Antagonist binding and induced conformational dynamics of GPCR A2A adenosine receptor

The A_2A adenosine receptor (A_2AAR) is a unique G‐protein coupled receptor (GPCR), because besides agonist, its antagonist could also lead to therapeutic relevance. Based on A_2AAR‐antagonist crystal structure, we have studied the binding mechanism of two distinct antagonists, ZM241385 and KW6002, and dynamic behaviors of A_2AAR induced by antagonist binding. Key residues interacting with both antagonists and residues specifically binding to one of them are identified. ZM241385 specifically bound to S67^2.65, M177^5.38, and N253^6.55, while KW6002 binds to F62^2.60, A81^3.29, and H264^7.29. Moreover, interactions with L167^5.28 are found for both antagonists, which were not reported in agonist binding. The dynamic behaviors of antagonist bound holo‐A_2AARs were found to be different from the apo‐A_2AAR in three typical functional switches, (i) the “ionic lock” was in equilibrium between formation and breakage in apo‐A_2AAR, but stayed broken in holo‐A_2AARs; (ii) the “rotamer toggle switch,” T88^3.36/F242^6.44/W246^6.48, adopted different rotameric conformations in apo‐A_2AAR and holo‐A_2AARs; (iii) apo‐A_2AAR preferred α‐helical intracellular loop (IC)2 and flexible IC3, while holo‐A_2AARs had a flexible IC2 and α‐helical IC3. Our results indicated that antagonist binding induced different conformational rearrangements of these characteristic functional switches in apo‐A_2AAR and holo‐A_2AARs. Proteins 2013; 81:1399–1410. © 2013 Wiley Periodicals, Inc.     

Characterization of a human peptide deformylase: implications for antibacterial drug design

Ribosomal protein synthesis in eubacteria and eukaryotic organelles initiates with an N-formylmethionyl-tRNA(i), resulting in N-terminal formylation of all nascent polypeptides. Peptide deformylase (PDF) catalyzes the subsequent removal of the N-terminal formyl group from the majority of bacterial proteins. Deformylation was for a long time thought to be a feature unique to the prokaryotes, making PDF an attractive target for designing novel antibiotics. However, recent genomic sequencing has revealed PDF-like sequences in many eukaryotes, including man. In this work, the cDNA encoding Homo sapiens PDF (HsPDF) has been cloned and a truncated form that lacks the N-terminal 58-amino-acid targeting sequence was overexpressed in Escherichia coli. The recombinant, Co(2+)-substituted protein is catalytically active in deformylating N-formylated peptides, shares many of the properties of bacterial PDF, and is strongly inhibited by specific PDF inhibitors. Expression of HsPDF fused to the enhanced green fluorescence protein in human embryonic kidney cells revealed its location in the mitochondrion. However, HsPDF is much less active than its bacterial counterpart, providing a possible explanation for the apparent lack of deformylation in the mammalian mitochondria. The lower catalytic activity is at least partially due to mutation of a highly conserved residue (Leu-91 in E. coli PDF) in mammalian PDF. PDF inhibitors had no detectable effect on two different human cell lines. These results suggest that HsPDF is likely an evolutional remnant without any functional role in protein formylation/deformylation and validates PDF as an excellent target for antibacterial drug design.     

Optimization of the human adenosine A2a receptor yields in Saccharomyces cerevisiae

G-protein coupled receptors (GPCRs) have been implicated in many human diseases and have emerged as important drug targets. Despite their medical relevance, knowledge about GPCR structure is limited, mainly due to difficulties associated with producing large amounts of functional protein and isolating this protein in functional form. However, our previous results indicate that when the human adenosine A(2)a receptor (A(2)aR) is expressed in Saccharomyces cerevisiae, high yields can be achieved. In light of these initial results and in anticipation of future purification efforts, experiments were conducted to optimize the system for maximum total protein yield. Emphasis was placed on not only producing large quantities of A(2)aR in each cell but also achieving high cell density in batch culture. Therefore, temperature, media pH, inducer concentration in the media, and induction cell density were tested for their effects on both cell growth (as measured by optical density, OD(600)) and per cell A(2)aR expression levels. For these studies, the A(2)aR expression levels were determined using a previously described A(2)aR-green fluorescent protein (GFP) fusion, so that expression could be monitored by fluorescence. Overall the data indicate that at late times ( approximately 60 h of expression) approximately 75% higher total batch protein yields can be achieved using lower expression temperatures or 60% higher using elevated induction cell density. The highest yields correspond to approximately 28 mg per liter of culture of total A(2)aR. Amounts of functional receptor were shown to increase on a per cell basis by decreasing expression temperature up to 25 h of expression, but at late time points ( approximately 60 h) functional yields did not appreciably improve. When compared to other reports of GPCR expression in yeast it is clear that this system is among those producing the highest GPCR protein yields per culture both before and after optimization.     

Characterization of Dahl salt-sensitive rats with genetic disruption of the A2B adenosine receptor gene: implications for A2B adenosine receptor signaling during hypertension

The A_2B adenosine receptor (AR) has emerged as a unique member of the AR family with contrasting roles during acute and chronic disease states. We utilized zinc-finger nuclease technology to create A_2BAR gene (Adora2b)-disrupted rats on the Dahl salt-sensitive (SS) genetic background. This strategy yielded a rat strain (SS-Adora2b mutant rats) with a 162-base pair in-frame deletion of Adora2b that included the start codon. Disruption of A_2BAR function in SS-Adora2b mutant rats was confirmed by loss of agonist (BAY 60-6583 or NECA)-induced cAMP accumulation and loss of interleukin-6 release from isolated fibroblasts. In addition, BAY 60-6583 produced a dose-dependent increase in glucose mobilization that was absent in SS-Adora2b mutants. Upon initial characterization, SS-Adora2b mutant rats were found to exhibit increased body weight, a transient delay in glucose clearance, and reduced proinflammatory cytokine production following challenge with lipopolysaccharide (LPS). In addition, blood pressure was elevated to a greater extent (∼15–20 mmHg) in SS-Adora2b mutants as they aged from 7 to 21 weeks. In contrast, hypertension augmented by Ang II infusion was attenuated in SS-Adora2b mutant rats. Despite differences in blood pressure, indices of renal and cardiac injury were similar in SS-Adora2b mutants during Ang II-augmented hypertension. We have successfully created and validated a new animal model that will be valuable for investigating the biology of the A_2BAR. Our data indicate varying roles for A_2BAR signaling in regulating blood pressure in SS rats, playing both anti- and prohypertensive roles depending on the pathogenic mechanisms that contribute to blood pressure elevation.     

The extracellular regions of PSMA and the transferrin receptor contain an aminopeptidase domain: implications for drug design

The Aeromonas proteolytica aminopeptidase (AMP), Pseudomonas sp. (RS-16) carboxypeptidase G2 (CPG2), and Streptomyces griseus aminopeptidase (SGAP) are zinc dependent proteolytic enzymes with cocatalytic zinc ion centers and a conserved aminopeptidase fold. A BLAST search with the sequence of the solved AMP structure indicated that a similar domain could be found in prostate-specific membrane antigen (PSMA) and the transferrin receptor (TfR). When the PSMA or TfR sequence was input into the THREADER program, the top structural matches were SGAP and AMP confirming that these are structurally conserved domains. Optimal sequence alignment of PSMA and TfR using the known three-dimensional structures of AMP, CPG2, and SGAP shows that the critical amino acids involved in forming the catalytic pocket are conserved in PSMA but absent in the TfR. The specificity pocket in AMP is formed from four aromatic side chains and the equivalent region in CPG2/PSMA has a changed sequence pattern. Since CPG2 and PSMA are folate hydrolases, the changed specificity pocket leaves space to accommodate the large pteroate moiety of folic acid. In contrast, no enzyme function has been ascribed to the TfR.     

Molecular basis for anti-insomnia drug design from structure of lemborexant-bound orexin 2 receptor

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GPCR screening via ERK 1/2: a novel platform for screening G protein-coupled receptors

Discovery of novel agonists and antagonists for G protein-coupled receptors (GPCRs) relies heavily on cell-based assays because determination of functional consequences of receptor engagement is often desirable. Currently, there are several key parameters measured to achieve this, including mobilization of intracellular Ca2+ and formation of cyclic adenosine monophosphate or inositol triphosphate. However, no single assay platform is suitable for all situations, and all of the assays have limitations. The authors have developed a new high-throughput homogeneous assay platform for GPCR discovery as an alternative to current assays, which employs detection of phosphorylation of the key signaling molecule p42/44 MAP kinase (ERK 1/2). The authors show that ERK 1/2 is consistently activated in cells stimulated by Gq-coupled GPCRs and provides a new high-throughput platform for screening GPCR drug candidates. The activation of ERK 1/2 in Gq-coupled GPCR systems generates comparable pharmacological data for receptor agonist and antagonist data obtained by other GPCR activation measurement techniques.     

Probing the structure and function of human glutaminase-interacting protein: a possible target for drug design

PDZ domains are one of the most ubiquitous protein-protein interaction modules found in living systems. Glutaminase interacting protein (GIP), also known as Tax interacting protein 1 (TIP-1), is a PDZ domain-containing protein, which plays pivotal roles in many aspects of cellular signaling, protein scaffolding and modulation of tumor growth. We report here the overexpression, efficient refolding, single-step purification, and biophysical characterization of recombinant human GIP with three different C-terminal target protein recognition sequence motifs by CD, fluorescence, and high-resolution solution NMR methods. It is clear from our NMR analysis that GIP contains 2 alpha-helices and 6 beta-strands. The three target protein C-terminal recognition motifs employed in our interaction studies are glutaminase, beta-catenin and FAS. This is the first report of GIP recognition of the cell surface protein FAS, which belongs to the tumor necrosis factor (TNF) receptor family and mediates cell apoptosis. The dissociation constant ( K D) values for the binding of GIP with different interacting partners as measured by fluorescence spectroscopy range from 1.66 to 2.64 microM. Significant chemical shift perturbations were observed upon titration of GIP with above three ligands as monitored by 2D {(1)H, (15)N}-HSQC NMR spectroscopy. GIP undergoes a conformational change upon ligand binding.     

Pharmacophore modeling of human adenosine receptor A2A antagonists

Three-dimensional pharmacophore models of human adenosine receptor A_2A antagonists were developed based on 23 diverse compounds selected from a large number of A_2A antagonists. The best pharmacophore model, Hypo1, contained five features: one hydrogen bond donor , three hydrophobic points and one ring aromatic. Its correlation coefficient, root mean square deviation, and cost difference values were 0.955, 0.921 and 84.4, respectively, suggested that the Hypo1 model was reasonable and reliable. This model was validated by three methods: a test set of 106 diverse compounds, a simulated virtual screening, and superimposition with the crystal structure of A_2A receptor. The results showed that Hypo1 was not only in agreement with the A_2A crystal structure and literature reports, but also well identified active A_2A antagonists from the virtual database. This methodology provides helpful information and a robust tool for the discovery of potent A_2A antagonists.     

New tools for G-protein coupled receptor (GPCR) drug discovery: combination of baculoviral expression system and solid state NMR

Biotechnology using molecular biology, biochemistry, biophysics, and computational approaches provides an alternative approach for classical pharmacological screening to look at ligand-receptor interactions and receptor specificity, which should support the design of selective drugs based on detailed structural principles. This review addresses specific approaches to study function, structure and relevance of a major pharmaceutical target, namely the G-Protein Coupled Receptors (GPCRs). The main aim of this review has been to exploit and combine GPCR over-expression in a baculoviral expression system with solid-state MAS NMR (ssNMR) approaches for the elucidation of electronic structures of the coordinating ligands/drugs and their modes of interactions with the GPCRs. This review summarizes the approaches, possible future experiments and developments using the above combination of tools for GPCR drug discovery.     

New A2A adenosine receptor antagonists: a structure-based upside-down interaction in the receptor cavity

Adenosine receptor antagonists are generally based on heterocyclic core structures presenting substituents of various volumes and chemical-physical profiles. Adenine and purine-based adenosine receptor antagonists have been reported in literature. In this work we combined various substituents in the 2, 6, and 8-positions of 9-ethylpurine to depict a structure-affinity relationship analysis at the human adenosine receptors. Compounds were rationally designed trough molecular modeling analysis and then synthesized and evaluated at radioligand binding studies at human adenosine receptors. The new compounds showed affinity for the human adenosine receptors, with some derivatives endowed with low nanomolar K_i data, in particular at the A_2AAR subtype. The purine core proves to be a versatile core structure for the development of novel adenosine receptor antagonists with nanomolar affinity for these membrane proteins.     

The concept of a changing receptor concentration: implications for the theory of drug action

Drugs are considered to produce their effects on biological tissues either by altering some physical property of cells or by interacting with specific cellular components, called receptors. Most drugs and endogenous neurotransmitters act on highly selective receptors located on the outer surface membrane of cells. These receptors were believed, until recently, to be stationary on the cell surface and to be present in unvarying numbers. Consequently, most early theorists modeled the drug-receptor interaction on the basis of stationary and static receptor molecules. The substantial advances in our understanding of drug action based on these models have partly justified this view. However, recent electron microscopic studies have revealed the presence of structures, including "coated" pits and vesicles, that appear to provide a mechanism by which cell surface receptors might be internalized in a process of endocytosis. The precise intracellular fate of these internalized receptors is unknown, but based on present understanding, it seems reasonable to believe that some are destroyed intracellularly whereas others are recycled to the cell surface. The importance of such processes to pharmacologic theory is a new awareness of a cellular pathway that is capable of internalizing drugs, receptors, or both. The implications of such a process to the theory of drug action extends to some unexplained drug phenomena such as down regulation, drug tolerance, tachyphyllaxis, and partial agonism. We present herein the theoretical framework for a model of drug action that incorporates the possibility of receptor internalization and subsequent degradation, recycling, or replacement.     

The existence of a second allosteric site on the M1 muscarinic acetylcholine receptor and its implications for drug design

Fully flexible docking of KT5720, an allosteric modulator of the muscarinic receptors, was performed on a dynamic model of the M(1) muscarinic acetylcholine receptor. The results confirmed the existence of a second allosteric site, located on the intracellular face of the receptor. These results would be beneficial for the design of modulators of this receptor to be used as an effective alternative against the Alzheimer's disease.     

2-Aryl tryptamines: selective high-affinity antagonists for the h5-HT2A receptor

A series of 2-aryl tryptamines have been identified as high-affinity h5-HT_2A antagonists. Structure–activity relationship studies have shown that h5-HT_2A affinity can be attained via modifications to the tryptamine side chain and that selectivity over h5-HT_2C and hD₂ receptors can be controlled by suitable C-2 aryl groups.     

Evidence for an adenosine A2/Ra receptor on human basophils

5'-N-ethylcarboxamideadenosine (NECA) greater than 2-chloroadenosine greater than adenosine greater than (-)-N6-(R-phenyl-isopropyl)-adenosine greater than (+)-N6-(S-phenylisopropyl)-adenosine, in that order of potency, inhibited in vitro antigen-induced histamine release from human basophils in a dose-dependent fashion. Inhibition occurred only during the first stage of antigen-induced histamine release and the nucleosides failed to inhibit the release caused by the Ca2+ ionophore, A23187. 6-nitrobenzylthioinosine and dipyridamole, which inhibit adenosine uptake, and erythro-9-(2-hydroxy-3-nonyl)adenine, which blocks adenosine metabolism, did not impair the inhibition caused by NECA and adenosine. 8-phenyltheophylline and theophylline, two competitive antagonists of adenosine receptors, blocked the inhibition caused by NECA and adenosine. These data suggest that NECA and other adenosine analogs activate a specific cell surface adenosine receptor which possesses properties similar to those of an adenosine A2/Ra receptor.     

A major role for adenosine A2A receptor in the interaction between astrocytes and myelinated neurons: possible implications for the therapy of neurodegenerative disorders

Purinergic Signalling -     

Structure-affinity relationships of the affinity of 2-pyrazolyl adenosine analogues for the adenosine A2A receptor

The structure-affinity relationships of two novel 2-substituted adenosine series containing a substituted pyrazole attached at the N-1 or C-4 position for the adenosine (ADO) A2A receptor are described. Compounds in the 2-(N-1-pyrazolyl) adenosine series IV provided the highest affinity for the ADO A2A receptor as compared to the 2-(C-4-pyrazolyl) series V. The main structural differences between the two series point to the N-1 nitrogen of series IV imparting more favorable binding interactions with the receptor than those of series V.     

First crystallographic structure of a xylanase from glycoside hydrolase family 5: implications for catalysis

The room-temperature structure of xylanase (EC 3.2.1.8) from the bacterial plant pathogen Erwinia chrysanthemi expressed in Escherichia coli, a 45 kDa, 413-amino acid protein belonging to glycoside hydrolase family 5, has been determined by multiple isomorphous replacement and refined to a resolution of 1.42 A. This represents the first structure of a xylanase not belonging to either glycoside hydrolase family 10 or family 11. The enzyme is composed of two domains similar to most family 10 xylanases and the alpha-amylases. The catalytic domain (residues 46-315) has a (beta/alpha)(8)-barrel motif with a binding cleft along the C-terminal side of the beta-barrel. The catalytic residues, Glu165 and Glu253, determined by correspondence to other family 5 and family 10 glycoside hydrolases, lie inside this cleft on the C-terminal ends of beta-strands 4 and 7, respectively, with an O(epsilon)2...O(epsilon)1 distance of 4.22 A. The smaller domain (residues 31-43 and 323-413) has a beta(9)-barrel motif with five of the strands interfacing with alpha-helices 7 and 8 of the catalytic domain. The first 13 N-terminal residues form one beta-strand of this domain. Residues 44, 45, and 316-322 form the linkers between this domain and the catalytic domain.