RESUMO
Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Desenho de Fármacos , Receptor Muscarínico M1/agonistas , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/patologia , Doença de Alzheimer/complicações , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Animais , Pressão Sanguínea/efeitos dos fármacos , Células CHO , Inibidores da Colinesterase/farmacologia , Cricetulus , Cristalização , Modelos Animais de Doenças , Cães , Donepezila/farmacologia , Eletroencefalografia , Feminino , Células HEK293 , Frequência Cardíaca/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Modelos Moleculares , Simulação de Dinâmica Molecular , Degeneração Neural/complicações , Degeneração Neural/patologia , Primatas , Ratos , Receptor Muscarínico M1/química , Transdução de Sinais , Homologia Estrutural de ProteínaRESUMO
The complement system is a crucial component of the host response to infection and tissue damage. Activation of the complement cascade generates anaphylatoxins including C5a and C3a. C5a exerts a pro-inflammatory effect via the G-protein-coupled receptor C5a anaphylatoxin chemotactic receptor 1 (C5aR1, also known as CD88) that is expressed on cells of myeloid origin. Inhibitors of the complement system have long been of interest as potential drugs for the treatment of diseases such as sepsis, rheumatoid arthritis, Crohn's disease and ischaemia-reperfusion injuries. More recently, a role of C5a in neurodegenerative conditions such as Alzheimer's disease has been identified. Peptide antagonists based on the C5a ligand have progressed to phase 2 trials in psoriasis and rheumatoid arthritis; however, these compounds exhibited problems with off-target activity, production costs, potential immunogenicity and poor oral bioavailability. Several small-molecule competitive antagonists for C5aR1, such as W-54011 and NDT9513727, have been identified by C5a radioligand-binding assays. NDT9513727 is a non-peptide inverse agonist of C5aR1, and is highly selective for the primate and gerbil receptors over those of other species. Here, to study the mechanism of action of C5a antagonists, we determine the structure of a thermostabilized C5aR1 (known as C5aR1 StaR) in complex with NDT9513727. We found that the small molecule bound between transmembrane helices 3, 4 and 5, outside the helical bundle. One key interaction between the small molecule and residue Trp2135.49 seems to determine the species selectivity of the compound. The structure demonstrates that NDT9513727 exerts its inverse-agonist activity through an extra-helical mode of action.
Assuntos
Benzodioxóis/química , Benzodioxóis/metabolismo , Imidazóis/química , Imidazóis/metabolismo , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/química , Animais , Benzodioxóis/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Agonismo Inverso de Drogas , Células HEK293 , Humanos , Imidazóis/farmacologia , Modelos Moleculares , Mutação , Estabilidade Proteica , Estrutura Secundária de Proteína , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismoRESUMO
This corrects the article DOI: 10.1038/nature22800.
RESUMO
Glucagon-like peptide 1 (GLP-1) regulates glucose homeostasis through the control of insulin release from the pancreas. GLP-1 peptide agonists are efficacious drugs for the treatment of diabetes. To gain insight into the molecular mechanism of action of GLP-1 peptides, here we report the crystal structure of the full-length GLP-1 receptor bound to a truncated peptide agonist. The peptide agonist retains an α-helical conformation as it sits deep within the receptor-binding pocket. The arrangement of the transmembrane helices reveals hallmarks of an active conformation similar to that observed in class A receptors. Guided by this structural information, we design peptide agonists with potent in vivo activity in a mouse model of diabetes.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/química , Peptídeos/química , Peptídeos/farmacologia , Animais , Sítios de Ligação , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Humanos , Masculino , Camundongos , Modelos Moleculares , Peptídeos/metabolismo , Conformação Proteica , Ratos , Receptores de Hormônio Liberador da Corticotropina/química , Receptores de Glucagon/químicaRESUMO
Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.
Assuntos
Receptor PAR-2/química , Receptor PAR-2/metabolismo , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Anticorpos Bloqueadores/química , Anticorpos Bloqueadores/farmacologia , Benzimidazóis/química , Benzimidazóis/farmacologia , Benzodioxóis/química , Benzodioxóis/farmacologia , Álcoois Benzílicos/química , Álcoois Benzílicos/farmacologia , Cristalografia por Raios X , Humanos , Imidazóis/química , Imidazóis/farmacologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/farmacologia , Cinética , Ligantes , Modelos Moleculares , Receptor PAR-2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
Chemokines and their G-protein-coupled receptors play a diverse role in immune defence by controlling the migration, activation and survival of immune cells. They are also involved in viral entry, tumour growth and metastasis and hence are important drug targets in a wide range of diseases. Despite very significant efforts by the pharmaceutical industry to develop drugs, with over 50 small-molecule drugs directed at the family entering clinical development, only two compounds have reached the market: maraviroc (CCR5) for HIV infection and plerixafor (CXCR4) for stem-cell mobilization. The high failure rate may in part be due to limited understanding of the mechanism of action of chemokine antagonists and an inability to optimize compounds in the absence of structural information. CC chemokine receptor type 9 (CCR9) activation by CCL25 plays a key role in leukocyte recruitment to the gut and represents a therapeutic target in inflammatory bowel disease. The selective CCR9 antagonist vercirnon progressed to phase 3 clinical trials in Crohn's disease but efficacy was limited, with the need for very high doses to block receptor activation. Here we report the crystal structure of the CCR9 receptor in complex with vercirnon at 2.8 Å resolution. Remarkably, vercirnon binds to the intracellular side of the receptor, exerting allosteric antagonism and preventing G-protein coupling. This binding site explains the need for relatively lipophilic ligands and describes another example of an allosteric site on G-protein-coupled receptors that can be targeted for drug design, not only at CCR9, but potentially extending to other chemokine receptors.
Assuntos
Receptores CCR/antagonistas & inibidores , Receptores CCR/química , Sulfonamidas/química , Sulfonamidas/farmacologia , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico/efeitos dos fármacos , Sítio Alostérico/genética , Sequência Conservada , Cristalografia por Raios X , Citoplasma/metabolismo , Desenho de Fármacos , Proteínas Heterotriméricas de Ligação ao GTP/antagonistas & inibidores , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Ligantes , Modelos Moleculares , Mutagênese , Receptores CCR/genética , Receptores CCR5/química , Receptores CXCR4/químicaRESUMO
Glucagon is a 29-amino-acid peptide released from the α-cells of the islet of Langerhans, which has a key role in glucose homeostasis. Glucagon action is transduced by the class B G-protein-coupled glucagon receptor (GCGR), which is located on liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreas cells, and this receptor has been considered an important drug target in the treatment of diabetes. Administration of recently identified small-molecule GCGR antagonists in patients with type 2 diabetes results in a substantial reduction of fasting and postprandial glucose concentrations. Although an X-ray structure of the transmembrane domain of the GCGR has previously been solved, the ligand (NNC0640) was not resolved. Here we report the 2.5 Å structure of human GCGR in complex with the antagonist MK-0893 (ref. 4), which is found to bind to an allosteric site outside the seven transmembrane (7TM) helical bundle in a position between TM6 and TM7 extending into the lipid bilayer. Mutagenesis of key residues identified in the X-ray structure confirms their role in the binding of MK-0893 to the receptor. The unexpected position of the binding site for MK-0893, which is structurally similar to other GCGR antagonists, suggests that glucagon activation of the receptor is prevented by restriction of the outward helical movement of TM6 required for G-protein coupling. Structural knowledge of class B receptors is limited, with only one other ligand-binding site defined--for the corticotropin-releasing hormone receptor 1 (CRF1R)--which was located deep within the 7TM bundle. We describe a completely novel allosteric binding site for class B receptors, providing an opportunity for structure-based drug design for this receptor class and furthering our understanding of the mechanisms of activation of these receptors.
Assuntos
Pirazóis/metabolismo , Receptores de Glucagon/antagonistas & inibidores , Receptores de Glucagon/química , beta-Alanina/análogos & derivados , Sítio Alostérico/efeitos dos fármacos , Cristalografia por Raios X , Glucagon/metabolismo , Glucagon/farmacologia , Humanos , Ligantes , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Conformação Proteica/efeitos dos fármacos , Pirazóis/química , Pirazóis/farmacologia , Receptores de Hormônio Liberador da Corticotropina/química , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Receptores de Glucagon/classificação , Receptores de Glucagon/metabolismo , beta-Alanina/química , beta-Alanina/metabolismo , beta-Alanina/farmacologiaRESUMO
Metabotropic glutamate receptors are class C G-protein-coupled receptors which respond to the neurotransmitter glutamate. Structural studies have been restricted to the amino-terminal extracellular domain, providing little understanding of the membrane-spanning signal transduction domain. Metabotropic glutamate receptor 5 is of considerable interest as a drug target in the treatment of fragile X syndrome, autism, depression, anxiety, addiction and movement disorders. Here we report the crystal structure of the transmembrane domain of the human receptor in complex with the negative allosteric modulator, mavoglurant. The structure provides detailed insight into the architecture of the transmembrane domain of class C receptors including the precise location of the allosteric binding site within the transmembrane domain and key micro-switches which regulate receptor signalling. This structure also provides a model for all class C G-protein-coupled receptors and may aid in the design of new small-molecule drugs for the treatment of brain disorders.
Assuntos
Modelos Moleculares , Receptor de Glutamato Metabotrópico 5/química , Motivos de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Células HEK293 , Humanos , Estrutura Terciária de Proteína , Rodopsina/químicaRESUMO
We present a robust protocol based on iterations of free energy perturbation (FEP) calculations, chemical synthesis, biophysical mapping and X-ray crystallography to reveal the binding mode of an antagonist series to the A2A adenosine receptor (AR). Eight A2A AR binding site mutations from biophysical mapping experiments were initially analyzed with sidechain FEP simulations, performed on alternate binding modes. The results distinctively supported one binding mode, which was subsequently used to design new chromone derivatives. Their affinities for the A2A AR were experimentally determined and investigated through a cycle of ligand-FEP calculations, validating the binding orientation of the different chemical substituents proposed. Subsequent X-ray crystallography of the A2A AR with a low and a high affinity chromone derivative confirmed the predicted binding orientation. The new molecules and structures here reported were driven by free energy calculations, and provide new insights on antagonist binding to the A2A AR, an emerging target in immuno-oncology.
Assuntos
Antagonistas de Receptores Purinérgicos P1/química , Receptor A2A de Adenosina/química , Termodinâmica , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Estrutura Molecular , Antagonistas de Receptores Purinérgicos P1/farmacologia , Receptor A2A de Adenosina/metabolismoRESUMO
A series of novel allosteric antagonists of the GLP-1 receptor (GLP-1R), exemplified by HTL26119, are described. SBDD approaches were employed to identify HTL26119, exploiting structural understanding of the allosteric binding site of the closely related Glucagon receptor (GCGR) (Jazayeri et al., 2016) and the homology relationships between GCGR and GLP-1R. The region around residue C3476.36b of the GLP-1R receptor represents a key difference from GCGR and was targeted for selectivity for GLP-1R.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/antagonistas & inibidores , Compostos Heterocíclicos/química , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico , Sequência de Aminoácidos , Desenho de Fármacos , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Receptores de Glucagon/antagonistas & inibidores , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Structural analysis of class B G-protein-coupled receptors (GPCRs), cell-surface proteins that respond to peptide hormones, has been restricted to the amino-terminal extracellular domain, thus providing little understanding of the membrane-spanning signal transduction domain. The corticotropin-releasing factor receptor type 1 is a class B receptor which mediates the response to stress and has been considered a drug target for depression and anxiety. Here we report the crystal structure of the transmembrane domain of the human corticotropin-releasing factor receptor type 1 in complex with the small-molecule antagonist CP-376395. The structure provides detailed insight into the architecture of class B receptors. Atomic details of the interactions of the receptor with the non-peptide ligand that binds deep within the receptor are described. This structure provides a model for all class B GPCRs and may aid in the design of new small-molecule drugs for diseases of brain and metabolism.
Assuntos
Receptores de Hormônio Liberador da Corticotropina/química , Receptores de Hormônio Liberador da Corticotropina/classificação , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminopiridinas/química , Aminopiridinas/metabolismo , Aminopiridinas/farmacologia , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Receptores de Dopamina D3/antagonistas & inibidores , Receptores de Dopamina D3/química , Receptores de Dopamina D3/classificaçãoRESUMO
Rapid release of prepublication data has served the field of genomics well. Attendees at a workshop in Toronto recommend extending the practice to other biological data sets.
Assuntos
Acesso à Informação , Guias como Assunto , Editoração , Pesquisa , Comportamento Cooperativo , Projeto Genoma Humano , Humanos , Ontário , Editoração/ética , Editoração/normas , Pesquisa/normas , Pesquisadores/ética , Pesquisadores/normasRESUMO
Since the publication of the first X-ray structure of a GPCR (G-protein couple receptor) in 2000, the rate at which subsequent ones have appeared has steadily increased. This has required the development of new methodology to overcome the challenges presented by instability of isolated GPCRs, combined with a systematic optimization of existing approaches for protein expression, purification and crystallization. In addition, quality control measures that are predictive of successful outcomes have been identified. Repeated attempts at solving the structures of GPCRs have highlighted experimental approaches that are most likely to lead to success, and have allowed definition of a first-pass protocol for new receptors.
Assuntos
Receptores Acoplados a Proteínas G/isolamento & purificação , Cristalografia por Raios X , Descoberta de Drogas , Eletroforese em Gel de Poliacrilamida , Conformação Proteica , Receptores Acoplados a Proteínas G/químicaRESUMO
In this study, we determined the crystal structure of an engineered human adenosine A2A receptor bound to a partial agonist and compared it to structures cocrystallized with either a full agonist or an antagonist/inverse agonist. The interaction between the partial agonist, belonging to a class of dicyanopyridines, and amino acids in the ligand binding pocket inspired us to develop a small library of derivatives and assess their affinity in radioligand binding studies and potency and intrinsic activity in a functional, label-free, intact cell assay. It appeared that some of the derivatives retained the partial agonist profile, whereas other ligands turned into inverse agonists. We rationalized this remarkable behavior with additional computational docking studies.
Assuntos
Agonistas do Receptor A2 de Adenosina/metabolismo , Aminopiridinas/metabolismo , Pirimidinas/metabolismo , Receptor A2A de Adenosina/metabolismo , Aminopiridinas/síntese química , Animais , Sítios de Ligação , Células CHO , Cricetulus , Cristalografia por Raios X , Agonismo Inverso de Drogas , Agonismo Parcial de Drogas , Células HEK293 , Humanos , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Pirimidinas/síntese química , Bibliotecas de Moléculas Pequenas/metabolismoRESUMO
The orexin system, which consists of the two G protein-coupled receptors OX1 and OX2, activated by the neuropeptides OX-A and OX-B, is firmly established as a key regulator of behavioral arousal, sleep, and wakefulness and has been an area of intense research effort over the past two decades. X-ray structures of the receptors in complex with 10 new antagonist ligands from diverse chemotypes are presented, which complement the existing structural information for the system and highlight the critical importance of lipophilic hotspots and water molecules for these peptidergic GPCR targets. Learnings from the structural information regarding the utility of pharmacophore models and how selectivity between OX1 and OX2 can be achieved are discussed.
Assuntos
Antagonistas dos Receptores de Orexina/metabolismo , Receptores de Orexina/metabolismo , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Células HEK293 , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Antagonistas dos Receptores de Orexina/química , Receptores de Orexina/químicaRESUMO
The four adenosine receptors (ARs), A1, A2A, A2B, and A3, constitute a subfamily of G protein-coupled receptors (GPCRs) with exceptional foundations for structure-based ligand design. The vast amount of mutagenesis data, accumulated in the literature since the 1990s, has been recently supplemented with structural information, currently consisting of several inactive and active structures of the A2A and inactive conformations of the A1 ARs. We provide the first integrated view of the pharmacological, biochemical, and structural data available for this receptor family, by mapping onto the relevant crystal structures all site-directed mutagenesis data, curated and deposited at the GPCR database (available through http://www.gpcrdb.org). This analysis provides novel insights into ligand binding, allosteric modulation, and signaling of the AR family.
Assuntos
Mutação , Agonistas do Receptor Purinérgico P1/farmacologia , Antagonistas de Receptores Purinérgicos P1/farmacologia , Receptores Purinérgicos P1/química , Transdução de Sinais , Sítio Alostérico , Animais , Humanos , Ligação Proteica , Agonistas do Receptor Purinérgico P1/química , Antagonistas de Receptores Purinérgicos P1/química , Receptores Purinérgicos P1/genética , Receptores Purinérgicos P1/metabolismoRESUMO
Here we report an efficient method to generate multiple co-structures of the A2A G protein-coupled receptor (GPCR) with small-molecules from a single preparation of a thermostabilised receptor crystallised in Lipidic Cubic Phase (LCP). Receptor crystallisation is achieved following purification using a low affinity "carrier" ligand (theophylline) and crystals are then soaked in solutions containing the desired (higher affinity) compounds. Complete datasets to high resolution can then be collected from single crystals and seven structures are reported here of which three are novel. The method significantly improves structural throughput for ligand screening using stabilised GPCRs, thereby actively driving Structure-Based Drug Discovery (SBDD).
Assuntos
Receptor A2A de Adenosina/química , Receptores Acoplados a Proteínas G/química , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Desdobramento de Proteína , Receptor A2A de Adenosina/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
The discovery of ligands via affinity-mediated selection of DNA-encoded chemical libraries is driven by the quality and concentration of the protein target. G-protein-coupled receptors (GPCRs) and other membrane-bound targets can be difficult to isolate in their functional state and at high concentrations, and therefore have been challenging for affinity-mediated selection. Here, we report a successful selection campaign against protease-activated receptor 2 (PAR2). Using a thermo-stabilized mutant of PAR2, we conducted affinity selection using our >100-billion-compound DNA-encoded library. We observed a number of putative ligands enriched upon selection, and subsequent cellular profiling revealed these ligands to comprise both agonists and antagonists. The agonist series shared structural similarity with known agonists. The antagonists were shown to bind in a novel allosteric binding site on the PAR2 protein. This report serves to demonstrate that cell-free affinity selection against GPCRs can be achieved with mutant stabilized protein targets.
Assuntos
DNA/genética , Mutação/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Sítio Alostérico/efeitos dos fármacos , Linhagem Celular , Células HEK293 , Humanos , Ligantes , Proteínas/genética , Receptor PAR-2 , Receptores Acoplados a Proteínas G/genéticaRESUMO
The adenosine A1 and A2A receptors belong to the purinergic family of G protein-coupled receptors, and regulate diverse functions of the cardiovascular, respiratory, renal, inflammation, and CNS. Xanthines such as caffeine and theophylline are weak, non-selective antagonists of adenosine receptors. Here we report the structure of a thermostabilized human A1 receptor at 3.3 Å resolution with PSB36, an A1-selective xanthine-based antagonist. This is compared with structures of the A2A receptor with PSB36 (2.8 Å resolution), caffeine (2.1 Å), and theophylline (2.0 Å) to highlight features of ligand recognition which are common across xanthines. The structures of A1R and A2AR were analyzed to identify the differences that are important selectivity determinants for xanthine ligands, and the role of T2707.35 in A1R (M2707.35 in A2AR) in conferring selectivity was confirmed by mutagenesis. The structural differences confirmed to lead to selectivity can be utilized in the design of new subtype-selective A1R or A2AR antagonists.
Assuntos
Cafeína/farmacologia , Receptor A1 de Adenosina/química , Receptor A2A de Adenosina/química , Teofilina/farmacologia , Sítios de Ligação , Cafeína/química , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Receptor A1 de Adenosina/metabolismo , Receptor A2A de Adenosina/metabolismo , Especificidade por Substrato , Teofilina/químicaRESUMO
Ligand-protein binding kinetic rates are growing in importance as parameters to consider in drug discovery and lead optimization. In this study we analysed using surface plasmon resonance (SPR) the transition state (TS) properties of a set of six adenosine A2A receptor inhibitors, belonging to both the xanthine and the triazolo-triazine scaffolds. SPR highlighted interesting differences among the ligands in the enthalpic and entropic components of the TS energy barriers for the binding and unbinding events. To better understand at a molecular level these differences, we developed suMetaD, a novel molecular dynamics (MD)-based approach combining supervised MD and metadynamics. This method allows simulation of the ligand unbinding and binding events. It also provides the system conformation corresponding to the highest energy barrier the ligand is required to overcome to reach the final state. For the six ligands evaluated in this study their TS thermodynamic properties were linked in particular to the role of water molecules in solvating/desolvating the pocket and the small molecules. suMetaD identified kinetic bottleneck conformations near the bound state position or in the vestibule area. In the first case the barrier is mainly enthalpic, requiring the breaking of strong interactions with the protein. In the vestibule TS location the kinetic bottleneck is instead mainly of entropic nature, linked to the solvent behaviour.