RESUMO
Acinetobacter baumannii, a commonly multidrug-resistant Gram-negative bacterium responsible for large numbers of bloodstream and lung infections worldwide, is increasingly difficult to treat and constitutes a growing threat to human health. Structurally novel antibacterial chemical matter that can evade existing resistance mechanisms is essential for addressing this critical medical need. Herein, we describe our efforts to inhibit the essential A. baumannii lipooligosaccharide (LOS) ATP-binding cassette (ABC) transporter MsbA. An unexpected impurity from a phenotypic screening was optimized as a series of dimeric compounds, culminating with 1 (cerastecin D), which exhibited antibacterial activity in the presence of human serum and a pharmacokinetic profile sufficient to achieve efficacy against A. baumannii in murine septicemia and lung infection models.
RESUMO
The Gs protein-coupled adenosine A2A receptor (A2AAR) represents an emerging drug target for cancer immunotherapy. The clinical candidate Etrumadenant was developed as an A2AAR antagonist with ancillary blockade of the A2BAR subtype. It constitutes a unique chemotype featuring a poly-substituted 2-amino-4-phenyl-6-triazolylpyrimidine core structure. Herein, we report two crystal structures of the A2AAR in complex with Etrumadenant, obtained with differently thermostabilized A2AAR constructs. This led to the discovery of an unprecedented interaction, a hydrogen bond of T883.36 with the cyano group of Etrumadenant. T883.36 is mutated in most A2AAR constructs used for crystallization, which has prevented the discovery of its interactions. In-vitro characterization of Etrumadenant indicated low selectivity versus the A1AR subtype, which can be rationalized by the structural data. These results will facilitate the future design of AR antagonists with desired selectivity. Moreover, they highlight the advantages of the employed A2AAR crystallization construct that is devoid of ligand binding site mutations.
RESUMO
Lipopolysaccharides are major constituents of the extracellular leaflet in the bacterial outer membrane and form an effective physical barrier for environmental threats and for antibiotics in Gram-negative bacteria. The last step of LPS insertion via the Lpt pathway is mediated by the LptD/E protein complex. Detailed insights into the architecture of LptDE transporter complexes have been derived from X-ray crystallography. However, no structure of a laterally open LptD transporter, a transient state that occurs during LPS release, is available to date. Here, we report a cryo-EM structure of a partially opened LptDE transporter in complex with rigid chaperones derived from nanobodies, at 3.4 Å resolution. In addition, a subset of particles allows to model a structure of a laterally fully opened LptDE complex. Our work offers insights into the mechanism of LPS insertion, provides a structural framework for the development of antibiotics targeting LptD and describes a highly rigid chaperone scaffold to enable structural biology of challenging protein targets.
Assuntos
Proteínas de Escherichia coli , Lipopolissacarídeos , Proteínas da Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Microscopia Crioeletrônica , Cristalografia por Raios X , Proteínas de Escherichia coli/metabolismo , Bactérias Gram-Negativas/metabolismo , Lipopolissacarídeos/metabolismoRESUMO
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
We determined two crystal structures of the chemokine receptor CCR2A in complex with the orthosteric antagonist MK-0812. Full-length CCR2A, stabilized by rubredoxin and a series of five mutations were resolved at 3.3 Å. An N- and C-terminally truncated CCR2A construct was crystallized in an alternate crystal form, which yielded a 2.7 Å resolution structure using serial synchrotron crystallography. Our structures provide a clear structural explanation for the observed key role of residue E2917.39 in high-affinity binding of several orthosteric CCR2 antagonists. By combining all the structural information collected, we generated models of co-structures for the structurally diverse pyrimidine amide class of CCR2 antagonists. Even though the representative Ex15 overlays well with MK-0812, it also interacts with the non-conserved H1213.33, resulting in a significant selectivity over CCR5. Insights derived from this work will facilitate drug discovery efforts directed toward highly selective CCR2 antagonists with potentially superior efficacy.
Assuntos
Naftiridinas/farmacologia , Receptores CCR2/química , Receptores CCR2/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Células HEK293 , Humanos , Modelos Moleculares , Mutação , Naftiridinas/química , Conformação Proteica , Estabilidade Proteica , Receptores CCR2/antagonistas & inibidores , Receptores CCR2/genética , Rubredoxinas/farmacologia , Células THP-1RESUMO
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
Past decades have shown the impact of structural information derived from complexes of drug candidates with their protein targets to facilitate the discovery of safe and effective medicines. Despite recent developments in single particle cryo-electron microscopy, X-ray crystallography has been the main method to derive structural information. The unique properties of X-ray free electron laser (XFEL) with unmet peak brilliance and beam focus allow X-ray diffraction data recording and successful structure determination from smaller and weaker diffracting crystals shortening timelines in crystal optimization. To further capitalize on the XFEL advantage, innovations in crystal sample delivery for the X-ray experiment, data collection and processing methods are required. This development was a key contributor to serial crystallography allowing structure determination at room temperature yielding physiologically more relevant structures. Adding the time resolution provided by the femtosecond X-ray pulse will enable monitoring and capturing of dynamic processes of ligand binding and associated conformational changes with great impact to the design of candidate drug compounds.
Assuntos
Descoberta de Drogas/métodos , Elétrons , Lasers , Proteínas/química , Bibliotecas de Moléculas Pequenas/química , Cristalografia por Raios X , Coleta de Dados/métodos , Ligantes , Proteínas/ultraestrutura , Síncrotrons , Temperatura , Difração de Raios XRESUMO
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
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
The structural analysis of class B G protein-coupled receptors (GPCR), cell surface proteins responding to peptide hormones, has until recently been restricted to the extracellular domain (ECD). Corticotropin-releasing factor receptor type 1 (CRF1R) is a class B receptor mediating stress response and also considered a drug target for depression and anxiety. Here we report the crystal structure of the transmembrane domain of human CRF1R in complex with the small-molecule antagonist CP-376395 in a hexagonal setting with translational non-crystallographic symmetry. Molecular dynamics and metadynamics simulations on this novel structure and the existing TMD structure for CRF1R provides insight as to how the small molecule ligand gains access to the induced-fit allosteric binding site with implications for the observed selectivity against CRF2R. Furthermore, molecular dynamics simulations performed using a full-length receptor model point to key interactions between the ECD and extracellular loop 3 of the TMD providing insight into the full inactive state of multidomain class B GPCRs.
Assuntos
Receptores de Hormônio Liberador da Corticotropina/química , Sítio Alostérico , Aminopiridinas/farmacologia , Sítios de Ligação , Cristalografia por Raios X/métodos , Humanos , Simulação de Dinâmica Molecular , Conformação Proteica , Receptores de Hormônio Liberador da Corticotropina/antagonistas & inibidores , Receptores de Hormônio Liberador da Corticotropina/metabolismoRESUMO
The association and dissociation kinetics of ligands binding to proteins vary considerably, but the mechanisms behind this variability are poorly understood, limiting their utilization for drug discovery. This is particularly so for G protein-coupled receptors (GPCRs) where high resolution structural information is only beginning to emerge. Engineering the human A2A adenosine receptor has allowed structures to be solved in complex with the reference compound ZM241385 and four related ligands at high resolution. Differences between the structures are limited, with the most pronounced being the interaction of each ligand with a salt bridge on the extracellular side of the receptor. Mutagenesis experiments confirm the role of this salt bridge in controlling the dissociation kinetics of the ligands from the receptor, while molecular dynamics simulations demonstrate the ability of ligands to modulate salt bridge stability. These results shed light on a structural determinant of ligand dissociation kinetics and identify a means by which this property may be optimized.
Assuntos
Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/metabolismo , Triazinas/química , Triazinas/farmacologia , Triazóis/química , Triazóis/farmacologia , Células Cultivadas , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Engenharia de Proteínas , Receptor A2A de Adenosina/genética , 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
Tissue transglutaminase 2 (TG2) is a multifunctional protein primarily known for its calcium-dependent enzymatic protein cross-linking activity via isopeptide bond formation between glutamine and lysine residues. TG2 overexpression and activity have been found to be associated with Huntington's disease (HD); specifically, TG2 is up-regulated in the brains of HD patients and in animal models of the disease. Interestingly, genetic deletion of TG2 in two different HD mouse models, R6/1 and R6/2, results in improved phenotypes including a reduction in neuronal death and prolonged survival. Starting with phenylacrylamide screening hit 7d, we describe the SAR of this series leading to potent and selective TG2 inhibitors. The suitability of the compounds as in vitro tools to elucidate the biology of TG2 was demonstrated through mode of inhibition studies, characterization of druglike properties, and inhibition profiles in a cell lysate assay.
Assuntos
Acrilamidas/síntese química , Proteínas de Ligação ao GTP/antagonistas & inibidores , Doença de Huntington/tratamento farmacológico , Sulfonamidas/síntese química , Transglutaminases/antagonistas & inibidores , Acrilamidas/química , Acrilamidas/farmacologia , Animais , Células CACO-2 , Permeabilidade da Membrana Celular , Células HEK293 , Humanos , Técnicas In Vitro , Masculino , Camundongos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Piperazinas/síntese química , Piperazinas/química , Piperazinas/farmacologia , Proteína 2 Glutamina gama-Glutamiltransferase , Piridinas/síntese química , Piridinas/química , Piridinas/farmacologia , Pirimidinas/síntese química , Pirimidinas/química , Pirimidinas/farmacologia , Ratos , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
(1,1-Dioxo-2H-[1,2,4]benzothiadiazin-3-yl) azolo[1,5-a]pyridine and azolo[1,5-a]pyrimidine derivatives have been investigated as potential anti-HCV drugs. Their synthesis, HCV NS5B polymerase inhibition, and replicon activity are discussed.
Assuntos
Antivirais/síntese química , Azóis/síntese química , Benzotiadiazinas/síntese química , Inibidores Enzimáticos/síntese química , Hepatite C/tratamento farmacológico , Piridinas/síntese química , Pirimidinas/síntese química , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/farmacologia , Azóis/farmacologia , Benzotiadiazinas/farmacologia , Química Farmacêutica/métodos , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Hepacivirus/metabolismo , Técnicas In Vitro , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Modelos Químicos , Estrutura Molecular , Piridinas/farmacologia , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Proteínas não Estruturais Virais/químicaRESUMO
Heat shock protein 90 (Hsp90) plays a key role in stress response and protection of the cell against the effects of mutation. Herein we report the identification of an Hsp90 inhibitor identified by fragment screening using a high-concentration biochemical assay, as well as its optimisation by in silico searching coupled with a structure-based drug design (SBDD) approach.