Novel Mutants of the Human β1-Adrenergic Receptor Reveal Amino Acids Relevant for Receptor Activation

Activation of G protein-coupled receptors like the beta(1)-adrenergic receptor results in conformational changes that ultimately lead to signal propagation through a G protein to an effector like adenylyl cyclase. In this study we identified amino acids that seem to be critical for activation of the human beta(1)-adrenergic receptor. Activation patterns of mutant receptors were analyzed using two structurally different ligands for beta-adrenergic receptors that both are mixed agonist/antagonists. Broxaterol and terbutaline are agonists at beta(2)- and beta(3)-receptors; however, they act as antagonists at the beta(1)-subtype. We reasoned that this functional selectivity may be reflected by a corresponding sequence pattern in the receptor subtypes. Therefore, we exchanged single amino acids of the beta(1)-adrenergic receptor for residues that were identical in the beta(2)- and beta(3)-subtypes but different in the beta(1)-receptor. Pharmacological characterization of such receptor mutants revealed that binding of a panel of agonists and antagonists including broxaterol and terbutaline was unaltered. However, two of the mutants (I185V and D212N) were activated by broxaterol and terbutaline, which acted as antagonists at the wild-type receptor. Two additional mutants (V120L and K253R) could be activated by terbutaline alone, which is structurally more closely related to endogenous catecholamines like epinephrine than to broxaterol. A model of the human beta(1)-adrenergic receptor showed that the four gain-of-function mutations are outside of the putative ligand-binding domain substantiating the lack of an effect of the mutations on binding characteristics. These results support the notion that Val-120, Ile-185, Asp-212, and Lys-253 are critically involved in conformational changes occurring during receptor activation

The current status of pharmacotherapy for the treatment of Parkinson's disease: transition from single-target to multitarget therapy

Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of dopaminergic neurons. Motor features such as tremor, rigidity, bradykinesia and postural instability are common traits of PD. Current treatment options provide symptomatic relief to the condition but are unable to reverse disease progression. The conventional single-target therapeutic approach might not always induce the desired effect owing to the multifactorial nature of PD. Hence, multitarget strategies have been proposed to simultaneously target multiple proteins involved in the development of PD. Herein, we provide an overview of the pathogenesis of PD and the current pharmacotherapies. Furthermore, rationales and examples of multitarget approaches that have been tested in preclinical trials for the treatment of PD are also discussed

Discovery of Novel Adenosine Receptor Antagonists through a Combined Structure- and Ligand-Based Approach Followed by Molecular Dynamics Investigation of Ligand Binding Mode

An intense effort is made by pharmaceutical and academic research laboratories to identify and develop selective antagonists for each adenosine receptor (AR) subtype as potential clinical candidates for "soft" treatment of various diseases. Crystal structures of subtypes A2A and A1ARs offer exciting opportunities for structure-based drug design. In the first part of the present work, Maybridge HitFinder library of 14400 compounds was utilized to apply a combination of structure-based against the crystal structure of A2AAR and ligand-based methodologies. The docking poses were rescored by CHARMM energy minimization and calculation of the desolvation energy using Poisson-Boltzmann equation electrostatics. Out of the eight selected and tested compounds, five were found positive hits (63% success). Although the project was initially focused on targeting A2AAR, the identified antagonists exhibited low micromolar or micromolar affinity against A2A/A3, ARs, or A3AR, respectively. Based on these results, 19 compounds characterized by novel chemotypes were purchased and tested. Sixteen of them were identified as AR antagonists with affinity toward combinations of the AR family isoforms (A2A/A3, A1/A3, A1/A2A/A3, and A3). The second part of this work involves the performance of hundreds of molecular dynamics (MD) simulations of complexes between the ARs and a total of 27 ligands to resolve the binding interactions of the active compounds, which were not achieved by docking calculations alone. This computational work allowed the prediction of stable and unstable complexes which agree with the experimental results of potent and inactive compounds, respectively. Of particular interest is that the 2-amino-thiophene-3-carboxamides, 3-acylamino-5-aryl-thiophene-2-carboxamides, and carbonyloxycarboximidamide derivatives were found to be selective and possess a micromolar to low micromolar affinity for the A3 receptor

Targeting G protein-coupled receptors with magnetic carbon nanotubes:The Case of the A ₃ Adenosine Receptor

A 3 adenosine receptor (AR) is a G protein-coupled receptor (GPCR) overexpressed in the membrane of specific cancer cells. Thus, the development of nanosystems targeting this receptor could be a strategy to both treat and diagnose cancer. Fe-filled carbon nanotubes (CNTs) are an optimal platform for theranostic purposes, and the use of a magnetic field can be exploited for cancer magnetic cell sorting and thermal therapy. In this work, we have conjugated an A 3 AR ligand on the surface of Fe-filled CNTs with the aim to target cells overexpressing A 3 ARs. In particular, two conjugates bearing PEG linkers of different length were designed. A docking analysis on the A 3 AR showed that both CNT and linker do not interfere with ligand binding to the receptor, that was confirmed by in vitro preliminary radioligand competition assays on A 3 AR. Encouraged by this result, magnetic cell sorting was applied to a mixture of cells overexpressing or not the A 3 AR where our compound resulted to not be selective for A 3 AR-cancer cells. Despite this, it is the first time that a GPCR ligand was anchored to a magnetic nanosystem, thus it opens the door to new applications for cancer treatment

5'-C-ethyl-tetrazolyl-N 6-substituted adenosine and 2-chloro-adenosine derivatives as highly potent dual acting A1 adenosine receptor agonists and A3 adenosine receptor antagonists

A series of N(6)-substituted-5'-C-(2-ethyl-2H-tetrazol-5-yl)-adenosine and 2-chloro-adenosine derivatives was synthesized as novel, highly potent dual acting hA1AR agonists and hA3AR antagonists, potentially useful in the treatment of glaucoma and other diseases. The best affinity and selectivity profiles were achieved by N(6)-substitution with a 2-fluoro-4-chloro-phenyl- or a methyl- group. Through an in silico receptor-driven approach, the molecular bases of the hA1- and hA3AR recognition and activation of this series of 5'-C-ethyl-tetrazolyl derivatives were explained

Adenosine Receptors as Mediators of Both Cell Proliferation and Cell Death of Cultured Human Melanoma Cells

Adenosine displays contradictory effects on cell growth: it improves cell proliferation, but it may also induce apoptosis and impair cell survival. Following the pharmacologic characterization of adenosine receptor expression on the human melanoma cell line A375, we chose A375 as our cellular model to define how the extracellular adenosine signals are conveyed from each receptor. By using selective adenosine receptor agonists or antagonists, we found that A2A stimulation reduced cell viability and cell clone formation, whereas, at the same time, it improved cell proliferation. In support of this finding we demonstrated that the stimulation of A2A adenosine receptors stably expressed in Chinese hamster ovary cell clone reproduced deleterious effects observed in human melanoma cells. A3 stimulation counteracted A2A-induced cell death but also reduced cell proliferation. Furthermore, we found that A3 stimulation ensures cell survival. We demonstrated that adenosine triggers a survival signal via A3 receptor activation and it kills the cell through A2A receptor inducing a signaling pathway that involves protein kinase C and mitogen-activated protein kinases

Adenosine receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Adenosine receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Adenosine Receptors [103]) are activated by the endogenous ligand adenosine (potentially inosine also at A3 receptors). Crystal structures for the antagonist-bound [146, 305, 213, 55], agonist-bound [362, 196, 198] and G protein-bound A2A adenosine receptors [43] have been described. The structures of an antagonist-bound A1 receptor [123] and an adenosine-bound A1 receptor-Gi complex [80] have been resolved by cryo-electronmicroscopy. Another structure of an antagonist-bound A1 receptor obtained with X-ray crystallography has also been reported [51]

Adenosine receptors in GtoPdb v.2021.2

Adenosine receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Adenosine Receptors [110]) are activated by the endogenous ligand adenosine (potentially inosine also at A3 receptors). Crystal structures for the antagonist-bound [153, 313, 221, 61], agonist-bound [375, 203, 204] and G protein-bound A2A adenosine receptors [49] have been described. The structures of an antagonist-bound A1 receptor [128] and an adenosine-bound A1 receptor-Gi complex [86] have been resolved by cryo-electronmicroscopy. Another structure of an antagonist-bound A1 receptor obtained with X-ray crystallography has also been reported [57]. caffeine is a nonselective antagonist for adenosine receptors, while istradefylline, a selective A2A receptor antagonist, is on the market for the treatment of Parkinson's disease

Adenosine receptors in GtoPdb v.2023.1

Adenosine receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on Adenosine Receptors [112]) are activated by the endogenous ligand adenosine (potentially inosine also at A3 receptors). Crystal structures for the antagonist-bound [155, 316, 224, 62], agonist-bound [379, 205, 206] and G protein-bound A2A adenosine receptors [49] have been described. The structures of an antagonist-bound A1 receptor [130] and an adenosine-bound A1 receptor-Gi complex [87] have been resolved by cryo-electronmicroscopy. Another structure of an antagonist-bound A1 receptor obtained with X-ray crystallography has also been reported [58]. The structure of the A2B receptor has also been elucidated [57]. caffeine is a nonselective antagonist for adenosine receptors, while istradefylline, a selective A2A receptor antagonist, is on the market for the treatment of Parkinson's disease

5,7-Disubstituted-[1,2,4]triazolo[1,5-a][1,3,5]triazines as pharmacological tools to explore the antagonist selectivity profiles toward adenosine receptors

The structureeactivity relationship of new 5,7-disubstituted-[1,2,4]triazolo[1,5-a][1,3,5]triazines as adenosine receptors (ARs) antagonists has been explored. The introduction of a benzylamino group at C5 with a free amino group at C7 increases the affinity toward all the ARs subtypes (10: KihA1 \ubc 94.6 nM; KihA2A \ubc 1.11 nM; IC50hA2B \ubc 2214 nM; KihA3 \ubc 30.8 nM). Replacing the free amino group at C7 with a phenylureido moiety yields a potent and quite selective hA2A AR antagonist (14: hA2A AR Ki \ubc 1.44 nM; hA1/hA2A \ubc 216.0; hA3/hA2A \ubc 20.6). This trend diverges from the analysis on the pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine series previously reported. With the help of an in silico receptor-driven approach, we have rationalized these observations and elucidated from a molecular point of view the role of the benzylamino group at C5 in determining affinity toward the hA2A AR