ORCID Profile
0000-0002-7361-7875
Current Organisation
University of Nottingham
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Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 29-11-2012
Abstract: Designer receptors exclusively activated by designer drugs (DREADDs) are chemogenetically modified muscarinic acetylcholine receptors (mAChRs) that have minimal responsiveness to acetylcholine (ACh) but are potently and efficaciously activated by an otherwise inert synthetic ligand, clozapine-N-oxide (CNO). DREADDs have been used as tools for selectively modulating signal transduction pathways in vitro and in vivo. Recent comprehensive studies have validated how the pharmacology of a CNO-bound DREADD mirrors that of an ACh-bound wild-type (WT) mAChR. However, nothing is known about whether this equivalence extends to the allosteric modulation of DREADDs by small molecules. To address this, we investigated the actions at an M(1) DREADD of benzyl quinolone carboxylic acid (BQCA), a positive allosteric modulator of ACh binding and function that is known to behave according to a simple two-state mechanism at the WT receptor. We found that allosteric modulation of the CNO-bound DREADD receptor is not equivalent to the corresponding modulation of the ACh-bound WT receptor. We also found that BQCA engenders stimulus bias at the M(1) DREADD, having differential types of cooperativity depending on the signaling pathway. Furthermore, the modulation of ACh itself by BQCA at the DREADD is not compatible with the two-state model that we previously applied to the M(1) WT receptor.
Publisher: American Chemical Society (ACS)
Date: 30-05-2014
DOI: 10.1021/JM500457X
Abstract: Biased agonism at GPCRs highlights the potential for the discovery and design of pathway-selective ligands and may confer therapeutic advantages to ligands targeting the dopamine D2 receptor (D2R). We investigated the determinants of efficacy, affinity, and bias for three privileged structures for the D2R, exploring changes to linker length and incorporation of a heterocyclic unit. Profiling the compounds in two signaling assays (cAMP and pERK1/2) allowed us to identify and quantify determinants of biased agonism at the D2R. Substitution on the phenylpiperazine privileged structures (2-methoxy vs 2,3-dichloro) influenced bias when the thienopyridine heterocycle was absent. Upon inclusion of the thienopyridine unit, the substitution pattern (4,6-dimethyl vs 5-chloro-6-methoxy-4-methyl) had a significant effect on bias that overruled the effect of the phenylpiperazine substitution pattern. This latter observation could be reconciled with an extended binding mode for these compounds, whereby the interaction of the heterocycle with a secondary binding pocket may engender bias.
Publisher: American Chemical Society (ACS)
Date: 21-02-2013
DOI: 10.1021/CN400005T
Publisher: Future Science Ltd
Date: 07-2016
Abstract: The dopamine D 2 receptor (D 2 R) has been implicated in the symptomology of disorders such as schizophrenia and Parkinson's disease. Multivalent ligands provide useful tools to investigate emerging concepts of G protein-coupled receptor drug action such as allostery, bitopic binding and receptor dimerization. This review focuses on the approaches taken toward the development of multivalent ligands for the D 2 R recently and highlights the challenges associated with each approach, their utility in probing D 2 R function and approaches to develop new D 2 R-targeting drugs. Furthermore, we extend our discussion to the possibility of designing multitarget ligands. The insights gained from such studies may provide the basis for improved therapeutic targeting of the D 2 R.
Publisher: Elsevier BV
Date: 04-2019
DOI: 10.1016/J.EJMECH.2019.01.061
Abstract: We recently described a structurally novel series of negative allosteric modulators (NAMs) of the dopamine D
Publisher: Elsevier BV
Date: 04-2019
Publisher: Elsevier BV
Date: 10-2016
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 14-09-2016
Abstract: Current antipsychotics are effective in treating the positive symptoms associated with schizophrenia, but they remain suboptimal in targeting cognitive dysfunction. Recent studies have suggested that positive allosteric modulation of the M
Publisher: American Chemical Society (ACS)
Date: 28-08-2015
DOI: 10.1021/ACS.JMEDCHEM.5B00585
Abstract: Recently, we have demonstrated that N-((trans)-4-(2-(7-cyano-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)cyclohexyl)-1H-indole-2-carboxamide (SB269652) (1) adopts a bitopic pose at one protomer of a dopamine D2 receptor (D2R) dimer to negatively modulate the binding of dopamine at the other protomer. The 1H-indole-2-carboxamide moiety of 1 extends into a secondary pocket between the extracellular ends of TM2 and TM7 within the D2R protomer. To target this putative allosteric site, we generated and characterized fragments that include and extend from the 1H-indole-2-carboxamide moiety of 1. N-Isopropyl-1H-indole-2-carboxamide (3) displayed allosteric pharmacology and sensitivity to mutations of the same residues at the top of TM2 as was observed for 1. Using 3 as an "allosteric lead", we designed and synthesized an extensive fragment library to generate novel SAR and identify N-butyl-1H-indole-2-carboxamide (11d), which displayed both increased negative cooperativity and affinity for the D2R. These data illustrate that fragmentation of extended compounds can expose fragments with purely allosteric pharmacology.
Publisher: Elsevier BV
Date: 05-2011
DOI: 10.1016/J.BMCL.2010.11.091
Abstract: Nicotinic acid (niacin) has been used for decades as an antidyslipidemic drug in man. Its main target is the hydroxy-carboxylic acid receptor HCA2 (GPR109A), a G protein-coupled receptor. Other acids and esters such as methyl fumarate also interact with the receptor, which constituted the basis for the current study. We synthesized a novel series of substituted propenoic acids, such as fumaric acid esters, fumaric acid amides and cinnamic acid derivatives, and determined their affinities for the HCA2 receptor. We observed a rather restricted binding pocket on the receptor with trans-cinnamic acid being the largest planar ligand in our series with appreciable affinity for the receptor. Molecular modeling and analysis of the structure-activity relationships in the series suggest a planar trans-propenoic acid pharmacophore with a maximum length of 8 Å and out-of-plane orientation of the larger substituents.
Publisher: Elsevier BV
Date: 2010
DOI: 10.1016/J.BCP.2009.07.024
Abstract: Studies of endogenous cannabinoid agonists, such as 2-arachidonylglycerol (2-AG), have revealed their potential to exert modulatory actions on other receptor systems in addition to their ability to activate cannabinoid receptors. This study investigated the effect of cannabinoid ligands on the human adenosine A(3) (hA(3)R) receptor. The endocannabinoid 2-AG was able to inhibit agonist ([125I]N(6)-(4-amino-3-iodobenzyl) adenosine-5'-(N-methyluronamide)--[125I] AB MECA) binding at the hA(3)R. This inhibition occurred over a narrow range of ligand concentration and was characterized by high Hill coefficients suggesting a non-competitive interaction. Furthermore, in the presence of 2-AG, the rate of [125I] AB MECA dissociation was increased, consistent with an action as a negative allosteric modulator of the hA(3)R. Moreover, by measuring intracellular cAMP levels, we demonstrate that 2-AG decreases both the potency of an agonist at the hA(3)R and the basal signalling of this receptor. Since the hA(3)R has been shown to be expressed in astrocytes and microglia, these findings may be particularly relevant in certain pathological states such as cerebral ischemia where levels of 2-AG and anandamide are raised.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 21-12-2012
Abstract: The recent publication of both the antagonist- and agonist-bound structures of the adenosine A(2A) receptor have revealed much about how a ligand may bind to a receptor and cause the conformational changes associated with agonist-mediated activation. In particular, the agonist-bound structure revealed key interactions between the ribose group of adenosine-derived agonists and amino acids in the receptor binding pocket that lead to receptor activation. However, agonists without a ribose group also exist, and we wondered whether such compounds occupy the same agonist binding site. Therefore we used a mutagenesis approach in this study to investigate the mode of binding of 2-amino-4-(4-hydroxyphenyl)- 6-(1H-imidazol-2-ylmethylsulfanyl)pyridine-3,5-dicarbonitrile (LUF5834), a potent partial agonist without a ribose moiety, compared with the adenosine-derived reference agonist 2-[p-(2-carboxyethyl)phenyl-ethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680). Mutation of the orthosteric residue Phe168 to alanine abrogated the function of both agonists. However, mutation to alanine of residues Thr88 and Ser277 shown by the crystal structures to interact with the ribose group of adenosine-like ligands had no effect on the potency of LUF5834. Furthermore, alanine mutation of Asn253, which makes a hydrogen-bonding interaction with the exocyclic nitrogen of the adenine ring, had minimal effect on LUF5834 affinity but removed agonist activity of this ligand. Mutation of other residues, such as the highly conserved Trp246 or Glu13, had significant deleterious effects on the function of CGS21680 but little effect on LUF5834. In summary, our findings suggest that this class of agonist interacts with distinct residues to activate the receptor compared with classic adenosine derived agonists.
Publisher: American Chemical Society (ACS)
Date: 10-02-2017
Publisher: American Chemical Society (ACS)
Date: 27-03-2012
DOI: 10.1021/JM300164Q
Abstract: A number of pyrazolopyrimidines were synthesized and tested for their positive allosteric modulation of the HCA(2) receptor (GPR109A). Compound 24, an efficacious and potent agonist and allosteric enhancer of nicotinic acid's action, was the basis for most other compounds. Interestingly, some of the compounds were found to increase the efficacy of the endogenous ligand 3-hydroxybutyrate and enhance its potency almost 10-fold. This suggests that the pyrazolopyrimidines may have therapeutic value when given alone.
Publisher: Elsevier BV
Date: 10-2007
DOI: 10.1016/J.COPH.2007.06.007
Abstract: Single, bi-functional polypeptides consisting of a G-protein-coupled receptor (GPCR) linked directly to a G protein alpha subunit have been employed for a number of years to study many aspects of signal initiation, including the roles of post-translational modifications, effects of mutations in both receptor and G protein and in the de-orphanisation of novel G-protein-coupled receptors. Recently, they have been used to improve signal-to-background in ligand assay screens and to study both agonist-directed signal trafficking and distinct conformational states of receptors. As well as such novel concepts in pharmacology, G-protein-coupled receptor-G protein fusions have recently been employed to examine receptor homo-dimerisation and hetero-dimerisation and are beginning to be used to explore allosteric effects within GPCR hetero-dimers.
Publisher: Wiley
Date: 22-08-2023
DOI: 10.1111/BPH.16199
Abstract: Opioid‐induced respiratory depression limits the use of μ‐opioid receptor agonists in clinical settings and is the main cause of opioid overdose fatalities. The relative potential of different opioid agonists to induce respiratory depression at doses exceeding those producing analgesia is understudied despite its relevance to assessments of opioid safety. Here we evaluated the respiratory depressant and anti‐nociceptive effects of three novel opioids and relate these measurements to their in vitro efficacy. Respiration was measured in awake, freely moving male CD‐1 mice using whole body plethysmography. Anti‐nociception was measured using the hot plate test. Morphine, oliceridine and tianeptine were administered intraperitoneally, whereas methadone, oxycodone and SR‐17018 were administered orally. Receptor activation and arrestin‐3 recruitment were measured in HEK293 cells using BRET assays. Across the dose ranges examined, all opioids studied depressed respiration in a dose‐dependent manner, with similar effects at the highest doses, and with tianeptine and oliceridine showing reduced duration of effect, when compared with morphine, oxycodone, methadone and SR‐17018. When administered at doses that induced similar respiratory depression, all opioids induced similar anti‐nociception, with tianeptine and oliceridine again showing reduced duration of effect. These data were consistent with the in vitro agonist activity of the tested compounds. In addition to providing effective anti‐nociception, the novel opioids, oliceridine, tianeptine and SR‐17018 depress respiration in male mice. However, the different potencies and kinetics of effect between these novel opioids may be relevant to their therapeutic application in different clinical settings.
Publisher: Elsevier BV
Date: 04-2010
Publisher: American Chemical Society (ACS)
Date: 26-03-2010
DOI: 10.1021/JM901252A
Abstract: Many G protein-coupled receptors (GPCRs), including the adenosine A(1) receptor (A(1)AR), have been shown to be allosterically modulated by small molecule ligands. So far, in the absence of structural information, the exact location of the allosteric site on the A(1)AR is not known. We synthesized a series of bivalent ligands (4) with an increasing linker length between the orthosteric and allosteric pharmacophores and used these as tools to search for the allosteric site on the A(1)AR. The compounds were tested in both equilibrium radioligand displacement and functional assays in the absence and presence of a reference allosteric enhancer, (2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluoromethyl)phenyl]methanone, PD81,723 (1). Bivalent ligand N(6)-[2-amino-3-(3,4-dichlorobenzoyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-6-yl-9-nonyloxy-4-phenyl]-adenosine 4h (LUF6258) with a 9 carbon atom spacer did not show significant changes in affinity or potency in the presence of 1, indicating that this ligand bridged both sites on the receptor. Furthermore, 4h displayed an increase in efficacy, but not potency, compared to the parent, monovalent agonist 2. From molecular modeling studies, we speculate that the allosteric site of the A(1)AR is located in the proximity of the orthosteric site, possibly within the boundaries of the second extracellular loop of the receptor.
Publisher: Elsevier BV
Date: 2013
DOI: 10.1016/J.TIPS.2012.10.003
Abstract: Although classical approaches to G-protein-coupled receptor (GPCR) drug design have targeted the orthosteric binding site, potentially all GPCRs possess druggable allosteric sites. In addition, it is clear that GPCRs can adopt multiple active states linked to distinct functional outcomes that can be stabilized by both allosteric and orthosteric ligands. Recent studies have begun to explore the possibilities of linking orthosteric and allosteric pharmacophores to yield 'bitopic' ligands as an approach to achieve improved receptor affinity or selectivity. Furthermore, it is possible that previously identified functionally selective drugs may represent unappreciated bitopic ligands at this important class of drug targets. Here we discuss both the potential of bitopic ligands in GPCR drug discovery and the challenges associated with the design of such ligands.
Publisher: MDPI AG
Date: 22-06-2021
DOI: 10.3390/MOLECULES26133799
Abstract: (1) Background: Two first-in-class racemic dopamine D1 receptor (D1R) positive allosteric modulator (PAM) chemotypes (1 and 2) were identified from a high-throughput screen. In particular, due to its selectivity for the D1R and reported lack of intrinsic activity, compound 2 shows promise as a starting point toward the development of small molecule allosteric modulators to ameliorate the cognitive deficits associated with some neuropsychiatric disease states (2) Methods: Herein, we describe the enantioenrichment of optical isomers of 2 using chiral auxiliaries derived from (R)- and (S)-3-hydroxy-4,4-dimethyldihydrofuran-2(3H)-one (d- and l-pantolactone, respectively) (3) Results: We confirm both the racemate and enantiomers of 2 are active and selective for the D1R, but that the respective stereoisomers show a significant difference in their affinity and magnitude of positive allosteric cooperativity with dopamine (4) Conclusions: These data warrant further investigation of asymmetric syntheses of optically pure analogues of 2 for the development of D1R PAMs with superior allosteric properties.
Publisher: Springer Science and Business Media LLC
Date: 10-06-2010
Abstract: G protein-coupled receptors (GPCRs) represent a family of well-characterized drug targets with significant therapeutic value. Phylogenetic classifications may help to understand the characteristics of in idual GPCRs and their subtypes. Previous phylogenetic classifications were all based on the sequences of receptors, adding only minor information about the ligand binding properties of the receptors. In this work, we compare a sequence-based classification of receptors to a ligand-based classification of the same group of receptors, and evaluate the potential to use sequence relatedness as a predictor for ligand interactions thus aiding the quest for ligands of orphan receptors. We present a classification of GPCRs that is purely based on their ligands, complementing sequence-based phylogenetic classifications of these receptors. Targets were hierarchically classified into phylogenetic trees, for both sequence space and ligand (substructure) space. The overall organization of the sequence-based tree and substructure-based tree was similar in particular, the adenosine receptors cluster together as well as most peptide receptor subtypes ( e.g . opioid, somatostatin) and adrenoceptor subtypes. In ligand space, the prostanoid and cannabinoid receptors are more distant from the other targets, whereas the tachykinin receptors, the oxytocin receptor, and serotonin receptors are closer to the other targets, which is indicative for ligand promiscuity. In 93% of the receptors studied, de-orphanization of a simulated orphan receptor using the ligands of related receptors performed better than random (AUC 0.5) and for 35% of receptors de-orphanization performance was good (AUC 0.7). We constructed a phylogenetic classification of GPCRs that is solely based on the ligands of these receptors. The similarities and differences with traditional sequence-based classifications were investigated: our ligand-based classification uncovers relationships among GPCRs that are not apparent from the sequence-based classification. This will shed light on potential cross-reactivity of GPCR ligands and will aid the design of new ligands with the desired activity profiles. In addition, we linked the ligand-based classification with a ligand-focused sequence-based classification described in literature and proved the potential of this method for de-orphanization of GPCRs.
Publisher: Elsevier BV
Date: 02-2014
Publisher: American Chemical Society (ACS)
Date: 03-03-2016
DOI: 10.1021/ACSCHEMNEURO.6B00018
Abstract: Benzoquinazolinone 1 is a positive allosteric modulator (PAM) of the M1 muscarinic acetylcholine receptor (mAChR), which is significantly more potent than the prototypical PAM, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (BQCA). In this study, we explored the structural determinants that underlie the activity of 1 as a PAM of the M1 mAChR. We paid particular attention to the importance of the tricyclic scaffold of compound 1, for the activity of the molecule. Complete deletion of the peripheral fused benzene ring caused a significant decrease in affinity and binding cooperativity with acetylcholine (ACh). This loss of affinity was rescued with the addition of either one or two methyl groups in the 7- and/or 8-position of the quinazolin-4(3H)-one core. These results demonstrate that the tricyclic benzo[h]quinazolin-4(3H)-one core could be replaced with a quinazolin-4(3H)-one core and maintain functional affinity. As such, the quinazolin-4(3H)-one core represents a novel scaffold to further explore M1 mAChR PAMs with improved physicochemical properties.
Publisher: American Chemical Society (ACS)
Date: 03-10-2019
DOI: 10.1021/ACS.JMEDCHEM.9B00864
Abstract: Haloperidol is a typical antipsychotic drug (APD) associated with an increased risk of extrapyramidal side effects (EPSs) and hyperprolactinemia relative to atypical APDs such as clozapine. Both drugs are dopamine D
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 07-02-2007
Abstract: A range of ligands displayed agonism at the long isoform of the human dopamine D(2) receptor, whether using receptor-G protein fusions or membranes of cells in which pertussis toxin-resistant mutants of in idual Galpha(i)-family G proteins could be expressed in an inducible fashion. Varying degrees of efficacy were observed for in idual ligands as monitored by their capacity to load [(35)S]GTPgammaS onto each of Galpha(i1),Galpha(i2),Galpha(i3), and Galpha(o1). By contrast, (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine was a partial agonist when Galpha(o1) was the target G protein but an antagonist/inverse agonist at Galpha(i1),Galpha(i2), and Galpha(i3). In ligand binding assays, dopamine identified both high- and low-affinity states at each of the dopamine D(2) receptor-G protein fusion proteins, and the high-affinity state was eliminated by guanine nucleotide. (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine bound to an apparent single state of the constructs in which the D(2) receptor was fused to Galpha(i1),Galpha(i2), or Galpha(i3). However, it bound to distinct high- and low-affinity states of the D(2) receptor-Galpha(o1) fusion, with the high-affinity state being eliminated by guanine nucleotide. Likewise, although dopamine identified guanine nucleotide-sensitive high-affinity states of the D(2) receptor when expression of pertussis toxin-resistant forms of each of Galpha(i1), Galpha(i2), Galpha(i3), and Galpha(o1) was induced, (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine identified a high-affinity site only in the presence of Galpha(o1). p-Tyramine displayed a protean ligand profile similar to that of (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine but with lower potency. These results demonstrate (S)-(-)-3-(3-hydroxyphenyl)-N-propylpiperidine to be a protean agonist at the D(2) receptor and may explain in vivo actions of this ligand.
Publisher: Public Library of Science (PLoS)
Date: 16-01-2018
Publisher: Elsevier BV
Date: 2012
Publisher: American Chemical Society (ACS)
Date: 11-06-2018
DOI: 10.1021/ACS.JMEDCHEM.8B00192
Abstract: SB269652 (1) is a negative allosteric modulator of the dopamine D
Publisher: Elsevier BV
Date: 03-2019
Publisher: Springer Science and Business Media LLC
Date: 18-08-2017
Abstract: G-protein-coupled receptors (GPCRs) are one of the most tractable classes of drug targets. These dynamic proteins can adopt multiple active states that are linked to distinct functional outcomes. Such states can be differentially stabilized by ligands interacting with the endogenous agonist-binding orthosteric site and/or by ligands acting via spatially distinct allosteric sites, leading to the phenomena of 'biased agonism' or 'biased modulation'. These paradigms are having a major impact on modern drug discovery, but it is becoming increasingly apparent that 'kinetic context', at the level of both ligand-receptor and receptor-signal pathway kinetics, can have a profound impact on the observation and quantification of these phenomena. The concept of kinetic context thus represents an important new consideration that should be routinely incorporated into contemporary chemical biology and drug discovery studies of GPCR bias and allostery.
Publisher: Springer Science and Business Media LLC
Date: 23-03-2022
DOI: 10.1038/S42003-022-03191-5
Abstract: Heterotrimeric G proteins are the main signalling effectors for G protein-coupled receptors. Understanding the distinct functions of different G proteins is key to understanding how their signalling modulates physiological responses. Pertussis toxin, a bacterial AB 5 toxin, inhibits Gα i/o G proteins and has proven useful for interrogating inhibitory G protein signalling. Pertussis toxin, however, does not inhibit one member of the inhibitory G protein family, Gα z . The role of Gα z signalling has been neglected largely due to a lack of inhibitors. Recently, the identification of another Pertussis-like AB 5 toxin was described. Here we show that this toxin, that we call OZITX, specifically inhibits Gα i/o and Gα z G proteins and that expression of the catalytic S1 subunit is sufficient for this inhibition. We identify mutations that render Gα subunits insensitive to the toxin that, in combination with the toxin, can be used to interrogate the signalling of each inhibitory Gα G protein.
Publisher: MDPI AG
Date: 19-04-2021
DOI: 10.3390/IJMS22084232
Abstract: Chemokines interact with chemokine receptors in a promiscuous network, such that each receptor can be activated by multiple chemokines. Moreover, different chemokines have been reported to preferentially activate different signalling pathways via the same receptor, a phenomenon known as biased agonism. The human CC chemokine receptors (CCRs) CCR4, CCR7 and CCR10 play important roles in T cell trafficking and have been reported to display biased agonism. To systematically characterize these effects, we analysed G protein- and β-arrestin-mediated signal transduction resulting from stimulation of these receptors by each of their cognate chemokine ligands within the same cellular background. Although the chemokines did not elicit ligand-biased agonism, the three receptors exhibited different arrays of signaling outcomes. Stimulation of CCR4 by either CC chemokine ligand 17 (CCL17) or CCL22 induced β-arrestin recruitment but not G protein-mediated signaling, suggesting that CCR4 has the potential to act as a scavenger receptor. At CCR7, both CCL19 and CCL21 stimulated G protein signaling and β-arrestin recruitment, with CCL19 consistently displaying higher potency. At CCR10, CCL27 and CCL28(4-108) stimulated both G protein signaling and β-arrestin recruitment, whereas CCL28(1-108) was inactive, suggesting that CCL28(4-108) is the biologically relevant form of this chemokine. These comparisons emphasize the intrinsic abilities of different receptors to couple with different downstream signaling pathways. Comparison of these results with previous studies indicates that differential agonism at these receptors may be highly dependent on the cellular context.
Publisher: Springer Science and Business Media LLC
Date: 24-02-2016
DOI: 10.1038/NCOMMS10842
Abstract: Biased agonism describes the ability of ligands to stabilize different conformations of a GPCR linked to distinct functional outcomes and offers the prospect of designing pathway-specific drugs that avoid on-target side effects. This mechanism is usually inferred from pharmacological data with the assumption that the confounding influences of observational (that is, assay dependent) and system (that is, cell background dependent) bias are excluded by experimental design and analysis. Here we reveal that ‘kinetic context’, as determined by ligand-binding kinetics and the temporal pattern of receptor-signalling processes, can have a profound influence on the apparent bias of a series of agonists for the dopamine D 2 receptor and can even lead to reversals in the direction of bias. We propose that kinetic context must be acknowledged in the design and interpretation of studies of biased agonism.
Publisher: Elsevier BV
Date: 08-2014
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 24-06-2013
Abstract: Recent interest in the M₁ muscarinic acetylcholine (ACh) receptor (mAChR) has led to the discovery of various selective agonists for the receptor. The novel selective agonist 1-(1'-(2-methylbenzyl)-1,4'-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-1 (TBPB) displays unprecedented functional selectivity at the M₁ mAChR. This functional selectivity has been described to stem from sole interaction with an allosteric site, although the evidence for such a mechanism is equivocal. To delineate TBPB's mechanism of action, several truncated variants of TBPB were synthesized and characterized. Binding experiments with [³H]N-methylscopolamine at the M₁, M₂, M₃, and M₄ mAChRs revealed radioligand displacement in a manner consistent with a competitive binding mode at the orthosteric site by TBPB and fragment derivatives. Cell-based functional assays of fragment derivatives of TBPB identified both agonistic and antagonistic moieties, one of which, 1-(1-cyclohexylpiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-1 (VCP794), lost agonistic selectivity for the M₁ mAChR. Further interaction experiments between TBPB or its antagonist fragments with ACh also indicated a mechanism consistent with competitive binding at mAChRs. However, interaction with an allosteric site by an antagonist fragment of TBPB was demonstrated via its ability to retard radioligand dissociation. To reconcile this dual orthosteric/allosteric pharmacological behavior, we propose that TBPB is a bitopic ligand, interacting with both the orthosteric site and an allosteric site, at the M₁ mAChR. This mechanism may also be the case for other selective agonists for mAChRs, and should be taken into consideration in the profiling and classification of new novel selective agonists for this receptor family.
Publisher: American Chemical Society (ACS)
Date: 22-01-2010
DOI: 10.1021/JM901647P
Publisher: Wiley
Date: 17-11-2015
DOI: 10.1096/FJ.15-277798
Publisher: Springer Science and Business Media LLC
Date: 15-04-2021
DOI: 10.1038/S41598-021-87417-2
Abstract: The dopamine D 2 receptor (D 2 R) is the target of drugs used to treat the symptoms of Parkinson’s disease and schizophrenia. The D 2 R is regulated through its interaction with and phosphorylation by G protein receptor kinases (GRKs) and interaction with arrestins. More recently, D 2 R arrestin-mediated signaling has been shown to have distinct physiological functions to those of G protein signalling. Relatively little is known regarding the patterns of D 2 R phosphorylation that might control these processes. We aimed to generate antibodies specific for intracellular D 2 R phosphorylation sites to facilitate the investigation of these mechanisms. We synthesised double phosphorylated peptides corresponding to regions within intracellular loop 3 of the hD 2 R and used them to raise phosphosite-specific antibodies to capture a broad screen of GRK-mediated phosphorylation. We identify an antibody specific to a GRK2/3 phosphorylation site in intracellular loop 3 of the D 2 R. We compared measurements of D 2 R phosphorylation with other measurements of D 2 R signalling to profile selected D 2 R agonists including previously described biased agonists. These studies demonstrate the utility of novel phosphosite-specific antibodies to investigate D 2 R regulation and signalling.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 10-09-2013
Publisher: American Chemical Society (ACS)
Date: 20-05-2021
Publisher: American Chemical Society (ACS)
Date: 31-12-2015
DOI: 10.1021/ACS.JMEDCHEM.5B01562
Abstract: Positive allosteric modulators (PAMs) of the M1 muscarinic acetylcholine receptor (M1 mAChR) are a promising strategy for the treatment of the cognitive deficits associated with diseases including Alzheimer's and schizophrenia. Herein, we report the design, synthesis, and characterization of a novel family of M1 mAChR PAMs. The most active compounds of the 4-phenylpyridin-2-one series exhibited comparable binding affinity to the reference compound, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (BQCA) (1), but markedly improved positive cooperativity with acetylcholine, and retained exquisite selectivity for the M1 mAChR. Furthermore, our pharmacological characterization revealed ligands with a erse range of activities, including modulators that displayed both high intrinsic efficacy and PAM activity, those that showed no detectable agonism but robust PAM activity and ligands that displayed robust allosteric agonism but little modulatory activity. Thus, the 4-phenylpyridin-2-one scaffold offers an attractive starting point for further lead optimization.
Publisher: American Chemical Society (ACS)
Date: 24-07-2019
Publisher: Elsevier BV
Date: 06-2013
Publisher: Elsevier BV
Date: 10-2010
DOI: 10.1016/J.BCP.2010.06.041
Abstract: The adenosine A(1) receptor is a promising therapeutic target for neurological disorders such as cognition deficits and is involved in cardiovascular preconditioning. Classically adenosine receptor agonists were all derivatives of adenosine, and thought to require a D-ribose moiety. More recently, however, the discovery of non-adenosine agonists for the human adenosine A(1) receptor (hA(1)R) has challenged this dogma (Beukers et al., 2004). In this study we characterize the tritiated form of one of these compounds, [(3)H]LUF5834, as the first non-ribose partial agonist radioligand with nanomolar affinity for the hA(1)R. Due to its partial agonist efficacy, [(3)H]LUF5834 labeled both G protein-coupled and uncoupled receptors with a similar high affinity. Using [(3)H]LUF5834 we performed competition binding experiments to characterize a range of A(1)R ligands varying in efficacy from the full agonist CPA to the inverse agonist DPCPX. Surprisingly, in the control condition both agonists and inverse agonists displayed biphasic isotherms. With the addition of 1mM GTP the high affinity isotherm of agonists or the low affinity isotherm of inverse agonists was lost revealing the mechanism of action of such inverse agonists at the A(1)R. Consequently, [(3)H]LUF5834 represents a novel high affinity radioligand for the A(1)R and may prove a useful tool to provide estimates of inverse agonist efficacy at this receptor.
Publisher: Springer Science and Business Media LLC
Date: 19-01-2018
DOI: 10.1038/S41598-018-19642-1
Abstract: Sodium ions (Na + ) allosterically modulate the binding of orthosteric agonists and antagonists to many class A G protein-coupled receptors, including the dopamine D 2 receptor (D 2 R). Experimental and computational evidences have revealed that this effect is mediated by the binding of Na + to a conserved site located beneath the orthosteric binding site (OBS). SB269652 acts as a negative allosteric modulator (NAM) of the D 2 R that adopts an extended bitopic pose, in which the tetrahydroisoquinoline moiety interacts with the OBS and the indole-2-carboxamide moiety occupies a secondary binding pocket (SBP). In this study, we find that the presence of a Na + within the conserved Na + -binding pocket is required for the action of SB269652. Using fragments of SB269652 and novel full-length analogues, we show that Na + is required for the high affinity binding of the tetrahydroisoquinoline moiety within the OBS, and that the interaction of the indole-2-carboxamide moiety with the SBP determines the degree of Na + -sensitivity. Thus, we extend our understanding of the mode of action of this novel class of NAM by showing it acts synergistically with Na + to modulate the binding of orthosteric ligands at the D 2 R, providing opportunities for fine-tuning of modulatory effects in future allosteric drug design efforts.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 31-03-2020
DOI: 10.1126/SCISIGNAL.AAZ3140
Abstract: Low intrinsic efficacy can explain the reduced side effects of apparently biased μ-opioid receptor agonists.
Publisher: MDPI AG
Date: 15-05-2019
DOI: 10.3390/IJMS20102417
Abstract: Leukocyte migration, a hallmark of the inflammatory response, is stimulated by the interactions between chemokines, which are expressed in injured or infected tissues, and chemokine receptors, which are G protein-coupled receptors (GPCRs) expressed in the leukocyte plasma membrane. One mechanism for the regulation of chemokine receptor signaling is biased agonism, the ability of different chemokine ligands to preferentially activate different intracellular signaling pathways via the same receptor. To identify features of chemokines that give rise to biased agonism, we studied the activation of the receptor CCR1 by the chemokines CCL7, CCL8, and CCL15(Δ26). We found that, compared to CCL15(Δ26), CCL7 and CCL8 exhibited biased agonism towards cAMP inhibition and away from β-Arrestin 2 recruitment. Moreover, N-terminal substitution of the CCL15(Δ26) N-terminus with that of CCL7 resulted in a chimera with similar biased agonism to CCL7. Similarly, N-terminal truncation of CCL15(Δ26) also resulted in signaling bias between cAMP inhibition and β-Arrestin 2 recruitment signals. These results show that the interactions of the chemokine N-terminal region with the receptor transmembrane region play a key role in selecting receptor conformations coupled to specific signaling pathways.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 26-05-2015
Abstract: Biased agonism is having a major impact on modern drug discovery, and describes the ability of distinct G protein-coupled receptor (GPCR) ligands to activate different cell signaling pathways, and to result in different physiologic outcomes. To date, most studies of biased agonism have focused on synthetic molecules targeting various GPCRs however, many of these receptors have multiple endogenous ligands, suggesting that "natural" bias may be an unappreciated feature of these GPCRs. The μ-opioid receptor (MOP) is activated by numerous endogenous opioid peptides, remains an attractive therapeutic target for the treatment of pain, and exhibits biased agonism in response to synthetic opiates. The aim of this study was to rigorously assess the potential for biased agonism in the actions of endogenous opioids at the MOP in a common cellular background, and compare these to the effects of the agonist d-Ala2-N-MePhe4-Gly-ol enkephalin (DAMGO). We investigated activation of G proteins, inhibition of cAMP production, extracellular signal-regulated kinase 1 and 2 phosphorylation, β-arrestin 1/2 recruitment, and MOP trafficking, and applied a novel analytical method to quantify biased agonism. Although many endogenous opioids displayed signaling profiles similar to that of DAMGO, α-neoendorphin, Met-enkephalin-Arg-Phe, and the putatively endogenous peptide endomorphin-1 displayed particularly distinct bias profiles. These may represent ex les of natural bias if it can be shown that they have different signaling properties and physiologic effects in vivo compared with other endogenous opioids. Understanding how endogenous opioids control physiologic processes through biased agonism can reveal vital information required to enable the design of biased opioids with improved pharmacological profiles and treat diseases involving dysfunction of the endogenous opioid system.
Publisher: Springer Science and Business Media LLC
Date: 10-08-2014
Publisher: Wiley
Date: 09-03-2012
Publisher: American Chemical Society (ACS)
Date: 23-04-2018
DOI: 10.1021/ACS.JMEDCHEM.7B01565
Abstract: Recently, a novel negative allosteric modulator (NAM) of the D
Publisher: American Chemical Society (ACS)
Date: 30-01-2015
DOI: 10.1021/JM5013243
Abstract: Herein we describe the hybridization of a benzoxazinone M1 scaffold with D2 privileged structures derived from putative and clinically relevant antipsychotics to develop designed multiple ligands. The M1 mAChR is an attractive target for the cognitive deficits in key CNS disorders. Moreover, activity at D2 and 5-HT2A receptors has proven useful for antipsychotic efficacy. We identified 9 which retained functional activity at the target M1 mAChR and D2R and demonstrated high affinity for the 5-HT2AR.
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 05-08-2021
Publisher: Springer Science and Business Media LLC
Date: 25-11-2015
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 24-01-2008
Abstract: The human dopamine D(2L) receptor couples promiscuously to multiple members of the Galpha(i/o) subfamily. Despite the high homology of the D(2L) and D(3) receptors, the G protein coupling specificity of the human D(3) receptor is less clearly characterized. The primary aim of this study, then, was the parallel characterization of the G protein coupling specificity of the D(2L) and D(3) receptors. By using both receptor-G protein fusion proteins and stable cell lines in which pertussis toxin-resistant mutants of in idual Galpha(i)-family G proteins were expressed in an inducible fashion, we demonstrated highly selective coupling of the D(3) receptor to Galpha(o1). Furthermore, by using the fusion proteins to ensure identical stoichiometry of receptor to G protein for each pairing, a range of ligands displayed higher potency and, for partial agonists, higher efficacy at the D(3) receptor when coupled to Galpha(o1) compared with the D(2L) receptor. The second aim of this study was to investigate the molecular basis of the above differential G protein coupling specificity. The importance of a 12-amino acid sequence from the C-terminal end of the third intracellular loop of the D(2L) receptor in providing promiscuity in G protein coupling was demonstrated using a chimeric D(3)/D(2) receptor in which the equivalent region of the D(3) receptor was exchanged for this sequence. This chimera displayed D(3)-like affinity for [(3)H]spiperone and potency for agonists but gained D(2)-like ability to couple to each of Galpha(i1-3) as well as Galpha(o1).
Publisher: Springer Science and Business Media LLC
Date: 03-09-2018
Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)
Date: 08-2018
Publisher: Annual Reviews
Date: 10-02-2012
DOI: 10.1146/ANNUREV-PHARMTOX-010611-134514
Abstract: It is now acknowledged that G protein–coupled receptors, the largest class of drug targets, adopt multiple active states that can be preferentially stabilized by orthosteric ligands or allosteric modulators, thus giving rise to the phenomenon of pathway-biased signaling. In the past few years, researchers have begun to explore the potential of linking orthosteric and allosteric pharmacophores to yield bitopic hybrid ligands. This approach is an extension of the more traditional bivalent ligand concept and shares some of the same challenges, including the choice and role of the linker between the two pharmacophores and the validation of mechanism of action. Nonetheless, the promise of bitopic ligands is the generation of novel chemical tools that have improved affinity and/or selectivity profiles. Previously identified functionally selective compounds (and medicines) also may act via a bitopic mechanism, suggesting that the phenomenon is more widespread than currently appreciated.
Publisher: Springer Netherlands
Date: 06-11-2009
Publisher: Wiley
Date: 2006
DOI: 10.1002/BIT.20751
Abstract: A pilot scale whole cell process was developed for the enantioselective 1,2-reduction of prochiral alpha,beta-unsaturated ketone to (R) allylic alcohol using Candida chilensis. Initial development showed high enantiomeric excess (EE > 95%) but low product yield (10%). Process development, using a combination of statistically designed screening and optimization experiments, improved the desired alcohol yield to 90%. The fermentation growth stage, particularly medium composition and growth pH, had a significant impact on the bioconversion while process characterization identified erse challenges including the presence of multiple enzymes, substrate roduct toxicity, and biphasic cellular morphology. Manipulating the fermentation media allowed control of the whole cell morphology to a predominantly unicellular broth, away from the viscous pseudohyphae, which were detrimental to the bioconversion. The activity of a competing enzyme, which produced the undesired saturated ketone and (R) saturated alcohol, was minimized to 95%) and overall yield greater than 80%. This was the preferred route compared to a partially purified process using ultra centrifugation, which led to improved volumetric productivity but reduced yield (g/day). The whole cell approach proved to be a valuable alternative to chemical reduction routes, as an intermediate step for the asymmetric synthesis of an integrin receptor antagonist for the inhibition of bone resorption and treatment of osteoporosis.
Publisher: eLife Sciences Publications, Ltd
Date: 27-01-2020
DOI: 10.7554/ELIFE.52189
Abstract: By analyzing and simulating inactive conformations of the highly homologous dopamine D2 and D3 receptors (D2R and D3R), we find that eticlopride binds D2R in a pose very similar to that in the D3R/eticlopride structure but incompatible with the D2R/risperidone structure. In addition, risperidone occupies a sub-pocket near the Na+ binding site, whereas eticlopride does not. Based on these findings and our experimental results, we propose that the ergent receptor conformations stabilized by Na+-sensitive eticlopride and Na+-insensitive risperidone correspond to different degrees of inverse agonism. Moreover, our simulations reveal that the extracellular loops are highly dynamic, with spontaneous transitions of extracellular loop 2 from the helical conformation in the D2R/risperidone structure to an extended conformation similar to that in the D3R/eticlopride structure. Our results reveal previously unappreciated ersity and dynamics in the inactive conformations of D2R. These findings are critical for rational drug discovery, as limiting a virtual screen to a single conformation will miss relevant ligands.
Publisher: American Chemical Society (ACS)
Date: 24-09-2020
Publisher: Elsevier BV
Date: 08-2016
DOI: 10.1016/J.BCP.2016.05.014
Abstract: Biased agonism describes the ability of distinct G protein-coupled receptor (GPCR) ligands to stabilise distinct receptor conformations leading to the activation of different cell signalling pathways that can deliver different physiologic outcomes. This phenomenon is having a major impact on modern drug discovery as it offers the potential to design ligands that selectively activate or inhibit the signalling pathways linked to therapeutic effects with minimal activation or blockade of signalling pathways that are linked to the development of adverse on-target effects. However, the explosion in studies of biased agonism at multiple GPCR families in recombinant cell lines has revealed a high degree of variability on descriptions of biased ligands at the same GPCR and raised the question of whether biased agonism is a fixed attribute of a ligand in all cell types. The current study addresses this question at the mu-opioid receptor (MOP). Here, we have systematically assessed the impact of differential cellular protein complement (and cellular background), signalling kinetics and receptor species on our previous descriptions of biased agonism at MOP by several opioid peptides and synthetic opioids. Our results show that all these factors need to be carefully determined and reported when considering biased agonism. Nevertheless, our studies also show that, despite changes in overall signalling profiles, ligands that previously showed distinct bias profiles at MOP retained their uniqueness across different cell backgrounds.
Publisher: American Chemical Society (ACS)
Date: 20-01-2023
Publisher: American Chemical Society (ACS)
Date: 02-02-2012
DOI: 10.1021/JM201420S
Abstract: To date all typical and atypical antipsychotics target the dopamine D(2) receptor. Clozapine represents the best-characterized atypical antipsychotic, although it displays only moderate (submicromolar) affinity for the dopamine D(2) receptor. Herein, we present the design, synthesis, and pharmacological evaluation of three series of homobivalent ligands of clozapine, differing in the length and nature of the spacer and the point of attachment to the pharmacophore. Attachment of the spacer at the N4' position of clozapine yielded a series of homobivalent ligands that displayed spacer-length-dependent gains in affinity and activity for the dopamine D(2) receptor. The 16 and 18 atom spacer bivalent ligands were the highlight compounds, displaying marked low nanomolar receptor binding affinity (1.41 and 1.35 nM, respectively) and functional activity (23 and 44 nM), which correspond to significant gains in affinity (75- and 79-fold) and activity (9- and 5-fold) relative to the original pharmacophore, clozapine. As such these ligands represent useful tools with which to investigate dopamine receptor dimerization and the atypical nature of clozapine.
Publisher: Springer Science and Business Media LLC
Date: 02-10-2017
DOI: 10.1038/S41467-017-00716-Z
Abstract: Atypical antipsychotic drugs (APDs) have been hypothesized to show reduced extrapyramidal side effects (EPS) due to their rapid dissociation from the dopamine D 2 receptor. However, support for this hypothesis is limited to a relatively small number of observations made across several decades and under different experimental conditions. Here we show that association rates, but not dissociation rates, correlate with EPS. We measured the kinetic binding properties of a series of typical and atypical APDs in a novel time-resolved fluorescence resonance energy transfer assay, and correlated these properties with their EPS and prolactin-elevating liabilities at therapeutic doses. EPS are robustly predicted by a rebinding model that considers the microenvironment of postsynaptic D 2 receptors and integrates association and dissociation rates to calculate the net rate of reversal of receptor blockade. Thus, optimizing binding kinetics at the D 2 receptor may result in APDs with improved therapeutic profile.
Publisher: American Chemical Society (ACS)
Date: 23-05-2012
DOI: 10.1021/JM300280E
Abstract: We present the systematic prospective evaluation of a protein-based and a ligand-based virtual screening platform against a set of three G-protein-coupled receptors (GPCRs): the β-2 adrenoreceptor (ADRB2), the adenosine A(2A) receptor (AA2AR), and the sphingosine 1-phosphate receptor (S1PR1). Novel bioactive compounds were identified using a consensus scoring procedure combining ligand-based (frequent substructure ranking) and structure-based (Snooker) tools, and all 900 selected compounds were screened against all three receptors. A striking number of ligands showed affinity/activity for GPCRs other than the intended target, which could be partly attributed to the fuzziness and overlap of protein-based pharmacophore models. Surprisingly, the phosphodiesterase 5 (PDE5) inhibitor sildenafil was found to possess submicromolar affinity for AA2AR. Overall, this is one of the first published prospective chemogenomics studies that demonstrate the identification of novel cross-pharmacology between unrelated protein targets. The lessons learned from this study can be used to guide future virtual ligand design efforts.
Publisher: MDPI AG
Date: 02-11-2020
DOI: 10.3390/CELLS9112400
Abstract: Understanding the link between agonist-induced phosphorylation of the mu-opioid receptor (MOR) and the associated physiological effects is critical for the development of novel analgesic drugs and is particularly important for understanding the mechanisms responsible for opioid-induced tolerance and addiction. The family of G protein receptor kinases (GRKs) play a pivotal role in such processes, mediating phosphorylation of residues at the C-tail of opioid receptors. Numerous strategies, such as phosphosite specific antibodies and mass spectrometry have allowed the detection of phosphorylated residues and the use of mutant knock-in mice have shed light on the role of GRK regulation in opioid receptor physiology. Here we review our current understanding on the role of GRKs in the actions of opioid receptors, with a particular focus on the MOR, the target of most commonly used opioid analgesics such as morphine or fentanyl.
Publisher: American Chemical Society (ACS)
Date: 30-12-2022
Publisher: Cold Spring Harbor Laboratory
Date: 17-05-2019
DOI: 10.1101/640870
Abstract: Understanding how crystal structures reflect the range of possible G protein-coupled receptor (GPCR) states is critical for rational drug discovery (RDD). Combining computational simulations with mutagenesis and binding studies, we find that the structure of the dopamine D2 receptor (D 2 R)/risperidone complex captures an inactive receptor conformation that accommodates some but not all antagonist scaffolds. Indeed, we find that eticlopride binds D 2 R in a configuration very similar to that seen in the D 3 R structure, in a pose that is incompatible with the D 2 R/risperidone structure. Moreover, our simulations reveal that extracellular loops 1 and 2 (EL1 and EL2) are highly dynamic, with spontaneous transitions of EL2 from the helical conformation in the D 2 R/risperidone structure to an extended conformation similar to that in the D 3 R/eticlopride structure. Our results highlight previously unappreciated conformational ersity and dynamics in the inactive state of a GPCR with potential functional implications. These findings are also of paramount importance for RDD as limiting a virtual screen to one state will miss relevant ligands.
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C5MD00334B
Abstract: Positive allosteric modulators targeting the M 4 muscarinic acetylcholine receptor offer greater sub-type selectivity and unique potential as CNS agents through their novel mode of action to traditional orthosteric ligands.
Publisher: American Chemical Society (ACS)
Date: 19-07-2023
Publisher: American Association for the Advancement of Science (AAAS)
Date: 20-11-2012
DOI: 10.1126/SCISIGNAL.2003503
Abstract: Fluorescent probes reveal the roles of domain rearrangement and dimerization in GPCR activation.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 21-11-2008
Abstract: The adenosine class of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A 2A adenosine receptor, in complex with a high-affinity subtype-selective antagonist, ZM241385, to 2.6 angstrom resolution. Four disulfide bridges in the extracellular domain, combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures, define a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extended conformation, perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core, in ligand recognition by this class of GPCRs and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 16-02-2021
DOI: 10.1126/SCISIGNAL.ABF9803
Abstract: Incorrect data entries in tables in the paper and Supplementary Materials have been corrected.
Publisher: Elsevier
Date: 2011
Publisher: American Association for the Advancement of Science (AAAS)
Date: 23-05-2017
DOI: 10.1126/SCISIGNAL.AAI8529
Abstract: Chemokines and their receptors collectively orchestrate the trafficking of leukocytes in normal immune function and inflammatory diseases. Different chemokines can induce distinct responses at the same receptor. In comparison to monocyte chemoattractant protein-1 (MCP-1 also known as CCL2), the chemokines MCP-2 (CCL8) and MCP-3 (CCL7) are partial agonists of their shared receptor CCR2, a key regulator of the trafficking of monocytes and macrophages that contribute to the pathology of atherosclerosis, obesity, and type 2 diabetes. Through experiments with chimeras of MCP-1 and MCP-3, we identified the chemokine amino-terminal region as being the primary determinant of both the binding and signaling selectivity of these two chemokines at CCR2. Analysis of CCR2 mutants showed that the chemokine amino terminus interacts with the major subpocket in the transmembrane helical bundle of CCR2, which is distinct from the interactions of some other chemokines with the minor subpockets of their receptors. These results suggest the major subpocket as a target for the development of small-molecule inhibitors of CCR2.
Publisher: Springer Science and Business Media LLC
Date: 17-08-2018
Publisher: IOP Publishing
Date: 11-04-2017
Publisher: Wiley
Date: 16-01-2008
DOI: 10.1096/FJ.07-100388
Abstract: Production of antisera able to recognize in idual heterotrimeric G protein alpha subunits resulted in rapid expansion of information on their distribution and function. However, no antibodies that specifically recognize the active state have been available. Four-way primary screening of 763 hybridomas generated from mice immunized with guanosine 5'-O-(3-thio)triphosphate-loaded G alpha(i1) and isolated using an automated robotic colony picker identified three antibodies that interacted with the constitutively active, Q(204)L, mutant but neither the constitutively inactive, G(203)A, mutant nor wild-type G alpha(i1). This profile extended to other closely related G(i) family G proteins but not to the less closely related G alpha(s) and G alpha(q)/G alpha(11) families. Each antibody was, however, also able to identify wild-type, GDP-bound G(i) family G proteins in the presence of fluoroaluminate, which mimics the presence of the terminal phosphate of GTP and hence generates an active/transition state conformation. Stimulation of cells coexpressing a wild-type G alpha(i) subunit and the dopamine D2 receptor with the agonist ligand nor-apomorphine also allowed these conformationally selective antibodies to bind the G protein. Such reagents allow the specific identification of activated G proteins in a native environment and may allow the development of label-free screening assays for G protein-coupled receptor-mediated activation of G(i) family G proteins.
Publisher: Elsevier BV
Date: 11-2014
Publisher: Springer Science and Business Media LLC
Date: 13-10-2013
DOI: 10.1038/NATURE12595
Abstract: The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural ersity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15 Å from the classical, 'orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.
Publisher: Wiley
Date: 30-03-2022
DOI: 10.1111/BPH.15832
Abstract: Mitragynine, the major alkaloid in Mitragyna speciosa (kratom), is a partial agonist at the μ opioid receptor. CYP3A‐dependent oxidation of mitragynine yields the metabolite 7‐OH mitragynine, a more efficacious μ receptor agonist. While both mitragynine and 7‐OH mitragynine can induce anti‐nociception in mice, recent evidence suggests that 7‐OH mitragynine formed as a metabolite is sufficient to explain the anti‐nociceptive effects of mitragynine. However, the ability of 7‐OH mitragynine to induce μ receptor‐dependent respiratory depression has not yet been studied. Respiration was measured in awake, freely moving, male CD‐1 mice, using whole body plethysmography. Anti‐nociception was measured using the hot plate assay. Morphine, mitragynine, 7‐OH mitragynine and the CYP3A inhibitor ketoconazole were administered orally. The respiratory depressant effects of mitragynine showed a ceiling effect, whereby doses higher than 10 mg·kg −1 produced the same level of effect. In contrast, 7‐OH mitragynine induced a dose‐dependent effect on mouse respiration. At equi‐depressant doses, both mitragynine and 7‐OH mitragynine induced prolonged anti‐nociception. Inhibition of CYP3A reduced mitragynine‐induced respiratory depression and anti‐nociception without affecting the effects of 7‐OH mitragynine. Both the anti‐nociceptive effects and the respiratory depressant effects of mitragynine are partly due to its metabolic conversion to 7‐OH mitragynine. The limiting rate of conversion of mitragynine into its active metabolite results in a built‐in ceiling effect of the mitragynine‐induced respiratory depression. These data suggest that such ‘metabolic saturation’ at high doses may underlie the improved safety profile of mitragynine as an opioid analgesic.
Publisher: Elsevier BV
Date: 02-2018
Publisher: Cold Spring Harbor Laboratory
Date: 16-11-2022
DOI: 10.1101/2022.11.14.516475
Abstract: We have developed and characterized a novel D2R antagonist with exceptional GPCR selectivity – ML321. In functional profiling screens of 168 different GPCRs, ML321 showed little activity beyond potent inhibition of the D2R, and to a lesser extent the D3R, demonstrating excellent receptor selectivity. The D2R selectivity of ML321 may be related to the fact that, unlike other monoaminergic ligands, ML321 lacks a positively charged amine group and adopts a unique binding pose within the orthosteric binding site of the D2R. PET imaging studies in non-human primates demonstrated that ML321 penetrates the CNS and occupies the D2R in a dose-dependent manner. Behavioral paradigms in rats demonstrate that ML321 can selectively antagonize a D2R-mediated response (hypothermia) while not affecting a D3R-mediated response (yawning) using the same dose of drug, thus indicating exceptional in vivo selectivity. We also investigated the effects of ML321 in animal models that are predictive of antipsychotic efficacy in humans. We found that ML321 attenuates both hetamine- and phencyclidine-induced locomotor activity and restored pre-pulse inhibition (PPI) of acoustic startle in a dose-dependent manner. Surprisingly, using doses that were maximally effective in both the locomotor and PPI studies, ML321 was relatively ineffective in promoting catalepsy. Kinetic studies revealed that ML321 exhibits slow-on and fast-off receptor binding rates, similar to those observed with atypical antipsychotics with reduced extrapyramidal side effects. Taken together, these observations suggest that ML321, or a derivative thereof, may exhibit “atypical” antipsychotic activity in humans with significantly fewer side effects than observed with currently FDA-approved D2R antagonists.
Location: United States of America
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
Start Date: 2020
End Date: 2023
Funder: Biotechnology and Biological Sciences Research Council
View Funded ActivityStart Date: 2013
End Date: 2017
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2013
End Date: 2016
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2011
End Date: 2014
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2017
End Date: 2019
Funder: Columbia University
View Funded ActivityStart Date: 2016
End Date: 2019
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2013
End Date: End date not available
Funder: National Institutes of Health
View Funded ActivityStart Date: 2010
End Date: 2013
Funder: Nederlandse Organisatie voor Wetenschappelijk Onderzoek
View Funded ActivityStart Date: 2011
End Date: 2015
Funder: Monash University
View Funded ActivityStart Date: 2020
End Date: 2023
Funder: Biotechnology and Biological Sciences Research Council
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