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1.
Cell ; 172(4): 719-730.e14, 2018 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-29398112

RESUMO

Drugs frequently require interactions with multiple targets-via a process known as polypharmacology-to achieve their therapeutic actions. Currently, drugs targeting several serotonin receptors, including the 5-HT2C receptor, are useful for treating obesity, drug abuse, and schizophrenia. The competing challenges of developing selective 5-HT2C receptor ligands or creating drugs with a defined polypharmacological profile, especially aimed at G protein-coupled receptors (GPCRs), remain extremely difficult. Here, we solved two structures of the 5-HT2C receptor in complex with the highly promiscuous agonist ergotamine and the 5-HT2A-C receptor-selective inverse agonist ritanserin at resolutions of 3.0 Å and 2.7 Å, respectively. We analyzed their respective binding poses to provide mechanistic insights into their receptor recognition and opposing pharmacological actions. This study investigates the structural basis of polypharmacology at canonical GPCRs and illustrates how understanding characteristic patterns of ligand-receptor interaction and activation may ultimately facilitate drug design at multiple GPCRs.


Assuntos
Ergotamina/química , Receptor 5-HT2C de Serotonina/química , Ritanserina/química , Agonistas do Receptor 5-HT2 de Serotonina/química , Antagonistas do Receptor 5-HT2 de Serotonina/química , Células HEK293 , Humanos , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Domínios Proteicos , Receptor 5-HT2C de Serotonina/metabolismo , Esquizofrenia/tratamento farmacológico , Esquizofrenia/metabolismo , Relação Estrutura-Atividade , Transtornos Relacionados ao Uso de Substâncias/tratamento farmacológico , Transtornos Relacionados ao Uso de Substâncias/metabolismo
2.
Mol Cell ; 82(14): 2681-2695.e6, 2022 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-35714614

RESUMO

Serotonin (or 5-hydroxytryptamine, 5-HT) is an important neurotransmitter that activates 12 different G protein-coupled receptors (GPCRs) through selective coupling of Gs, Gi, or Gq proteins. The structural basis for G protein subtype selectivity by these GPCRs remains elusive. Here, we report the structures of the serotonin receptors 5-HT4, 5-HT6, and 5-HT7 with Gs, and 5-HT4 with Gi1. The structures reveal that transmembrane helices TM5 and TM6 alternate lengths as a macro-switch to determine receptor's selectivity for Gs and Gi, respectively. We find that the macro-switch by the TM5-TM6 length is shared by class A GPCR-G protein structures. Furthermore, we discover specific residues within TM5 and TM6 that function as micro-switches to form specific interactions with Gs or Gi. Together, these results present a common mechanism of Gs versus Gi protein coupling selectivity or promiscuity by class A GPCRs and extend the basis of ligand recognition at serotonin receptors.


Assuntos
Receptores Acoplados a Proteínas G , Serotonina , Proteínas de Ligação ao GTP/metabolismo , Ligantes , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo
3.
Nature ; 592(7854): 469-473, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33762731

RESUMO

Serotonin, or 5-hydroxytryptamine (5-HT), is an important neurotransmitter1,2 that activates the largest subtype family of G-protein-coupled receptors3. Drugs that target 5-HT1A, 5-HT1D, 5-HT1E and other 5-HT receptors are used to treat numerous disorders4. 5-HT receptors have high levels of basal activity and are subject to regulation by lipids, but the structural basis for the lipid regulation and basal activation of these receptors and the pan-agonism of 5-HT remains unclear. Here we report five structures of 5-HT receptor-G-protein complexes: 5-HT1A in the apo state, bound to 5-HT or bound to the antipsychotic drug aripiprazole; 5-HT1D bound to 5-HT; and 5-HT1E in complex with a 5-HT1E- and 5-HT1F-selective agonist, BRL-54443. Notably, the phospholipid phosphatidylinositol 4-phosphate is present at the G-protein-5-HT1A interface, and is able to increase 5-HT1A-mediated G-protein activity. The receptor transmembrane domain is surrounded by cholesterol molecules-particularly in the case of 5-HT1A, in which cholesterol molecules are directly involved in shaping the ligand-binding pocket that determines the specificity for aripiprazol. Within the ligand-binding pocket of apo-5-HT1A are structured water molecules that mimic 5-HT to activate the receptor. Together, our results address a long-standing question of how lipids and water molecules regulate G-protein-coupled receptors, reveal how 5-HT acts as a pan-agonist, and identify the determinants of drug recognition in 5-HT receptors.


Assuntos
Microscopia Crioeletrônica , Ligantes , Lipídeos , Receptores 5-HT1 de Serotonina/metabolismo , Receptores 5-HT1 de Serotonina/ultraestrutura , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Aripiprazol/metabolismo , Aripiprazol/farmacologia , Sítios de Ligação , Colesterol/farmacologia , Proteínas Heterotriméricas de Ligação ao GTP/química , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/ultraestrutura , Humanos , Modelos Moleculares , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatos de Fosfatidilinositol/farmacologia , Receptor 5-HT1A de Serotonina/química , Receptor 5-HT1A de Serotonina/metabolismo , Receptor 5-HT1A de Serotonina/ultraestrutura , Receptores 5-HT1 de Serotonina/química , Agonistas do Receptor 5-HT1 de Serotonina/química , Agonistas do Receptor 5-HT1 de Serotonina/metabolismo , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , Água/química
4.
Mol Pharmacol ; 100(1): 46-56, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33990405

RESUMO

Delta selective compound 2 (DS2; 4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridin-3-yl]benzamide) is one of the most widely used tools to study selective actions mediated by δ-subunit-containing GABAA receptors. DS2 was discovered over 10 years ago, but despite great efforts, the precise molecular site of action has remained elusive. Using a combination of computational modeling, site-directed mutagenesis, and cell-based pharmacological assays, we probed three potential binding sites for DS2 and analogs at α 4 ß 1 δ receptors: an α 4 (+) δ (-) interface site in the extracellular domain (ECD), equivalent to the diazepam binding site in αßγ 2 receptors, and two sites in the transmembrane domain (TMD) - one in the α 4 (+) ß 1 (-) and one in the α 4 (-) ß 1 (+) interface, with the α 4 (-) ß 1 (+) site corresponding to the binding site for etomidate and a recently disclosed low-affinity binding site for diazepam. We show that mutations in the ECD site did not abrogate DS2 modulation. However, mutations in the TMD α 4 (+) ß 1 (-) interface, either α 4(S303L) of the α 4 (+) side or ß 1(I289Q) of the ß 1 (-) side, convincingly disrupted the positive allosteric modulation by DS2. This was consistently demonstrated both in an assay measuring membrane potential changes and by whole-cell patch-clamp electrophysiology and rationalized by docking studies. Importantly, general sensitivity to modulators was not compromised in the mutated receptors. This study sheds important light on the long-sought molecular recognition site for DS2, refutes the misconception that the selectivity of DS2 for δ-containing receptors is caused by a direct interaction with the δ-subunit, and instead points toward a functional selectivity of DS2 and its analogs via a surprisingly well conserved binding pocket in the TMD. SIGNIFICANCE STATEMENT: δ-Containing GABAA receptors represent potential drug targets for the treatment of several neurological conditions with aberrant tonic inhibition, yet no drugs are currently in clinical use. With the identification of the molecular determinants responsible for positive modulation by the known compound delta selective compound 2, the ground is laid for design of ligands that selectively target δ-containing GABAA receptor subtypes, for better understanding of tonic inhibition, and ultimately, for rational development of novel drugs.


Assuntos
Benzamidas/farmacologia , Imidazóis/farmacologia , Mutagênese Sítio-Dirigida/métodos , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Regulação Alostérica , Benzamidas/química , Sítios de Ligação , Diazepam/farmacologia , Etomidato/farmacologia , Células HEK293 , Humanos , Imidazóis/química , Modelos Moleculares , Conformação Molecular , Simulação de Acoplamento Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Domínios Proteicos , Receptores de GABA-A/genética
5.
J Biol Chem ; 295(40): 13850-13861, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-32753482

RESUMO

Heterotrimeric G proteins are essential mediators of intracellular signaling of G protein-coupled receptors. The Gq/11 subfamily consists of Gq, G11, G14, and G16 proteins, of which all but G16 are inhibited by the structurally related natural products YM-254890 and FR900359. These inhibitors act by preventing the GDP/GTP exchange, which is necessary for activation of all G proteins. A homologous putative binding site for YM-254890/FR900359 can also be found in members of the other three G protein families, Gs, Gi/o, and G12/13, but none of the published analogs of YM-254890/FR900359 have shown any inhibitory activity for any of these. To explain why the YM-254890/FR900359 scaffold only inhibits Gq/11/14, the present study delineated the molecular selectivity determinants by exchanging amino acid residues in the YM-254890/FR900359-binding site in Gq and Gs We found that the activity of a Gs mutant with a Gq-like binding site for YM-254890/FR900359 can be inhibited by FR900359, and a minimum of three mutations are necessary to introduce inhibition in Gs In all, this suggests that although the YM-254890/FR900359 scaffold has proven unsuccessful to derive Gs, Gi/o, and G12/13 inhibitors, the mechanism of inhibition between families of G proteins is conserved, opening up the possibility of targeting by other, novel inhibitor scaffolds. In lack of a selective Gαs inhibitor, FR900359-sensitive Gαs mutants may prove useful in studies where delicate control over Gαs signaling would be of the essence.


Assuntos
Depsipeptídeos/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Subunidades alfa de Proteínas de Ligação ao GTP , Mutação , Subunidades alfa de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos
6.
J Am Chem Soc ; 143(2): 891-901, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33398998

RESUMO

There is an urgent need for novel therapeutic approaches to treat Alzheimer's disease (AD) with the ability to both alleviate the clinical symptoms and halt the progression of the disease. AD is characterized by the accumulation of amyloid-ß (Aß) peptides which are generated through the sequential proteolytic cleavage of the amyloid precursor protein (APP). Previous studies reported that Mint2, a neuronal adaptor protein binding both APP and the γ-secretase complex, affects APP processing and formation of pathogenic Aß. However, there have been contradicting results concerning whether Mint2 has a facilitative or suppressive effect on Aß generation. Herein, we deciphered the APP-Mint2 protein-protein interaction (PPI) via extensive probing of both backbone H-bond and side-chain interactions. We also developed a proteolytically stable, high-affinity peptide targeting the APP-Mint2 interaction. We found that both an APP binding-deficient Mint2 variant and a cell-permeable PPI inhibitor significantly reduced Aß42 levels in a neuronal in vitro model of AD. Together, these findings demonstrate a facilitative role of Mint2 in Aß formation, and the combination of genetic and pharmacological approaches suggests that targeting Mint2 is a promising therapeutic strategy to reduce pathogenic Aß levels.


Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Caderinas/antagonistas & inibidores , Proteínas do Tecido Nervoso/antagonistas & inibidores , Peptídeos/farmacologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Caderinas/metabolismo , Humanos , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/síntese química , Peptídeos/química , Ligação Proteica/efeitos dos fármacos
7.
J Biol Chem ; 294(15): 5747-5758, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30745359

RESUMO

Transmembrane signals initiated by a range of extracellular stimuli converge on members of the Gq family of heterotrimeric G proteins, which relay these signals in target cells. Gq family G proteins comprise Gq, G11, G14, and G16, which upon activation mediate their cellular effects via inositol lipid-dependent and -independent signaling to control fundamental processes in mammalian physiology. To date, highly specific inhibition of Gq/11/14 signaling can be achieved only with FR900359 (FR) and YM-254890 (YM), two naturally occurring cyclic depsipeptides. To further development of FR or YM mimics for other Gα subunits, we here set out to rationally design Gα16 proteins with artificial FR/YM sensitivity by introducing an engineered depsipeptide-binding site. Thereby we permit control of G16 function through ligands that are inactive on the WT protein. Using CRISPR/Cas9-generated Gαq/Gα11-null cells and loss- and gain-of-function mutagenesis along with label-free whole-cell biosensing, we determined the molecular coordinates for FR/YM inhibition of Gq and transplanted these to FR/YM-insensitive G16. Intriguingly, despite having close structural similarity, FR and YM yielded biologically distinct activities: it was more difficult to perturb Gq inhibition by FR and easier to install FR inhibition onto G16 than perturb or install inhibition with YM. A unique hydrophobic network utilized by FR accounted for these unexpected discrepancies. Our results suggest that non-Gq/11/14 proteins should be amenable to inhibition by FR scaffold-based inhibitors, provided that these inhibitors mimic the interaction of FR with Gα proteins harboring engineered FR-binding sites.


Assuntos
Depsipeptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Subunidades alfa de Proteínas de Ligação ao GTP , Peptídeos Cíclicos/farmacologia , Engenharia de Proteínas , Animais , Sistemas CRISPR-Cas , Subunidades alfa de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos
9.
Nucleic Acids Res ; 46(D1): D440-D446, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29155946

RESUMO

G protein-coupled receptors are the most abundant mediators of both human signalling processes and therapeutic effects. Herein, we report GPCRome-wide homology models of unprecedented quality, and roughly 150 000 GPCR ligands with data on biological activities and commercial availability. Based on the strategy of 'Less model - more Xtal', each model exploits both a main template and alternative local templates. This achieved higher similarity to new structures than any of the existing resources, and refined crystal structures with missing or distorted regions. Models are provided for inactive, intermediate and active states-except for classes C and F that so far only have inactive templates. The ligand database has separate browsers for: (i) target selection by receptor, family or class, (ii) ligand filtering based on cross-experiment activities (min, max and mean) or chemical properties, (iii) ligand source data and (iv) commercial availability. SMILES structures and activity spreadsheets can be downloaded for further processing. Furthermore, three recent landmark publications on GPCR drugs, G protein selectivity and genetic variants have been accompanied with resources that now let readers view and analyse the findings themselves in GPCRdb. Altogether, this update will enable scientific investigation for the wider GPCR community. GPCRdb is available at http://www.gpcrdb.org.


Assuntos
Bases de Dados de Proteínas , Anotação de Sequência Molecular , Medicamentos sob Prescrição/química , Receptores Acoplados a Proteínas G/química , Software , Homologia Estrutural de Proteína , Sequência de Aminoácidos , Sítios de Ligação , Gráficos por Computador , Humanos , Internet , Ligantes , Modelos Moleculares , Medicamentos sob Prescrição/farmacologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Transdução de Sinais
10.
Bioinformatics ; 33(8): 1116-1120, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28011766

RESUMO

Motivation: Class C G protein-coupled receptors (GPCRs) regulate important physiological functions and allosteric modulators binding to the transmembrane domain constitute an attractive and, due to a lack of structural insight, a virtually unexplored potential for therapeutics and the food industry. Combining pharmacological site-directed mutagenesis data with the recent class C GPCR experimental structures will provide a foundation for rational design of new therapeutics. Results: We uncover one common site for both positive and negative modulators with different amino acid layouts that can be utilized to obtain selectivity. Additionally, we show a large potential for structure-based modulator design, especially for four orphan receptors with high similarity to the crystal structures. Availability and Implementation: All collated mutagenesis data is available in the GPCRdb mutation browser at http://gpcrdb.org/mutations/ and can be analyzed online or downloaded in excel format. Contact: david.gloriam@sund.ku.dk. Supplementary information: Supplementary data are available at Bioinformatics online.


Assuntos
Sítio Alostérico , Mutação/genética , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Regulação Alostérica , Sítios de Ligação , Humanos , Ligantes , Mutagênese Sítio-Dirigida , Filogenia , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Receptor de Glutamato Metabotrópico 5/química , Receptor de Glutamato Metabotrópico 5/metabolismo
11.
Nucleic Acids Res ; 44(D1): D356-64, 2016 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-26582914

RESUMO

Recent developments in G protein-coupled receptor (GPCR) structural biology and pharmacology have greatly enhanced our knowledge of receptor structure-function relations, and have helped improve the scientific foundation for drug design studies. The GPCR database, GPCRdb, serves a dual role in disseminating and enabling new scientific developments by providing reference data, analysis tools and interactive diagrams. This paper highlights new features in the fifth major GPCRdb release: (i) GPCR crystal structure browsing, superposition and display of ligand interactions; (ii) direct deposition by users of point mutations and their effects on ligand binding; (iii) refined snake and helix box residue diagram looks; and (iii) phylogenetic trees with receptor classification colour schemes. Under the hood, the entire GPCRdb front- and back-ends have been re-coded within one infrastructure, ensuring a smooth browsing experience and development. GPCRdb is available at http://www.gpcrdb.org/ and it's open source code at https://bitbucket.org/gpcr/protwis.


Assuntos
Bases de Dados de Proteínas , Receptores Acoplados a Proteínas G/química , Sítios de Ligação , Humanos , Ligantes , Mutação , Filogenia , Receptores Acoplados a Proteínas G/classificação , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Alinhamento de Sequência , Software
12.
J Biol Chem ; 288(4): 2559-70, 2013 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-23229547

RESUMO

Deciphering which specific agonist-receptor interactions affect efficacy levels is of high importance, because this will ultimately aid in designing selective drugs. The novel compound NS3861 and cytisine are agonists of nicotinic acetylcholine receptors (nAChRs) and both bind with high affinity to heteromeric α3ß4 and α4ß2 nAChRs. However, initial data revealed that the activation patterns of the two compounds show very distinct maximal efficacy readouts at various heteromeric nAChRs. To investigate the molecular determinants behind these observations, we performed in-depth patch clamp electrophysiological measurements of efficacy levels at heteromeric combinations of α3- and α4-, with ß2- and ß4-subunits, and various chimeric constructs thereof. Compared with cytisine, which selectively activates receptors containing ß4- but not ß2-subunits, NS3861 displays the opposite ß-subunit preference and a complete lack of activation at α4-containing receptors. The maximal efficacy of NS3861 appeared solely dependent on the nature of the ligand-binding domain, whereas efficacy of cytisine was additionally affected by the nature of the ß-subunit transmembrane domain. Molecular docking to nAChR subtype homology models suggests agonist specific interactions to two different residues on the complementary subunits as responsible for the ß-subunit preference of both compounds. Furthermore, a principal subunit serine to threonine substitution may explain the lack of NS3861 activation at α4-containing receptors. In conclusion, our results are consistent with a hypothesis where agonist interactions with the principal subunit (α) primarily determine binding affinity, whereas interactions with key amino acids at the complementary subunit (ß) affect agonist efficacy.


Assuntos
Alcaloides/farmacologia , Compostos Azabicíclicos/farmacologia , Receptores Nicotínicos/metabolismo , Tiofenos/farmacologia , Animais , Azocinas/farmacologia , Clonagem Molecular , Relação Dose-Resposta a Droga , Desenho de Fármacos , Eletrofisiologia/métodos , Células HEK293 , Humanos , Ligantes , Modelos Químicos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Conformação Proteica , Estrutura Terciária de Proteína , Quinolizinas/farmacologia , Receptores Nicotínicos/química , Xenopus laevis
13.
J Biol Chem ; 288(37): 26521-32, 2013 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-23893416

RESUMO

The α4ß2 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain and are implicated in a variety of physiological processes. There are two stoichiometries of the α4ß2 nAChR, (α4)2(ß2)3 and (α4)3(ß2)2, with different sensitivities to acetylcholine (ACh), but their pharmacological profiles are not fully understood. Methyllycaconitine (MLA) is known to be an antagonist of nAChRs. Using the two-electrode voltage clamp technique and α4ß2 nAChRs in the Xenopus oocyte expression system, we demonstrate that inhibition by MLA occurs via two different mechanisms; that is, a direct competitive antagonism and an apparently insurmountable mechanism that only occurs after preincubation with MLA. We hypothesized an additional MLA binding site in the α4-α4 interface that is unique to this stoichiometry. To prove this, we covalently trapped a cysteine-reactive MLA analog at an α4ß2 receptor containing an α4(D204C) mutation predicted by homology modeling to be within reach of the reactive probe. We demonstrate that covalent trapping results in irreversible reduction of ACh-elicited currents in the (α4)3(ß2)2 stoichiometry, indicating that MLA binds to the α4-α4 interface of the (α4)3(ß2)2 and providing direct evidence of ligand binding to the α4-α4 interface. Consistent with other studies, we propose that the α4-α4 interface is a structural target for potential therapeutics that modulate (α4)3(ß2)2 nAChRs.


Assuntos
Aconitina/análogos & derivados , Antagonistas Nicotínicos/química , Receptores Nicotínicos/química , Aconitina/química , Animais , Sítios de Ligação , Cisteína/química , Escherichia coli/metabolismo , Feminino , Ligantes , Maleimidas/química , Mutagênese Sítio-Dirigida , Oócitos/citologia , Ligação Proteica , Conformação Proteica , Ratos , Receptores Nicotínicos/fisiologia , Proteínas Recombinantes/química , Xenopus laevis
14.
Neurochem Res ; 39(10): 1862-75, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24493625

RESUMO

Since its discovery in 1992, mGluR5 has attracted significant attention and been linked to several neurological and psychiatric diseases. Ligand development was initially focused on the orthosteric binding pocket, but lack of subtype selective ligands changed the focus to the transmembrane allosteric binding pocket. This strategy has resulted in several drug candidates in clinical testing. In the present article we explore the orthosteric and allosteric binding pockets in terms of structure and ligand recognition across the mGluR subtypes and groups, and discuss the clinical potential of ligands targeting these pockets. We have performed binding mode analyses of non- and group-selective orthosteric ligands based on molecular docking in mGluR crystal structures and models. For the analysis of the allosteric binding pocket we have combined data from all mGluR5-mutagenesis studies, collectively reporting five negative allosteric modulators and 47 unique mutations, and compared it to the closest related homolog, mGluR1.


Assuntos
Receptor de Glutamato Metabotrópico 5/metabolismo , Sítio Alostérico , Sequência de Aminoácidos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Receptor de Glutamato Metabotrópico 5/química , Homologia de Sequência de Aminoácidos
16.
J Biol Chem ; 287(30): 25241-54, 2012 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-22589534

RESUMO

The ligand-gated ion channels in the Cys-loop receptor superfamily mediate the effects of neurotransmitters acetylcholine, serotonin, GABA, and glycine. Cys-loop receptor signaling is susceptible to modulation by ligands acting through numerous allosteric sites. Here we report the discovery of a novel class of negative allosteric modulators of the 5-HT(3) receptors (5-HT(3)Rs). PU02 (6-[(1-naphthylmethyl)thio]-9H-purine) is a potent and selective antagonist displaying IC(50) values of ~1 µM at 5-HT(3)Rs and substantially lower activities at other Cys-loop receptors. In an elaborate mutagenesis study of the 5-HT(3)A receptor guided by a homology model, PU02 is demonstrated to act through a transmembrane intersubunit site situated in the upper three helical turns of TM2 and TM3 in the (+)-subunit and TM1 and TM2 in the (-)-subunit. The Ser(248), Leu(288), Ile(290), Thr(294), and Gly(306) residues are identified as important molecular determinants of PU02 activity with minor contributions from Ser(292) and Val(310), and we propose that the naphthalene group of PU02 docks into the hydrophobic cavity formed by these. Interestingly, specific mutations of Ser(248), Thr(294), and Gly(306) convert PU02 into a complex modulator, potentiating and inhibiting 5-HT-evoked signaling through these mutants at low and high concentrations, respectively. The PU02 binding site in the 5-HT(3)R corresponds to allosteric sites in anionic Cys-loop receptors, which emphasizes the uniform nature of the molecular events underlying signaling through the receptors. Moreover, the dramatic changes in the functional properties of PU02 induced by subtle changes in its binding site bear witness to the delicate structural discrimination between allosteric inhibition and potentiation of Cys-loop receptors.


Assuntos
Receptores 5-HT3 de Serotonina/metabolismo , Agonistas do Receptor 5-HT3 de Serotonina/farmacologia , Antagonistas do Receptor 5-HT3 de Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Substituição de Aminoácidos , Sítios de Ligação , Células HEK293 , Humanos , Mutagênese , Mutação de Sentido Incorreto , Mapeamento de Peptídeos , Estrutura Secundária de Proteína , Receptores 5-HT3 de Serotonina/genética , Agonistas do Receptor 5-HT3 de Serotonina/química , Antagonistas do Receptor 5-HT3 de Serotonina/química , Transdução de Sinais/genética
17.
J Biol Chem ; 287(6): 4248-59, 2012 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-22170047

RESUMO

The α4ß2 subtype of the nicotinic acetylcholine receptor has been pursued as a drug target for treatment of psychiatric and neurodegenerative disorders and smoking cessation aids for decades. Still, a thorough understanding of structure-function relationships of α4ß2 agonists is lacking. Using binding experiments, electrophysiology and x-ray crystallography we have investigated a consecutive series of five prototypical pyridine-containing agonists derived from 1-(pyridin-3-yl)-1,4-diazepane. A correlation between binding affinities at α4ß2 and the acetylcholine-binding protein from Lymnaea stagnalis (Ls-AChBP) confirms Ls-AChBP as structural surrogate for α4ß2 receptors. Crystal structures of five agonists with efficacies at α4ß2 from 21-76% were determined in complex with Ls-AChBP. No variation in closure of loop C is observed despite large efficacy variations. Instead, the efficacy of a compound appears tightly coupled to its ability to form a strong intersubunit bridge linking the primary and complementary binding interfaces. For the tested agonists, a specific halogen bond was observed to play a large role in establishing such strong intersubunit anchoring.


Assuntos
Azepinas/química , Agonistas Colinérgicos/química , Halogênios/química , Piridinas/química , Receptores Nicotínicos/química , Animais , Azepinas/metabolismo , Agonistas Colinérgicos/metabolismo , Cristalografia por Raios X , Células HEK293 , Halogênios/metabolismo , Humanos , Lymnaea , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Piridinas/metabolismo , Receptores Nicotínicos/metabolismo
18.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 9): 1645-52, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23999288

RESUMO

Positive allosteric modulators of the ionotropic glutamate receptor A2 (GluA2) can serve as lead compounds for the development of cognitive enhancers. Several benzamide-type (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor modulators such as aniracetam, CX516 and CX614 have been shown to inhibit the deactivation of AMPA receptors with a less pronounced effect on desensitization. Despite CX516 being an extensively investigated AMPA receptor modulator and one of the few clinically evaluated compounds, the binding mode of CX516 to AMPA receptors has not been reported. Here, the structures of a GluA2 ligand-binding domain mutant in complex with CX516 and the 3-methylpiperidine analogue of CX516 (Me-CX516) are reported. The structures show that the binding modes of CX516 and Me-CX516 are similar to those of aniracetam and CX614 and that there is limited space for substitution at the piperidine ring of CX516. The results therefore support that CX516, like aniracetam and CX614, modulates deactivation of AMPA receptors.


Assuntos
Dioxóis/química , Piperidinas/química , Receptores de AMPA/química , Regulação Alostérica/genética , Animais , Cristalografia por Raios X , Dioxóis/metabolismo , Ligantes , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Mutação , Oxazinas/química , Oxazinas/metabolismo , Piperidinas/metabolismo , Ligação Proteica/genética , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína/genética , Ratos , Receptores de AMPA/antagonistas & inibidores , Receptores de AMPA/genética , Receptores de AMPA/metabolismo
19.
ACS Med Chem Lett ; 14(3): 319-325, 2023 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-36923922

RESUMO

The N-benzylphenethylamines (NBOMes) are a class of ligands from which compounds with impressive selectivity for the serotonin 2A receptor (5-HT2AR) over the closely related serotonin 2C receptor (5-HT2CR) have emerged. These include 4-(2-((2-hydroxybenzyl)amino)ethyl)-2,5-dimethoxybenzonitrile (25CN-NBOH, 1) and 2-(2,5-dimethoxy-4-bromobenzyl)-6-(2-methoxyphenyl)piperidine (DMPMBB, 2). The present work entails the synthesis and characterization of ligands wherein the structures of these two molecules have been fused. The desired compounds were accessed by a six-step synthetic procedure followed by the chiral resolution of the resulting racemic mixtures, giving one active ((S,S)-3) and three essentially inactive stereoisomers. In silico experiments support that one of the four possible stereoisomers would be active. Further in silico investigations showed that 1, 2, and (S,S)-3 share a common binding mode, further supporting the shared stereochemistry between the active enantiomer ((S,S)-3) and 2.

20.
Trends Pharmacol Sci ; 44(12): 978-990, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37914598

RESUMO

Serotonin is a neurotransmitter regulating numerous physiological processes also modulated by drugs, for example, schizophrenia, depression, migraine, and obesity. However, these drugs typically have adverse effects caused by promiscuous binding across 12 serotonin and more than 20 homologous receptors. Recently, structures of the entire serotonin receptor family uncovered molecular ligand recognition. Here, we present a map of 19 'selectivity hotspots', that is, nonconserved binding site residues governing selectivity via favorable target interactions or repulsive 'off-target' contacts. Furthermore, we review functional rationale from observed ligand-binding affinities and mutagenesis effects. Unifying knowledge underlying specific probes and drugs is critical toward the functional characterization of different receptors and alleviation of adverse effects.


Assuntos
Transtornos de Enxaqueca , Receptores Acoplados a Proteínas G , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Serotonina , Ligantes , Sítios de Ligação
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