterodimer was generated using a combined experimental and computational approach. Regions involved in the heteromer interface were modeled based on the effects of peptides derived from the transmembrane (TM) helices on AR-DR receptor-receptor interactions in bioluminescence resonance energy transfer (BRET) and proximity ligation assays. Peptides corresponding to TM-IV and TM-V of the AR blocked heterodimer interactions and disrupted the allosteric effect of AR activation on DR agonist binding. Protein-protein docking was used to construct a m...

Light-operated drugs constitute a major target in drug discovery, since they may provide spatiotemporal resolution for the treatment of complex diseases (i.e. chronic pain). JF-NP-26 is an inactive photocaged derivative of the metabotropic glutamate type 5 (mGlu5) receptor negative allosteric modulator raseglurant. Violet light illumination of JF-NP-26 induces a photochemical reaction prompting the active-drug's release, which effectively controls mGlu5 receptor activity both in ectopic expressing systems and in striatal primary neurons. Systemic administration in mice followed by local light-emitting diode (LED)-based illumination, either of the thalamus or the peripheral tissues, induced JF-NP-26-mediated light-dependent analgesia both in neuropathic and in acute/tonic inflammatory pain models. These data offer the first example of optical control of analgesia in vivo using a photocaged mGlu5 receptor negative allosteric modulator. This approach shows potential for precisely t...

The A adenosine (AR) and D dopamine (DR) receptors form oligomers in the cell membrane and allosteric interactions across the AR-DR heteromer represent a target for development of drugs against central nervous system disorders. However, understanding of the molecular determinants of AR-DR heteromerization and the allosteric antagonistic interactions between the receptor protomers is still limited. In this work, a structural model of the AR-DR heterodimer was generated using a combined experimental and computational approach. Regions involved in the heteromer interface were modeled based on the effects of peptides derived from the transmembrane (TM) helices on AR-DR receptor-receptor interactions in bioluminescence resonance energy transfer (BRET) and proximity ligation assays. Peptides corresponding to TM-IV and TM-V of the AR blocked heterodimer interactions and disrupted the allosteric effect of AR activation on DR agonist binding. Protein-protein docking was used to construct a m...

This perspective article provides observations supporting the view that nigro-striatal dopamine neurons and meso-limbic dopamine neurons mainly communicate through short distance volume transmission in the um range with dopamine diffusing into extrasynaptic and synaptic regions of glutamate and GABA synapses. Based on this communication it is discussed how volume transmission modulates synaptic glutamate transmission onto the D1R modulated direct and D2R modulated indirect GABA pathways of the dorsal striatum. Each nigro-striatal dopamine neuron was first calculated to form large numbers of neostriatal DA nerve terminals and then found to give rise to dense axonal arborizations spread over the neostriatum, from which dopamine is released. These neurons can through DA volume transmission directly influence not only the striatal GABA projection neurons but all the striatal cell types in parallel. It includes the GABA nerve cells forming the island-/striosome GABA pathway to the nigral...

The FGFR1-5-HT1A heteroreceptor complexes are involved in neuroplasticity in the rat hippocampus and in the mesencephalic raphe 5-HT nerve cells. There exists a 5-HT1A protomer enhancement of FGFR1 protomer signaling. Acute and 10 day treatment with intracerebroventricular (i.c.v.) FGF-2 and the 5-HT1A agonist 8-OH-DPAT produced enhanced antidepressant effects in the forced swim test (FST). We studied in the current work the disturbances in the FGFR1-5-HT1A heterocomplexes in a genetic rat model of depression, the Flinders sensitive line (FSL) rats of Sprague-Dawley (SD) origin, by means of neurochemical, neurophysiological and behavioral techniques. In control SD rats, the FGFR1 agonist SUN11602 and FGF2 produced a significant reduction of G protein-coupled inwardly rectifying K(+) channel (GIRK) currents induced by 8-OH-DPAT in the CA1 area of the hippocampus. In FSL rats, only i.c.v. 8-OH-DPAT alone treatment produced a significant reduction in the immobility time. The combined i...

The G protein-coupled receptor heterocomplex network database (GPCR-hetnet) is a database designed to store information on GPCR heteroreceptor complexes and their allosteric receptor-receptor interactions. It is an expert-authored and peer-reviewed, curated collection of well-documented GPCR-GPCR interactions that span the gamut from classical GPCR-GPCR interactions to more complex receptorreceptor interactions (GPCR-Receptor Tyrosine Kinase and GPCR-ionotropic receptor/ligand gated ion channel). Although GPCR-hetnet contains interactions among GPCR from several different species, the curators have initially focused on receptor-receptor interactions in humans. Currently (August 2017) GPCR-hetnet contains information on 250 receptors (192 GPCR, 52 RTK, and 6 ionotropic receptors) and >1023 interactions. The GPCR-hetnet provides four searchable datasets: the hetnet, the non-hetnet, the rtknet, and the ionnet. Other supporting datasets include information about receptors that are present in GPCR-hetnet such as literature citations. This chapter describes in a basic protocol how to use, navigate, and browse through the GPCR-hetnet database to identify the clusters in which a receptor protomer of interest is involved, while further applicability are also described and introduced.

Acetylcholine7 nicotinic receptors are widely expressed in the brain, where they are involved in the central processing of pain, as well as in neuropsychiatric, neurodegenerative and inflammatory processes. Positive allosteric modulators (PAM) show the advantage of allowing the selective regulation of different subtypes of acetylcholine receptors without directly interacting with the agonist binding site. Here we report the preparation and biological activity of a fluoro-containing compound, 1-(2',5'-dihydroxyphenyl)-3-(2-fluoro-4-hydroxyphenyl)-1-propanone (8, RGM079), that behaves as a potent PAM of the 7 receptors, and has a balanced pharmacokinetic profile and antioxidant properties comparable or even higher than well-known natural polyphenols. In addition, compound RGM079 shows neuroprotective properties in AD-toxicity related models. Thus, it causes a concentration-dependent neuroprotective effect against the toxicity induced by okadaic acid (OA) in the human neuroblastoma cell line SH-SY5Y. Similarly, in primary cultures of rat cortical neurons, RGM079 is able to restore the cellular viability after exposure to OA and amyloid peptide Aβ1-42, with cell death almost completely prevented at 10 and 30 M, respectively. Finally, compound RGM079 shows in vivo analgesic activity in the CFA-induced paw inflammation model, after intraperitoneal administration.

Acetylcholine7 nicotinic receptors are widely expressed in the brain, where they are involved in the central processing of pain, as well as in neuropsychiatric, neurodegenerative and inflammatory processes. Positive allosteric modulators (PAM) show the advantage of allowing the selective regulation of different subtypes of acetylcholine receptors without directly interacting with the agonist binding site. Here we report the preparation and biological activity of a fluoro-containing compound, 1-(2',5'-dihydroxyphenyl)-3-(2-fluoro-4-hydroxyphenyl)-1-propanone (8, RGM079), that behaves as a potent PAM of the 7 receptors, and has a balanced pharmacokinetic profile and antioxidant properties comparable or even higher than well-known natural polyphenols. In addition, compound RGM079 shows neuroprotective properties in AD-toxicity related models. Thus, it causes a concentration-dependent neuroprotective effect against the toxicity induced by okadaic acid (OA) in the human neuroblastoma cell line SH-SY5Y. Similarly, in primary cultures of rat cortical neurons, RGM079 is able to restore the cellular viability after exposure to OA and amyloid peptide Aβ1-42, with cell death almost completely prevented at 10 and 30 M, respectively. Finally, compound RGM079 shows in vivo analgesic activity in the CFA-induced paw inflammation model, after intraperitoneal administration.

The G protein-coupled receptor heterocomplex network database (GPCR-hetnet) is a database designed to store information on GPCR heteroreceptor complexes and their allosteric receptor-receptor interactions. It is an expert-authored and peer-reviewed, curated collection of well-documented GPCR-GPCR interactions that span the gamut from classical GPCR-GPCR interactions to more complex receptorreceptor interactions (GPCR-Receptor Tyrosine Kinase and GPCR-ionotropic receptor/ligand gated ion channel). Although GPCR-hetnet contains interactions among GPCR from several different species, the curators have initially focused on receptor-receptor interactions in humans. Currently (August 2017) GPCR-hetnet contains information on 250 receptors (192 GPCR, 52 RTK, and 6 ionotropic receptors) and >1023 interactions. The GPCR-hetnet provides four searchable datasets: the hetnet, the non-hetnet, the rtknet, and the ionnet. Other supporting datasets include information about receptors that are present in GPCR-hetnet such as literature citations. This chapter describes in a basic protocol how to use, navigate, and browse through the GPCR-hetnet database to identify the clusters in which a receptor protomer of interest is involved, while further applicability are also described and introduced.

There is a current and pressing need for improved cancer therapies. The use of small molecule kinase inhibitors and their application in combinatorial regimens represent an approach to personalized targeted cancer therapy. A number of AGC kinases, including atypical Protein Kinase C enzymes (PKCs), are validated drug targets for cancer treatment. Most drug development programs for protein kinases focus on the development of drugs that bind at the ATP-binding site. Alternatively, allosteric drugs have great potential for the development of future innovative drugs. However, the rational development of allosteric drugs poses important challenges because the compounds not only must bind to a given site but also must stabilize forms of the protein with a desired effect at a distant site. Here we describe the development of a new class of compounds targeting a regulatory site (PIF-pocket) present in the kinase domain and provide biochemical and crystallographic data showing that these compounds allosterically inhibit the activity of atypical PKCs. PS432, a representative compound, decreased the rate of proliferation of non-small cell lung cancer cells more potently than aurothiomalate, an atypical PKCι inhibitor currently under evaluation in clinical trials, and significantly reduced tumor growth without side effects in a mouse xenograft model. The druglike chemical class provides ample possibilities for the synthesis of derivative compounds, with the potential to allosterically modulate the activity of atypical PKCs and other kinases.

This paper describes the allosteric communication between the polobox domain and the catalytic domain of PLK1 with the support of small compounds. The paper describes new mode-of-action compounds which enhance or inhibit the interaction of PLK1 with protein partners.

R are noted in various neurodegenerative diseases (NDDs). Since studies have indicated that flavonoids can target brain GPCRs and provide neuroprotection via inhibition of monoamine oxidases (hMAOs), our study explored the functional role of kurarinone, an abundant lavandulated flavonoid in Sophora flavescens, on dopamine receptor subtypes, V 1A R, 5-HT 1A R, and hMAOs. Radioligand binding assays revealed considerable binding of kurarinone on D 1 R, D 2L R, and D 4 R. Functional GPCR assays unfolded the compound's antagonist behavior on D 1 R (IC 50 42.1 ± 0.35 μM) and agonist effect on D 2L R and D 4 R (EC 50 22.4 ± 3.46 and 71.3 ± 4.94 μM, respectively). Kurarinone was found to inhibit hMAO isoenzymes in a modest and nonspecific manner. Molecular docking displayed low binding energies during the intermolecular interactions of kurarinone with the key residues of the deep orthosteric binding pocket and the extracellular loops of D 1 R, D 2L R, and D 4 R, validating substantial binding affinities to these prime targets. With appreciable D 2L R and D 4 R agonism and D 1 R antagonism, kurarinone might be a potential compound that can alleviate clinical symptoms of Parkinson's disease and other NDDs.

The ongoing and widespread emergence of resistance to the existing anti-nematodal pharmacopeia has made it imperative to develop new anthelminthic agents. Historically, plants have been important sources of therapeutic compounds and offer an alternative to synthetic drugs. Monoterpenoids are phytochemicals that have been shown to produce acute toxic effects in insects and nematodes. Previous studies have shown nicotinic acetylcholine receptors (nAChRs) to be possible targets for naturally occurring plant metabolites such as carvacrol and carveol. In this study we examined the effects of monoterpenoid compounds on a levamisole sensitive nAChR from Oesophagostomum dentatum and a nicotine sensitive nAChR from Ascaris suum. We expressed the receptors in Xenopus laevis oocytes and used two-electrode voltage-clamp to characterize the effect of various compounds on these cys-loop receptors. At 100 μM the majority of these compounds acted as antagonists. Interestingly, further experiments revealed that both 0.1 μM and 10 μM menthol potentiated acetylcholine and levamisole responses in the levamisole sensitive receptor but not the nicotine sensitive receptor. We also investigated the effects of 0.1 μM menthol on the contractility of A. suum somatic muscle strips. Menthol produced significant potentiation of peak contractions at each concentration of acetylcholine. The positive allosteric modulatory effects of menthol in both in vivo and in vitro experiments suggests menthol as a promising candidate for combination therapy with cholinergic anthelmintics.

Background: The hippocampus and its 5-hydroxytryptamine transmission plays an important role in depression related to its involvement in limbic circuit plasticity. Methods: The analysis was made with bioluminescence resonance energy transfer, co-immunoprecipitation, in situ proximity ligation assay, binding assay, in cell western and the forced swim test. Results: Using bioluminescence resonance energy transfer analysis, fibroblast growth factor receptor 1 (FGFR1)-5-hydroxytryptamine 1A (5-HT1A) receptor complexes have been demonstrated and their specificity and agonist modulation characterized. Their presence based on co-immunoprecipitation and proximity ligation assay has also been indicated in hippocampal cultures and rat dorsal hippocampal formation showing a neuronal location. In vitro assays on extracellular signal-regulated kinases 1 and 2 phosphorylation have shown synergistic increases in signaling on coactivation with fibroblast growth factor 2 (FGF2) and a 5-HT1A agonist, and dependent on the heteroreceptor interface. In vitro and in vivo studies also revealed a 5-HT1A agonist induced phosphorylation of FGFR1 and extracellular signal-regulated kinase 1/2 in rat hippocampus without changing FGF2 levels. Co-activation of the heteroreceptor also resulted in synergistic increases in extensions of PC12 cells and neurite densities and protrusions in primary hippocampal cultures dependent on the receptor interface. The combined acute and repeated intracerebroventricular treatment with FGF2 and 8-OH-DPAT was found to produce evidence of highly significant antidepressant actions in the forced swim test. Conclusions: The findings indicate that neurotrophic and antidepressant effects of 5-HT in brain may, in part, be mediated by activation of the 5-HT1A receptor protomer in the hippocampal FGFR1-5-HT1A receptor complex enhancing the FGFR1 signaling.

Dopamine D 2 and D 4 receptors partially codistribute in the dorsal striatum and appear to play a fundamental role in complex behaviors and motor function. The discovery of D 2 R-D 4.x R (D 4.2 R, D 4.4 R or D 4.7 R) heteromers has been made in cellular models using co-immunoprecipitation, in situ Proximity Ligation Assays and BRET 1 techniques with the D 2 R and D 4.7 R receptors being the least effective in forming heteromers. Allosteric receptor-receptor interactions in D 2 R-D 4.2 R and D 2 R-D 4.4 R heteromers were observed using the MAPK assays indicating the existence of an enhancing allosteric receptor-receptor interaction in the corresponding heteromers between the two orthosteric binding sites. The bioinformatic predictions suggest the existence of a basic set of common triplets (ALQ and LRA) in the two participating receptors that may contribute to the receptor-receptor interaction interfaces.

In view of the co-distribution of dopamine D 2L R and 5-hydroxytryptamine 5-HT 2A receptors (D 2L R and 5-HT 2A R, respectively) within inter alia regions of the dorsal and ventral striatum and their role as a target of antipsychotic drugs; in this study we assessed the potential existence of D 2L R-5-HT 2A R heteromers in living cells and the functional consequences of this interaction. Thus, by means of a proximity-based bioluminescence resonance energy transfer (BRET) approach we demonstrated that the D 2L R and the 5-HT 2A R form stable and specific heteromers when expressed in HEK293T mammalian cells. Furthermore, when the D 2L R-5-HT 2A R heteromeric signaling was analyzed we found that the 5-HT 2A R-mediated phospholipase C (PLC) activation was synergistically enhanced by the concomitant activation of the D 2L R as shown in a NFAT-luciferase reporter gene assay and a specific and significant rise of the intracellular calcium levels were observed when both receptors were simultaneously activated. Conversely, when the D 2L R-mediated adenylyl cyclase (AC) inhibition was assayed we showed that costimulation of D 2L R and 5-HT 2A R within the heteromer led to inhibition of the D 2L R functioning, thus suggesting the existence of a 5-HT 2A R-mediated D 2L R trans-inhibition phenomenon. Finally, a bioinformatics study reveals that the triplet amino acid homologies LLT (Leu-Leu-Thr) and AIS (Ala-Ile-Ser) in TM1 and TM3, respectively of the D 2 R-5-HT 2A R may be involved in the receptor interface. Overall, the presence of the D 2L R-5-HT 2A R heteromer in discrete brain regions is postulated based on the existence of D 2L R-5-HT 2A receptor-receptor interactions in living cells and their codistribution inter alia in striatal regions. Possible novel therapeutic strategies for treatment of schizophrenia should be explored by targeting this heteromer.

Activity of the A 3 adenosine receptor (AR) allosteric m odulators L UF600 0 (2-cyclohexyl-N-(3,4dichlorophenyl)-1H-imidazo [4,5-c]quinolin-4-amine) and LUF6096 (N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarbox-amide) was compared at four A 3 AR species homologs used in preclinical drug development. In guanosine 5′-[γ-[ 35 S]thio]triphosphate ([ 35 S]GTPγS) binding assays with cell membranes isolated from human embryonic kidney cells stably expressing recombinant A 3 ARs, both modulators substantially enhanced agonist efficacy at human, dog, and rabbit A 3 ARs but provided only weak activity at mouse A 3 ARs. For human, dog, and rabbit, both modulators increased the maximal efficacy of the A 3 AR agonist 2-chloro-N 6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide as well as adenosine > 2-fold, while slightly reducing potency in human and dog. Based on results from N 6-(4-amino-3-[ 125 I]iodobenzyl)adenosine-5′-N-methylcarboxamide ([ 125 I]I-AB-MECA) binding assays, we hypothesize that potency reduction is explained by an allosterically induced slowing in orthosteric ligand binding kinetics that reduces the rate of formation of ligand-receptor complexes. Mutation of four amino acid residues of the human A 3 AR to the murine sequence identified the extracellular loop 1 (EL1) region as being important in selectively controlling the allosteric actions of LUF6096 on [ 125 I]I-AB-MECA binding kinetics. Homology modeling suggested interaction between speciesvariable EL1 and agonist-contacting EL2. These results indicate that A 3 AR allostery is species-dependent and provide mechanistic insights into this therapeutically promising class of agents.

Metabotropic glutamate receptors (mGluRs) are important drug targets because of their involvement in several neurological diseases. Among mGluRs, mGlu5 is a particularly high-profile target because its positive or negative allosteric modulation can potentially treat schizophrenia or anxiety and chronic pain, respectively. Here, we computationally and experimentally probe the functional binding of a novel photoswitchable mGlu5 NAM, termed alloswitch-1, which loses its NAM functionality under violet light. We show alloswitch-1 binds deep in the allosteric pocket in a similar fashion to mavoglurant, the co-crystallized NAM in the mGlu5 transmembrane domain crystal structure. Alloswitch-1, like NAM2-Methyl-6-(phenylethynyl)pyridine(MPEP), is significantly affected by P655M mutation deep in the allosteric pocket, eradicating its functionality. In MD simulations, we show alloswitch-1 and MPEP stabilize the co-crystallized water molecule located at the bottom of the allosteric site that is seemingly characteristic of the inactive receptor state. Furthermore, both NAMs form H-bonds with S809 on helix 7, which may constitute an important stabilizing interaction for NAM-induced mGlu5 inactivation. Alloswitch-1, through isomerization of its amide group from trans to cis is able to form an additional interaction with N747 on helix 5. This may be an important interaction for amidecontaining mGlu5 NAMs, helping to stabilize their binding in a potentially unusual cis-amide state. Simulated conformational switching of alloswitch-1 in silico suggests photoisomerization of its azo group from trans to cis may be possible within the allosteric pocket. However, photoexcited alloswitch-1 binds in an unstable fashion, breaking H-bonds with the protein and destabilizing the co-crystallized water molecule. This suggests photoswitching may have destabilizing effects on mGlu5 binding and functionality.

The mild neuroinflammation hypothesis of schizophrenia was introduced by Bechter in 2001. It has been hypothesized that a hypofunction of glutamatergic signaling via N-methyl-D-aspartate receptors (NMDARs) and hyperactivation of dopamine D2 receptors play a role in schizophrenia. The triplet puzzle theory states that sets of triplet amino acid homologies guide two different receptors toward each other and contributes to the formation of a receptor heteromer. It is, therefore, proposed that putative NMDAR-CC chemokine receptor type 2 (CCR2), NMDAR-C-X-C chemokine receptor type 4 (CXCR4), and NMDAR-interleukin 1 receptor type II (IL1R2) heteromers can be formed in the neuronal networks in mild neuroinflammation due to demonstration of Gly-Leu-Leu (GLL), Val-Ser-Thr (VST), and/or Ser-Val-Ser (SVS) amino acid homologies between these receptor protomers. This molecular process may underlie the ability to produce symptoms of schizophrenia in mild neuroinflammation. In this state, volume transmission (VT) is increased involving increased extracellular vesicle-mediated VT from microglia and astroglia. These vesicles may contain CCR2, CXCR4, and/or IL1R2 as well as their ligands and upon internalization by endocytic pathways into neurons can form heteroreceptor complexes with NMDAR in the plasma membrane with pathological allosteric receptor-receptor interactions involving increased internalization and reduced NMDAR signaling. The triplet puzzle theory also suggests the formation of putative D2R-CCR2, D2R-CXCR4, and D2R-IL1R2 heteromers in mild neuroinflammation in view of their demonstrated sets of Leu-Tyr-Ser (LYS), Leu-Pro-Phe (LPF), and/or Ser-Leu-Ala (SLA) triplet homologies. These D2R heteroreceptor complexes may also contribute to schizophrenia-like symptoms in mild neuroinflammation by enhancing D2R protomer function.

The concept of receptor-receptor and receptor-protein interactions and their integration in GPCR heteroreceptor complexes of the CNS over 20 years ago gave a new dimension to neuropsychopharmacology. Allosteric receptor-receptor interaction made possible through receptor oligomerization leads to novel receptor dynamics and the receptor protomers change their recognition, pharmacology, signaling and trafficking and novel allosteric binding sites may develop. GPCR heteroreceptor complexes can involve ion channel receptors, RTKs, sets of G protein interacting proteins, ion channels and/or transmitter transporters etc. The allosteric interactions in such dynamic high order receptor complexes take place in an orchestrated spatio-temporal fashion and participate in learning and formation of molecular engrams for short and long term memory. They represent major integrative centers. The GPCR heteroreceptor complexes in the CNS have become exciting new targets for neurotherapeutics in Parkinson's disease, schizophrenia, drug addiction, anxiety and depression opening a new field in neuropsychopharmacology. Possible novel strategies for targeting heteroreceptor complexes e.g. addiction are combined treatment with drugs targeting two receptor protomers to enhance compliance of patients by developing fixed formulations of the two drugs which give optimal pharmacokinetics for a time window of long duration for the combined anti-abuse actions. Dual acting drugs targeting two protomers in the receptor complex can also be developed as well as dual acting pro-drugs as a potential and novel multitarget approach to treat addiction. Heterobivalent drugs are other options to selectively target heterodimers e.g. in cocaine addiction built up of D2 antagonist and A2A agonist pharmacophors targeting A2A-D2 heterodimers.

The G protein-coupled receptor heterocomplex network database (GPCR-hetnet) is a database designed to store information on GPCR heteroreceptor complexes and their allosteric receptor-receptor interactions. It is an expert-authored and peer-reviewed, curated collection of well-documented GPCR-GPCR interactions that span the gamut from classical GPCR-GPCR interactions to more complex receptorreceptor interactions (GPCR-Receptor Tyrosine Kinase and GPCR-ionotropic receptor/ligand gated ion channel). Although GPCR-hetnet contains interactions among GPCR from several different species, the curators have initially focused on receptor-receptor interactions in humans. Currently (August 2017) GPCR-hetnet contains information on 250 receptors (192 GPCR, 52 RTK, and 6 ionotropic receptors) and >1023 interactions. The GPCR-hetnet provides four searchable datasets: the hetnet, the non-hetnet, the rtknet, and the ionnet. Other supporting datasets include information about receptors that are present in GPCR-hetnet such as literature citations. This chapter describes in a basic protocol how to use, navigate, and browse through the GPCR-hetnet database to identify the clusters in which a receptor protomer of interest is involved, while further applicability are also described and introduced.

In the last 10 years, it has become increasingly clear that large numbers of axon collaterals extend from the oxytocin (OXT) hypothalamic axons, especially the parvocellular components, to other brain regions. Consequently, the OXT signaling system forms, like other monoamine axons, a rich functional network across several brain regions. In this manuscript, we review the recently indicated higher order G-protein coupled heteroreceptor complexes of the oxytocin receptor (OXTR), and how these, via allosteric receptor-receptor interactions modulate the recognition, signaling, and trafficking of the participating receptor protomers and their potential impact for brain and behavior. The major focus will be on complexes of the OXTR protomer with the dopamine D2 receptor (D2R) protomer and the serotonin 2A (5-HT2AR) and 2C (5-HT2CR) receptor protomers. Specifically, the existence of D2R-OXTR heterocomplexes in the nucleus accumbens and the caudate putamen of rats has led to a postulated function for this heteromer in social behavior. Next, a physical interaction between OXTRs and the growth hormone secretagogue or ghrelin receptor (GHS-R1a) was demonstrated, which consequently was able to attenuate OXTR-mediated Gαq signaling. This highlights the potential of ghrelintargeted therapies to modulate oxytocinergic signaling with relevance for appetite regulation, anxiety, depression, and schizophrenia. Similarly, evidence for 5-HT2AR-OXTR heteromerization in the pyramidal cell layer of CA2 and CA3 in the dorsal hippocampus and in the nucleus accumbens shell was demonstrated. This complex may offer new strategies for the treatment of both mental disease and social behavior. Finally, the 5-HT2CR-OXTR Frontiers in Molecular Neuroscience 01 frontiersin.org Borroto-Escuela et al. 10.3389/fnmol.2022.1055344 heterocomplexes were demonstrated in the CA1, CA2, and CA3 regions of the dorsal hippocampus. Future work should be done to investigate the precise functional consequence of region-specific OXTR heteromerization in the brain, as well across the periphery, and whether the integration of neuronal signals in the brain may also involve higher order OXTR-GHS-R1a heteroreceptor complexes including the dopamine (DA), noradrenaline (NA) or serotonin (5-HT) receptor protomers or other types of G-protein coupled receptors (GPCRs).

Among mental diseases, major depressive disorder (MDD) and anxiety deserve a special place due to their high prevalence and their negative impact both on society and patients suffering from these disorders. Consequently, the development of novel strategies designed to treat them quickly and efficiently, without or at least having limited side effects, is considered a highly important goal. Growing evidence indicates that emerging properties are developed on recognition, trafficking, and signaling of G-protein coupled receptors (GPCRs) upon their heteromerization with other types of GPCRs, receptor tyrosine kinases, and ionotropic receptors such as N-methyl-D-aspartate (NMDA) receptors. Therefore, to develop new treatments for MDD and anxiety, it will be important to identify the most vulnerable heteroreceptor complexes involved in MDD and anxiety. This review focuses on how GPCRs, especially serotonin, dopamine, galanin, and opioid heteroreceptor complexes, modulate synaptic and vol...

Our hypothesis is that allosteric receptor-receptor interactions in homo-and heteroreceptor complexes may form the molecular basis of learning and memory. This principle is illustrated by showing how cocaine abuse can alter the adenosine A2AR-dopamine D2R heterocomplexes and their receptor-receptor interactions and hereby induce neural plasticity in the basal ganglia. Studies with A2AR ligands using cocaine self-administration procedures indicate that antagonistic allosteric A2AR-D2R heterocomplexes of the ventral striatopallidal GABA antireward pathway play a significant role in reducing cocaine induced reward, motivation, and cocaine seeking. Anticocaine actions of A2AR agonists can also be produced at A2AR homocomplexes in these antireward neurons, actions in which are independent of D2R signaling. At the A2AR-D2R heterocomplex, they are dependent on the strength of the antagonistic allosteric A2AR-D2R interaction and the number of A2AR-D2R and A2AR-D2R-sigma1R heterocomplexes present in the ventral striatopallidal GABA neurons. It involves a differential cocaine-induced increase in sigma1Rs in the ventral versus the dorsal striatum. In contrast, the allosteric brake on the D2R protomer signaling in the A2AR-D2R heterocomplex of the dorsal striatopallidal GABA neurons is lost upon cocaine self-administration. This is potentially due to differences in composition and allosteric plasticity of these complexes versus those in the ventral striatopallidal neurons.

and stress-related disorders. Using large-scale microsecond simulations, we examined the open and closed PAC1R conformations interconnected within an ensemble of transitional states. The open-to-closed transition can be initiated by ''unzipping'' the extracellular domain (ECD) and the transmembrane domain (7TM), mediated by a unique segment within the beta3-beta4 loop. Transitions between the open and closed states are 10-30 times longer than those within the closed states, which clearly implicate allosteric effects propagating from the extracellular face of the receptor to the intracellular G proteinbinding site. By simulating the agonist PACAP1-38 and the antagonist PACAP6-38 bound to the PAC1R orthosteric site, we observed the ligandinduced conformational changes that are associated with activation and deactivation. In particular, our data demonstrate evidence for the key role of transmembrane helix 6 (TM6) in connecting the ligand-receptor interactions with the opening/closing of the intracellular G protein-binding site. Such allosteric dynamics provides structural and mechanistic insights for the activation and modulation of PAC1R and related Class B receptors, which will serve as the essential molecular basis for the discovery novel anti-stress therapeutic agents.

We assess the stability of two previously suggested binding modes for the neuropeptide orexin-A in the OX 2 receptor through extensive molecular dynamics simulations. As the activation determinants of the receptor remain unknown, we simulated an unliganded receptor and two small-molecular ligands, the antagonist suvorexant and the agonist Nag26 for comparison. Each system was simulated in pure POPC membrane as well as in the 25% cholesterol−POPC membrane. In total, we carried out 36 μs of simulations. Through this set of simulations, we report a stable binding mode for the C-terminus of orexin-A. In addition, we suggest interactions that would promote orexin receptor activation, as well as others that would stabilize the inactive state.

Among mental diseases, major depressive disorder (MDD) and anxiety deserve a special place due to their high prevalence and their negative impact both on society and patients suffering from these disorders. Consequently, the development of novel strategies designed to treat them quickly and efficiently, without or at least having limited side effects, is considered a highly important goal. Growing evidence indicates that emerging properties are developed on recognition, trafficking, and signaling of G-protein coupled receptors (GPCRs) upon their heteromerization with other types of GPCRs, receptor tyrosine kinases, and ionotropic receptors such as N-methyl-D-aspartate (NMDA) receptors. Therefore, to develop new treatments for MDD and anxiety, it will be important to identify the most vulnerable heteroreceptor complexes involved in MDD and anxiety. This review focuses on how GPCRs, especially serotonin, dopamine, galanin, and opioid heteroreceptor complexes, modulate synaptic and vol...

Acetylcholine7 nicotinic receptors are widely expressed in the brain, where they are involved in the central processing of pain, as well as in neuropsychiatric, neurodegenerative and inflammatory processes. Positive allosteric modulators (PAM) show the advantage of allowing the selective regulation of different subtypes of acetylcholine receptors without directly interacting with the agonist binding site. Here we report the preparation and biological activity of a fluoro-containing compound, 1-(2',5'-dihydroxyphenyl)-3-(2-fluoro-4-hydroxyphenyl)-1-propanone (8, RGM079), that behaves as a potent PAM of the 7 receptors, and has a balanced pharmacokinetic profile and antioxidant properties comparable or even higher than well-known natural polyphenols. In addition, compound RGM079 shows neuroprotective properties in AD-toxicity related models. Thus, it causes a concentration-dependent neuroprotective effect against the toxicity induced by okadaic acid (OA) in the human neuroblastoma cell line SH-SY5Y. Similarly, in primary cultures of rat cortical neurons, RGM079 is able to restore the cellular viability after exposure to OA and amyloid peptide Aβ1-42, with cell death almost completely prevented at 10 and 30 M, respectively. Finally, compound RGM079 shows in vivo analgesic activity in the CFA-induced paw inflammation model, after intraperitoneal administration.

Our previous findings indicate that A2A and D2 receptors are co-expressed on adult rat striatal astrocytes and on the astrocyte processes, and that A2A-D2 receptor–receptor interaction can control the release of glutamate from the processes. Functional evidence suggests that the receptor–receptor interaction was based on heteromerization of native A2A and D2 receptors at the plasma membrane of striatal astrocyte processes. We here provide biochemical and biophysical evidence confirming that receptor–receptor interaction between A2A and D2 receptors at the astrocyte plasma membrane is based on A2A-D2 heteromerization. To our knowledge, this is the first direct demonstration of the ability of native A2A and D2 receptors to heteromerize on glial cells. As striatal astrocytes are recognized to be involved in Parkinson’s pathophysiology, the findings that adenosine A2A and dopamine D2 receptors can form A2A-D2 heteromers on the astrocytes in the striatum (and that these heteromers can pl...

Patients affected by recurrent seizures frequently present increased homocysteine plasma levels in consequence of treatment with antiepileptic drugs. Homocysteine is proconvulsant and can affect the response to antiepileptic drugs. In addition, high homocysteine plasma levels represent a risk factor for cardiovascular and neurodegenerative diseases. To better define the role of increased homocysteine in epilepsy, we analyzed the effects of homocysteine pretreatment in the pilocarpine model of status epilepticus (SE), which is used to mimic temporal lobe epilepsy (TLE) in rodents. Precisely, we investigated whether a moderate hyperhomocysteinemia, unable to cause seizures, could sensitize rats to pilocarpine and cooperate in inducing brain lesions. We found that a subthreshold dose of pilocarpine (200 mg/kg) is sufficient to induce SE in the majority (approximately 90%) of rats pretreated with homocysteine for 2 weeks, whereas only 40% of saline-treated controls developed SE following the same pilocarpine dose. Furthermore, homocysteine pretreatment led to a significant increase in neuronal cell loss evaluated by counting toluidine blue-stained or Fluoro-Jade-positive cells in hippocampal and parahippocampal regions. Pilocarpine augmented amyloid beta expression in both animal groups. However, pretreatment with homocysteine favored the intraneuronal fibrillar conformation of amyloid beta, thus promoting neurodegeneration. These findings indicate that increased homocysteine levels enhance seizure activity and neurodegeneration in pilocarpine-treated rats and suggest that similar detrimental effects may occur in patients affected by TLE.

Ionotropic glutamate receptors of the kainate and AMPA subtypes share a number of structural features, both topographical and in terms of stoichiometry. In addition, AMPA and kainate receptors share similar pharmacological and biophysical properties in that they are activated by common agonists and display rapid activation and desensitization characteristics. However, we show here that in contrast to AMPA receptor-mediated responses (native or recombinant GluR3 receptor), the response of native and recombinant (GluR6) kainate receptors to glutamate was drastically reduced in the absence of extracellular Na+(i.e., when replaced by Cs+). Removal of Na+increases the rate of desensitization, indicating that external Na+modulates channel gating. Whereas the size of the substituting cation is important in mimicking the action of Na+(Li+>K+>Cs+), modulation was voltage independent. These results indicate the existence of different gating mechanisms for AMPA and kainate receptors. By ...

Adenosine A 2A receptors (A 2A Rs) and dopamine D 2 receptors (D 2 Rs) form constitutive heteromers in living cells and exhibit a strong functional antagonistic interaction. Recent findings give neurochemical evidence that extended cocaine self-administration in the rat give rise to an up-regulation of functional A 2A Rs in the nucleus accumbens that return to baseline expression levels during cocaine withdrawal. In the present work, the acute in vitro effects of a concentration of cocaine known to fully block the dopamine (DA) transporter without exerting any toxic actions were investigated on A 2A R and D 2L R formed heteromers in transiently co-transfected HEK-293T cells. In vitro treatment of cocaine was found to produce changes in D 2 R homodimers and in A 2A R-D 2 R heterodimers detected through bioluminescent energy transfer (BRET). Cocaine was found to produce a time-and concentration-dependent reduction in the BRET max between A 2A R-D 2L R heterodimers and D 2L R homodimers, but not A 2A R homodimers, indicating its effect on D 2 R. Cocaine was evaluated with regard to D 2 R binding using a human D 2L R stable expressing CHO cell line and was found to produce an increase in the affinity of hD 2L R for DA. At the level of G protein-coupling, cocaine produced a small, but significant increase in DA-stimulated binding of GTPcS. However, cocaine failed to modulate D 2 R agonist-induced inhibition of cAMP in stable hD 2L R CHO cells or the gating of GIRK channels in oocytes. Taken together, these results indicate a direct and specific effect of a moderate concentration of cocaine on the DA D 2L R, that results in enhanced agonist recognition, G protein-coupling and an altered conformational state of D 2 R homodimers and A 2A R-D 2 R heterodimers.

The chemokine receptor CXCR3 is a G protein-coupled receptor, which conveys extracellular signals into cells by changing its conformation upon agonist binding. To facilitate the mechanistic understanding of allosteric modulation of CXCR3, we combined computational modeling with the synthesis of novel chemical tools containing boronic acid moiety, site-directed mutagenesis, and detailed functional characterization. The design of boronic acid derivatives was based on the predictions from homology modeling and docking. The choice of the boronic acid moiety was dictated by its unique ability to interact with proteins in a reversible covalent way, thereby influencing conformational dynamics of target biomolecules. During the synthesis of the library we have developed a novel approach for the purification of drug-like boronic acids. To validate the predicted binding mode and to identify amino acid residues responsible for the transduction of signal through CXCR3, we conducted a site-directed mutagenesis study. With the use of allosteric radioligand RAMX3 we were able to establish the existence of a second allosteric binding pocket in CXCR3, which enables different binding modes of structurally closely related allosteric modulators of CXCR3. We have also identified residues Trp109 2.60 and Lys300 7.35 inside the transmembrane bundle of the receptor as crucial for the regulation of the G protein activation. Furthermore we report the boronic acid 14 as the first biased negative allosteric modulator of the receptor. Overall, our data demonstrate that boronic acid derivatives represent an outstanding tool for determination of key receptor-ligand interactions and induction of ligand-biased signaling.

Pentameric ligand-gated ion channels (pLGICs) or Cys-loop receptors are involved in fast synaptic signaling in the nervous system. Allosteric modulators bind to sites that are remote from the neurotransmitter binding site, but modify coupling of ligand binding to channel opening. In this study, we developed nanobodies (single domain antibodies), which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote (Erwinia) channel ELIC bound either to a positive or a negative allosteric modulator. The allosteric nanobody binding sites partially overlap with those of small molecule modulators, including a vestibule binding site that is not accessible in some pLGICs. Using mutagenesis, we extrapolate the functional importance of the vestibule binding site to the human 5-HT3 receptor, suggesting a common mechanism of modulation in this protein and ELIC. Thus we identify key elements of allosteric binding sites, and extend drug design possibilities ...

Protein phosphorylation plays critical roles in a variety of intracellular signaling pathways and physiological functions that are controlled by neurotransmitters and neuromodulators in the brain. Dysregulation of these signaling pathways has been implicated in neurodevelopmental disorders, including autism spectrum disorder, attention deficit hyperactivity disorder and schizophrenia. While recent advances in mass spectrometry-based proteomics have allowed us to identify approximately 280,000 phosphorylation sites, it remains largely unknown which sites are phosphorylated by which kinases. To overcome this issue, previously, we developed methods for comprehensive screening of the target substrates of given kinases, such as PKA and Rho-kinase, upon stimulation by extracellular signals and identified many candidate substrates for specific kinases and their phosphorylation sites. Here, we developed a novel online database to provide information about the phosphorylation signals identif...

The ongoing and widespread emergence of resistance to the existing anti-nematodal pharmacopeia has made it imperative to develop new anthelminthic agents. Historically, plants have been important sources of therapeutic compounds and offer an alternative to synthetic drugs. Monoterpenoids are phytochemicals that have been shown to produce acute toxic effects in insects and nematodes. Previous studies have shown nicotinic acetylcholine receptors (nAChRs) to be possible targets for naturally occurring plant metabolites such as carvacrol and carveol. In this study we examined the effects of monoterpenoid compounds on a levamisole sensitive nAChR from Oesophagostomum dentatum and a nicotine sensitive nAChR from Ascaris suum. We expressed the receptors in Xenopus laevis oocytes and used two-electrode voltage-clamp to characterize the effect of various compounds on these cys-loop receptors. At 100 μM the majority of these compounds acted as antagonists. Interestingly, further experiments revealed that both 0.1 μM and 10 μM menthol potentiated acetylcholine and levamisole responses in the levamisole sensitive receptor but not the nicotine sensitive receptor. We also investigated the effects of 0.1 μM menthol on the contractility of A. suum somatic muscle strips. Menthol produced significant potentiation of peak contractions at each concentration of acetylcholine. The positive allosteric modulatory effects of menthol in both in vivo and in vitro experiments suggests menthol as a promising candidate for combination therapy with cholinergic anthelmintics.

The diuretic drug amiloride and its analogues were found previously to be allosteric modulators of antagonist binding to A 2A adenosine receptors. In this study, the possibility of the allosteric modulation by amiloride analogues of antagonist binding at A 1 and A 3 receptors, as well as agonist binding at A 1 , A 2A , and A 3 receptors, was explored. Amiloride analogues increased the dissociation rates of two antagonist radioligands, [ 3 H]8-cyclopentyl-1,3 dipropylxanthine ([ 3 H]DPCPX) and [ 3 H]8-ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H imidazo[2,1-i]purin-5-one ([ 3 H]PSB-11), from A 1 and A 3 receptors, respectively. Amiloride and 5-(N,N-dimethyl)amiloride (DMA) were more potent at A 1 receptors than at A 3 receptors, while 5-(N,N-hexamethylene)amiloride (HMA) was more potent at A 3 receptors. Thus, amiloride analogues are allosteric inhibitors of antagonist binding at A 1 , A 2A , and A 3 adenosine receptor subtypes. In contrast to their effects on antagonist-occupied receptors, amiloride analogues did not affect the dissociation rates of the A 1 agonist [ 3 H]N 6-[(R)-phenylisopropyl]adenosine ([ 3 H]R PIA) from A 1 receptors or the A 2A agonist [ 3 H]2-[p-(2-carboxyethyl)phenyl-ethylamino]-5′-N ethylcarboxamidoadenosine ([ 3 H]CGS21680) from A 2A receptors. The dissociation rate of the A 3 agonist radioligand [ 125 I]N 6-(4-amino-3-iodobenzyl)-adenosine-5′-N-methyluronamide ([ 125 I]I AB-MECA) from A 3 receptors was decreased significantly by amiloride analogues. The binding modes of amiloride analogues at agonist-occupied and antagonist-occupied receptors differed markedly, which was demonstrated in all three subtypes of adenosine receptors tested in this study. The effects of the amiloride analogues on the action of the A 3 receptor agonist were explored further using a cyclic AMP functional assay in intact CHO cells expressing the human A 3 receptor. Both binding and functional assays support the allosteric interactions of amiloride analogues with A 3 receptors.