Human A2A Adenosine Receptor Research Articles

Design, Synthesis and Evaluation of New Indolylpyrimidylpiperazines for Gastrointestinal Cancer Therapy.

Human A3 adenosine receptor hA3AR has been implicated in gastrointestinal cancer, where its cellular expression has been found increased, thus suggesting its potential as a molecular target for novel anticancer compounds. Observation made in our previous work indicated the importance of the carbonyl group of amide in the indolylpyrimidylpiperazine (IPP) for its human A2A adenosine receptor (hA2AAR) subtype binding selectivity over the other AR subtypes. Taking this observation into account, we structurally modified an indolylpyrimidylpiperazine (IPP) scaffold, 1 (a non-selective adenosine receptors’ ligand) into a modified IPP (mIPP) scaffold by switching the position of the carbonyl group, resulting in the formation of both ketone and tertiary amine groups in the new scaffold. Results showed that such modification diminished the A2A activity and instead conferred hA3AR agonistic activity. Among the new mIPP derivatives (3–6), compound 4 showed potential as a hA3AR partial agonist, with an Emax of 30% and EC50 of 2.89 ± 0.55 μM. In the cytotoxicity assays, compound 4 also exhibited higher cytotoxicity against both colorectal and liver cancer cells as compared to normal cells. Overall, this new series of compounds provide a promising starting point for further development of potent and selective hA3AR partial agonists for the treatment of gastrointestinal cancers.

Revisiting the Allosteric Regulation of Sodium Cation on the Binding of Adenosine at the Human A2A Adenosine Receptor: Insights from Supervised Molecular Dynamics (SuMD) Simulations.

One of the most intriguing findings highlighted from G protein-coupled receptor (GPCR) crystallography is the presence, in many members of class A, of a partially hydrated sodium ion in the middle of the seven transmembrane helices (7TM) bundle. In particular, the human adenosine A2A receptor (A2A AR) is the first GPCR in which a monovalent sodium ion was crystallized in a distal site from the canonical orthosteric one, corroborating, from a structural point of view, its role as a negative allosteric modulator. However, the molecular mechanism by which the sodium ion influences the recognition of the A2A AR agonists is not yet fully understood. In this study, the supervised molecular dynamics (SuMD) technique was exploited to analyse the sodium ion recognition mechanism and how its presence influences the binding of the endogenous agonist adenosine. Due to a higher degree of flexibility of the receptor extracellular (EC) vestibule, we propose the sodium-bound A2A AR as less efficient in stabilizing the adenosine during the different steps of binding.

Abstract 4140: Discovery and characterization of novel highly potent A2A adenosine receptor antagonists for cancerimmunotherapy

Abstract Introduction: Extracellular adenosine produced at high concentrations within the tumor micro-environment (TME) and suppresses immune function via inhibition of immune cell activation. Targeting adenosine receptors has emerged as a novel method to activate anti-tumor immunity. In particular, A2A adenosine receptor (A2AR), one of the G-protein-coupled-receptors, exhibits immunosuppressive functions. Herein, we suggest novel A2AR antagonist lead compounds as a highly potent antagonist of A2AR for immunotherapy. Materials and Methods: Novel A2AR antagonist lead compounds were designed using CADD and synthesized as the active biologic inhibitory compound. The protein preparation and molecular docking were performed using Glide (Schrödinger). A radioligand binding assay was performed to evaluate the affinities of A2AR Antagonists for the human adenosine A2AR. The potencie of A2AR antagonists were determined by cAMP assay in HEK293-hA2AR cells and cAMP-mediated pCREB assay in human CD8+ T cells from whole blood. In vivo CT26 and MC38 syngeneic tumor models were used to assess the therapeutic effect of A2AR antagonists. Results: A2AR antagonist lead compounds showed strong binding affinities toward human A2AR. They also potently inhibited the NECA-mediated production of intracellular cAMP in HEK293 cells expressing human A2AR. Elevated intracellular cAMP following A2AR activation results in the phosphorylation of CREB (cAMP response element-binding protein). A2AR antagonist lead compounds treatment inhibited pCREB in NECA-stimulated HEK293-hA2AR and human CD8+ T cells. A2AR antagonist lead compounds inhibited tumor growth of mouse syngeneic tumor models as a single agent and combination with anti-PD-L1. In combination with anti-PD-L1, A2AR antagonist lead compounds had remarkable antitumor activities in multiple mouse tumor models, including restoration of immune responses in models that incompletely responded to anti-PD-L1 monotherapy. Conclusion: These results showed the potencies of A2AR antagonist lead compounds with high capability of A2AR inhibition. Blockade of the adenosine signaling pathway may be vital for enhancing anti-tumor responses in solid tumors that show an incomplete response to anti-PD-L1 therapy. A2AR antagonist lead compounds demonstrate a novel approach to anti-cancer immunotherapy. Citation Format: Kyungik Lee, Seungah Jun, EunYoung Byun, Hosun Lee, Yongtaek Lee, MiJin Moon, Yu-Yon Kim, Hyun Jeong Kang, YoungGil Ahn, YoungHoon Kim, Kwee Hyun Suh. Discovery and characterization of novel highly potent A2A adenosine receptor antagonists for cancerimmunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4140.

2-Aryladenine derivatives as a potent scaffold for A1, A3 and dual A1/A3 adenosine receptor antagonists: Synthesis and structure-activity relationships

From a collection containing more than 1500 academic compounds, in silico screening identified a hit for the human A1 adenosine receptor containing a new purine scaffold. To study the structure activity relationships of this new chemical series for adenosine receptors, a library of 24 purines was synthesized and tested in radioligand binding assays at human A1, A2A, A2B and A3 adenosine receptor subtypes. Fourteen molecules showed potent antagonism at A1, A3 or dual A1/A3 adenosine receptors. This purine scaffold is an important source for novel biochemical tools and/or therapeutic drugs.

Structural Plasticity of a Human G Protein‐Coupled Receptor Studied by NMR Spectroscopy

The human genome encodes for 826 G protein‐coupled receptors (GPCRs), which are involved in nearly every physiological process and comprise the largest class of “druggable” proteins. Using nuclear magnetic resonance (NMR) in solution, we provide a global view of structural plasticity underlying the function of the human A2Aadenosine receptor (A2AAR), a representative class A human GPCR. To apply NMR to study a human GPCR we developed a robust molecular biology pipeline for production and sample preparation of stable‐isotope labeled receptors, yielding reproducible high‐quality NMR data. From these data, assignment of endogenous residues provided numerous probes distributed over the receptor intracellular surface, extracellular surface, and transmembrane domain, enabling a global characterization of A2AAR responses to variable efficacy of bound drugs. The NMR probes also permitted novel insights into changes in the local conformation of conserved GPCR activation motifs. In addition, we observed multiple local efficacy‐related conformers at the intracellular surface, which we relate directly to G protein signaling. The distributed NMR probes provide novel information on the role of a highly conserved GPCR allosteric center, permitting us to study the effect of this center on signaling pathways in the receptor. Amino acid replacement of the allosteric center caused striking changes in conformational polymorphisms at the intracellular surface but did not alter response to drug efficacy at the extracellular surface. We correlated changes at the intracellular surface to structural rearrangements at the toggle switch tryptophan and a conserved activation motif, providing novel insights into a widespread signaling mechanism in class A GPCRs. The presented work opens the door to exciting future studies of GPCR function, including studies of GPCRs in native environments.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel 8-amino-1,2,4-triazolo[4,3-a]pyrazin-3-one derivatives as potent human adenosine A1 and A2A receptor antagonists. Evaluation of their protective effect against β-amyloid-induced neurotoxicity in SH-SY5Y cells

In this work, an enlarged series of 1,2,4-triazolo[4,3-a]pyrazin-3-ones was designed to target the human (h) A2A adenosine receptor (AR) or both hA1 and hA2A ARs. The novel 8-amino-1,2,4-triazolopyrazin-3-one derivatives 1–25 featured a phenyl or a benzyl pendant at position 2 while different aryl/heteroaryl substituents were placed at position 6. Two compounds (8 and 10) endowed with high affinity (Ki = 7.2 and 10.6 nM) and a complete selectivity for the hA2A AR were identified. Moreover, several derivatives possessed nanomolar affinity for both hA1 and hA2A ARs (both Ki < 20 nM) and different degrees of selectivity versus the hA3 AR. Two selected compounds (10 and 25) demonstrated ability in preventing β-amyloid peptide (25–35)-induced neurotoxicity in SH-SY5Y cells. Results of docking studies at the hA2A and hA1 AR crystal structures helped us to rationalize the observed affinity data and to highlight that the steric hindrance of the substituents at the 2- and 6-position of the bicyclic core affects the binding mode in the receptor cavity.

Dimerization of Human Adenosine A2AReceptor - Impact of the C-Terminus

It has long been recognized that the functions and activities of G protein-coupled receptors (GPCRs) can be regulated by oligomerization. Among this largest family of receptors, the human adenosine A2A receptor (A2AR) is capable of forming homo-oligomers and hetero-oligomers with other GPCRs, playing an impactful role in several CNS disorders. Crystal structures and previous studies of A2AR usually involve the truncation of the 95-residue long C-terminal tail to enhance conformational and thermostability of the receptor. However, there remain major unknowns in the role of this disordered C-terminus in the dimerization of A2AR. The ultimate goal of the project is to understand at the molecular level (1) the impact of the C-terminus to A2AR dimerization, (2) the dimeric interface, and (3) potential functional consequences. Our approach involves the use of size-exclusion chromatography (SEC), electron paramagnetic resonance (EPR), and cryogenic electron microscopy (cryo-EM).

Studying Structural Plasticity Underlying GPCR Function

The human genome encodes for 826 G protein-coupled receptors (GPCRs), which are involved in nearly every physiological process and comprise the largest class of “druggable” proteins. Using nuclear magnetic resonance (NMR) in solution, we provide a global view of structural plasticity underlying the function of the human A2A adenosine receptor (A2AAR), a representative class A human GPCR. To apply NMR to study a human GPCR we developed a robust molecular biology pipeline for production and sample preparation of stable-isotope labeled receptors, yielding reproducible high-quality NMR data. From these data, assignment of endogenous residues provided numerous probes distributed over the receptor intracellular surface, extracellular surface, and transmembrane domain, enabling a global characterization of A2AAR responses to variable efficacy of bound drugs. The NMR probes also permitted novel insights into changes in the local conformation of conserved GPCR activation motifs. In addition, we observed multiple local efficacy-related conformers at the intracellular surface, which we relate directly to G protein signaling. The distributed NMR probes provide novel information on the role of a highly conserved GPCR allosteric center, permitting us to study the effect of this center on signaling pathways in the receptor. Amino acid replacement of the allosteric center caused striking changes in conformational polymorphisms at the intracellular surface but did not alter response to drug efficacy at the extracellular surface. We correlated changes at the intracellular surface to structural rearrangements at the toggle switch tryptophan and a conserved activation motif, providing novel insights into a widespread signaling mechanism in class A GPCRs. The presented work opens the door to exciting future studies of GPCR function, including studies of GPCRs in native environments.

Isotopic Labeling of Eukaryotic Membrane Proteins for NMR Studies of Interactions and Dynamics.

Membrane proteins, and especially G-protein coupled receptors (GPCRs), are increasingly important targets of structural biology studies due to their involvement in many biomedically critical pathways in humans. These proteins are often highly dynamic and thus benefit from studies by NMR spectroscopy in parallel with complementary crystallographic and cryo-EM analyses. However, such studies are often complicated by a range of practical concerns, including challenges in preparing suitably isotopically labeled membrane protein samples, large sizes of protein/detergent or protein/lipid complexes, and limitations on sample concentrations and stabilities. Here we describe our approach to addressing these challenges via the use of simple eukaryotic expression systems and modified NMR experiments, using the human adenosine A2A receptor as an example. Protocols are provided for the preparation of U-2H (13C,1H-Ile δ1)-labeled membrane proteins from overexpression in the methylotrophic yeast Pichia pastoris, as well as techniques for studying the fast ns-ps sidechain dynamics of the methyl groups of such samples. We believe that, with the proper optimization, these protocols should be generalizable to other GPCRs and human membrane proteins.

A2A adenosine receptor functional states characterized by 19F-NMR.

The human proteome contains 826 G protein-coupled receptors (GPCR), which control a wide array of key physiological functions, making them important drug targets. GPCR functions are based on allosteric coupling from the extracellular orthosteric drug binding site across the cell membrane to intracellular binding sites for partners such as G proteins and arrestins. This signaling process is related to dynamic equilibria in conformational ensembles that can be observed by NMR in solution. A previous high-resolution NMR study of the A2A adenosine receptor (A2AAR) resulted in a qualitative characterization of a network of such local polymorphisms. Here, we used 19F-NMR experiments with probes at the A2AAR intracellular surface, which provides the high sensitivity needed for a refined description of different receptor activation states by ensembles of simultaneously populated conformers and the rates of exchange among them. We observed two agonist-stabilized substates that are not measurably populated in apo-A2AAR and one inactive substate that is not seen in complexes with agonists, suggesting that A2AAR activation includes both induced fit and conformational selection mechanisms. Comparison of A2AAR and a constitutively active mutant established relations between the 19F-NMR spectra and signaling activity, which enabled direct assessment of the difference in basal activity between the native protein and its variant.

Could the presence of sodium ion influence the accuracy and precision of the ligand-posing in the human A2A adenosine receptor orthosteric binding site using a molecular docking approach? Insights from Dockbench.

The allosteric modulation of G protein-coupled receptors (GPCRs) by sodium ions has received considerable attention as crystal structures of several receptors, in their inactive conformation, show a Na+ ion bound to specific residues which, in the human A2A adenosine receptor (hA2A AR), are Ser913.39, Trp2466.48, Asn2807.45, and Asn2847.49. A cluster of water molecules completes the coordination of the sodium ion in the putative allosteric site. It is absolutely consolidated that the progress made in the field of GPCRs structural determination has increased the adoption of docking-driven approaches for the identification or the optimization of novel potent and selective ligands. Despite the extensive use of docking protocols in virtual screening approaches, to date, almost any of these studies have been carried out without taking into account the presence of the sodium cation and its first solvation shell in the putative allosteric binding site. In this study, we have focused our attention on determining how the presence of sodium ion binding and additionally its first hydration sphere, in hA2AAR could influence the ligand positioning accuracy during molecular docking simulations for most of the available resting and activated hA2A AR crystal structures, using DockBench as a comparative benchmarking tool and implementing a new correlation coefficient (EM). This work provides indications on the evidence that the posing performance (accuracy and/or precision) of the docking protocols in reproducing the crystallographic poses of different hA2A AR antagonists is generally increased in the presence of the sodium cation and its first solvation shell, in agreement with experimental observations. Consequently, the inclusion of sodium ion and its first solvation shell should be considered in order to facilitate the selection of new potential ligands in all molecular docking-based virtual screening protocols that aim to find novel GPCRs antagonists and inverse agonists.

PBF509, an Adenosine A2A Receptor Antagonist With Efficacy in Rodent Models of Movement Disorders.

Adenosine A2A receptor (A2AR) antagonists have emerged as complementary non-dopaminergic drugs to alleviate Parkinson’s disease (PD) symptomatology. Here, we characterize a novel non-xhantine non-furan A2AR antagonist, PBF509, as a potential pro-dopaminergic drug for PD management. First, PBF509 was shown to be a highly potent ligand at the human A2AR, since it antagonized A2AR agonist-mediated cAMP accumulation and impedance responses with KB values of 72.8 ± 17.4 and 8.2 ± 4.2 nM, respectively. Notably, these results validated our new A2AR-based label-free assay as a robust and sensitive approach to characterize A2AR ligands. Next, we evaluated the efficacy of PBF509 reversing motor impairments in several rat models of movement disorders, including catalepsy, tremor, and hemiparkinsonism. Thus, PBF509 (orally) antagonized haloperidol-mediated catalepsy, reduced pilocarpine-induced tremulous jaw movements and potentiated the number of contralateral rotations induced by L-3,4-dihydroxyphenylalanine (L-DOPA) in unilaterally 6-OHDA-lesioned rats. Moreover, PBF509 (3 mg/kg) inhibited L-DOPA-induced dyskinesia (LID), showing not only its efficacy on reversing parkinsonian motor impairments but also acting as antidyskinetic agent. Overall, here we describe a new orally selective A2AR antagonist with potential utility for PD treatment, and for some of the side effects associated to the current pharmacotherapy (i.e., dyskinesia).

1,2,4]Triazolo[1,5-c]pyrimidines as adenosine receptor antagonists: Modifications at the 8 position to reach selectivity towards A3 adenosine receptor subtype

[1,2,4]Triazolo[1,5-c]pyrimidine is a promising platform to develop adenosine receptor antagonists. Here, we tried to investigate the effect of the substituent at the 8 position of [1,2,4]triazolo[1,5-c]pyrimidine derivatives on affinity and selectivity at the human A3 adenosine receptor subtype. In particular, we have introduced both esters and amides, principally with a benzylic nature. In addition, a small series of 5-substituted [1,2,4]triazolo[1,5-c]pyrimidines was designed in order to complete the structure-activity relationship analysis. Several of these new compounds showed affinity towards human A3 adenosine receptor in the low nanomolar range, with the most potent derivative of the series bringing a 4-ethylbenzylester at the 8 position (compound 18, hA3AR Ki = 1.21 nM). Docking studies performed on the synthesized compounds inside models of human A1, A2A and A3 adenosine receptors showed similar binding modes, comparable with the typical crystallographic binding mode of the inverse agonist ZM-241,385.

An Affinity-Based Probe for the Human Adenosine A2A Receptor.

Using activity-based protein profiling (ABPP), functional proteins can be interrogated in their native environment. Despite their pharmaceutical relevance, G protein-coupled receptors (GPCRs) have been difficult to address through ABPP. In the current study, we took the prototypical human adenosine A2A receptor (hA2AR) as the starting point for the construction of a chemical toolbox allowing two-step affinity-based labeling of GPCRs. First, we equipped an irreversibly binding hA2AR ligand with a terminal alkyne to serve as probe. We showed that our probe irreversibly and concentration-dependently labeled purified hA2AR. Click-ligation with a sulfonated cyanine-3 fluorophore allowed us to visualize the receptor on SDS-PAGE. We further demonstrated that labeling of the purified hA2AR by our probe could be inhibited by selective antagonists. Lastly, we showed successful labeling of the receptor in cell membranes overexpressing hA2AR, making our probe a promising affinity-based tool compound that sets the stage for the further development of probes for GPCRs.

Probing Substituents in the 1- and 3-Position: Tetrahydropyrazino-Annelated Water-Soluble Xanthine Derivatives as Multi-Target Drugs With Potent Adenosine Receptor Antagonistic Activity.

Tetrahydropyrazino-annelated theophylline (1,3-dimethylxanthine) derivatives have previously been shown to display increased water-solubility as compared to the parent xanthines due to their basic character. In the present study, we modified this promising scaffold by replacing the 1,3-dimethyl residues by a variety of alkyl groups including combinations of different substituents in both positions. Substituted benzyl or phenethyl residues were attached to the N8 of the resulting 1,3-dialkyl-tetrahydropyrazino[2,1-f ]purinediones with the aim to obtain multi-target drugs that block human A1 and A2A adenosine receptors (ARs) and monoaminoxidase B (MAO-B). 1,3-Diethyl-substituted derivatives showed high affinity for A1 ARs, e.g., 15d (PSB-18339, 8-m-bromobenzyl-substituted) displayed a Ki value of 13.6 nM combined with high selectivity. 1-Ethyl-3-propargyl-substituted derivatives exhibited increased A2A AR affinity. The 8-phenethyl derivative 20h was selective for the A2A AR (Ki 149 nM), while the corresponding 8-benzyl-substituted compound 20e (PSB-1869) blocked A1 and A2A ARs with equal potency (Ki A1, 180 nM; A2A, 282 nM). The 1-ethyl-3-methyl-substituted derivative 16a (PSB-18405) bearing a m,p-dichlorobenzyl residue at N8 blocked all three targets, A1 ARs (Ki 396 nM), A2A ARs (Ki 1,620 nM), and MAO-B (IC50 106 nM) with high selectivity vs. the other subtypes (A2B and A3 ARs, MAO-A), and can thus be considered as a multi-target drug. Our findings were rationalized by molecular docking studies based on previously published X-ray structures of the protein targets. The new drugs have potential for the treatment of neurodegenerative diseases, in particular Parkinson's disease.

Structure-activity relationship studies and pharmacological characterization of N5-heteroarylalkyl-substituted-2-(2-furanyl)thiazolo[5,4-d]pyrimidine-5,7-diamine-based derivatives as inverse agonists at human A2A adenosine receptor

This paper describes the synthesis and characterization of N5-(hetero)arylalkyl-substituted-thiazolo [5,4-d]pyrimidine-5,7-diamine derivatives (4–19) as novel human (h) A2A adenosine receptor (AR) inverse agonists. Competition binding and cyclic AMP assays indicate that the examined compounds behave as hA2A AR inverse agonists showing binding affinity values in the nanomolar or subnanomolar range. Notably, compounds 4, 5, 6 and 11 showed two affinity values for the hA2A ARs with the highest (KH) falling in the femtomolar range and the lowest (KL) of the nanomolar order. In addition, in cyclic AMP assays, compounds 4, 5, 6 and 11 exhibited potency (IC50) values in the picomolar range.This study has confirmed that 2-(2-furanyl)thiazolo [5,4-d]pyrimidine-5,7-diamine-based derivatives represent a unique new class of hA2A AR inverse agonists.

Extrinsic Tryptophans as NMR Probes of Allosteric Coupling in Membrane Proteins: Application to the A2A Adenosine Receptor.

Tryptophan indole 15N-1H signals are well separated in nuclear magnetic resonance (NMR) spectra of proteins. Assignment of the indole 15N-1H signals therefore enables one to obtain site-specific information on complex proteins in supramacromolecular systems, even when extensive assignment of backbone 15N-1H resonances is challenging. Here we exploit the unique indole 15N-1H chemical shift by introducing extrinsic tryptophan reporter residues at judiciously chosen locations in a membrane protein for increased coverage of structure and function by NMR. We demonstrate this approach with three variants of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor, each containing a single extrinsic tryptophan near the receptor intracellular surface, in helix V, VI, or VII, respectively. We show that the native A2AAR global protein fold and ligand binding activity are preserved in these A2AAR variants. The indole 15N-1H signals from the extrinsic tryptophan reporter residues show different responses to variable efficacy of drugs bound to the receptor orthosteric cavity, and the indole 15N-1H chemical shift of the tryptophan introduced at the intracellular end of helix VI is sensitive to conformational changes resulting from interactions with a polypeptide from the carboxy terminus of the GαS intracellular partner protein. Introducing extrinsic tryptophans into proteins in complex supramolecular systems thus opens new avenues for NMR investigations in solution.

Tuning Vector Stability and Integration Frequency Elevates Functional GPCR Production and Homogeneity in Saccharomyces cerevisiae.

Membrane proteins play a valuable role in biotechnology, yet the difficulty of producing high yields of functional membrane protein limits their use in synthetic biology. The practical application of G protein-coupled receptors in whole cell biosensors, for example, is restricted to those that are functionally produced at the cell surface in the chosen host, limiting the range of detectable molecules. Here, we present a facile approach to significantly improve the yield and homogeneity of functional membrane proteins in Saccharomyces cerevisiae by altering only the choice of expression vector. Expression of a model GPCR, the human adenosine A2a receptor, from commonly used centromeric and episomal vectors leads to low yields and cellular heterogeneity due to plasmid loss in 20-90% of the cell population. In contrast, homogeneous production of GPCR is attained using a multisite integrating vector or a novel, modified high copy vector that does not require genomic integration or addition of any selection agents. Finally, we introduce a FACS-based screen, which enables rapid isolation of cells with 4- to 15-fold increases in gene dosage and up to a 9-fold increase in functional protein yield without loss of homogeneity compared to a strain isolated through conventional, low-throughput methods. These results can be extended to improve the cellular homogeneity and yield of other membrane proteins, expanding the repertoire of useful receptors for synthetic biology applications.

Identification of novel thiazolo[5,4-d]pyrimidine derivatives as human A1 and A2A adenosine receptor antagonists/inverse agonists

In this study a new set of thiazolo[5,4-d]pyrimidine derivatives was synthesized. These derivatives bear different substituents at positions 2 and 5 of the thiazolopyrimidine core while maintaining a free amino group at position-7. The new compounds were tested for their affinity and potency at human (h) A1, A2A, A2B and A3 adenosine receptors expressed in CHO cells. The results reveal that the higher affinity of these new set of thiazolopyrimidines is toward the hA1 and hA2A adenosine receptors subtypes and is tuned by the substitution pattern at both the 2 and 5 positions of the thiazolopyrimidine nucleus. Functional studies evidenced that the compounds behaved as dual A1/A2A antagonists/inverse agonists. Compound 3, bearing a 5-((2-methoxyphenyl) methylamino) group and a phenyl moiety at position 2, displayed the highest affinity (hA1 Ki = 10.2 nM; hA2A Ki = 4.72 nM) and behaved as a potent A1/A2A antagonist/inverse agonist (hA1 IC50 = 13.4 nM; hA2A IC50 = 5.34 nM).

Enabling STD-NMR fragment screening using stabilized native GPCR: A case study of adenosine receptor

Structural studies of integral membrane proteins have been limited by the intrinsic conformational flexibility and the need to stabilize the proteins in solution. Stabilization by mutagenesis was very successful for structural biology of G protein-coupled receptors (GPCRs). However, it requires heavy protein engineering and may introduce structural deviations. Here we describe the use of specific calixarenes-based detergents for native GPCR stabilization. Wild type, full length human adenosine A2A receptor was used to exemplify the approach. We could stabilize native, glycosylated, non-aggregated and homogenous A2AR that maintained its ligand binding capacity. The benefit of the preparation for fragment screening, using the Saturation-Transfer Difference nuclear magnetic resonance (STD-NMR) experiment is reported. The binding of the agonist adenosine and the antagonist caffeine were observed and competition experiments with CGS-21680 and ZM241385 were performed, demonstrating the feasibility of the STD-based fragment screening on the native A2A receptor. Interestingly, adenosine was shown to bind a second binding site in the presence of the agonist CGS-21680 which corroborates published results obtained with molecular dynamics simulation. Fragment-like compounds identified using STD-NMR showed antagonistic effects on A2AR in the cAMP cellular assay. Taken together, our study shows that stabilization of native GPCRs represents an attractive approach for STD-based fragment screening and drug design.