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

[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

8[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 ¼ 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.partially_openembargoed_20200820Federico, Stephanie; Margiotta, Enrico; Salmaso, Veronica; Pastorin, Giorgia; Kachler, Sonja; Klotz, Karl-Norbert; Moro, Stefano; Spalluto, Giampiero*Federico, Stephanie; Margiotta, Enrico; Salmaso, Veronica; Pastorin, Giorgia; Kachler, Sonja; Klotz, Karl-Norbert; Moro, Stefano; Spalluto, Giampier

2-Substituted N-ethylcarboxamidoadenosine derivatives as high affinity agonists at human A(3) adenosine receptors

A number of 2-substituted 5'-N-ethylcarboxamidoadenosine (NECA) derivatives was investigated for their affinity and selectivity at human A3 adenosine receptors. The compounds were tested in radioligand competition studies and modulation of adenylyl cyclase activity on membranes from CHO cell lines stably transfected with the four human adenosine receptor subtypes. In binding studies the most potent compound, 2-(3-hydroxy-3-phenyl)propyn-1-yl-NECA (PHPNECA), exhibited a subnanomolar affinity for A3 adenosine receptors with a Ki value of 0.4 nM. As opposed to the limited A3 selectivity of PHPNECA, a 100-fold selectivity compared to both A1 and A2A receptors was found for 2-(2-phenyl)ethynyl-NECA (PENECA; Ki 6 nM). The EC50 values for activation of adenylyl cyclase via A2A adenosine receptors were in good agreement with the respective Ki values from binding experiments. In contrast, IC50 values for A1 and A3 receptor-mediated inhibition of adenylyl cyclase were shifted to higher values compared to the respective affinities determined in radioligand competition studies. Similar discrepancies between binding and functional data have been observed for the inhibitory A1 adenosine receptor in previous studies. Therefore, the same A3 selectivity of PENECA compared to A1 receptors was found in binding and adenylyl cyclase inhibition whereas the selectivity compared to A2A receptors that was detected in ligand binding was obscured in the functional assay. The series of compounds presented in this study identifies 2-substitution of the purine system as a promising target for the development of A3-selective high-affinity ligands

Adenosine receptor ligands: coumarin−chalcone hybrids as modulating agents on the activity of hARs

Adenosine receptors (ARs) play an important role in neurological and psychiatric disorders such as Alzheimer's disease, Parkinson's disease, epilepsy and schizophrenia. The different subtypes of ARs and the knowledge on their densities and status are important for understanding the mechanisms underlying the pathogenesis of diseases and for developing new therapeutics. Looking for new scaffolds for selective AR ligands, coumarin–chalcone hybrids were synthesized (compounds 1–8) and screened in radioligand binding (hA$_1$, hA$_{2A}$ and hA$_3$) and adenylyl cyclase (hA$_{2B}$) assays in order to evaluate their affinity for the four human AR subtypes (hARs). Coumarin–chalcone hybrid has been established as a new scaffold suitable for the development of potent and selective ligands for hA$_1$ or hA$_3$ subtypes. In general, hydroxy-substituted hybrids showed some affinity for the hA$_1$, while the methoxy counterparts were selective for the hA$_3$. The most potent hA$_1$ ligand was compound 7 (K$_i$ = 17.7 µM), whereas compound 4 was the most potent ligand for hA$_3$ (K$_i$ = 2.49 µM). In addition, docking studies with hA$_1$ and hA$_3$ homology models were established to analyze the structure–function relationships. Results showed that the different residues located on the protein binding pocket could play an important role in ligand selectivity

Pyrazolo-triazolo-pyrimidine Scaffold as a Molecular Passepartout for the Pan-Recognition of Human Adenosine Receptors

Adenosine receptors are largely distributed in our organism and are promising therapeutic targets for the treatment of many pathologies. In this perspective, investigating the structural features of the ligands leading to affinity and/or selectivity is of great interest. In this work, we have focused on a small series of pyrazolo-triazolo-pyrimidine antagonists substituted in positions 2, 5, and N8, where bulky acyl moieties at the N5 position and small alkyl groups at the N8 position are associated with affinity and selectivity at the A3 adenosine receptor even if a good affinity toward the A2B adenosine receptor has also been observed. Conversely, a free amino function at the 5 position induces high affinity at the A2A and A1 receptors with selectivity vs. the A3 subtype. A molecular modeling study suggests that differences in affinity toward A1, A2A, and A3 receptors could be ascribed to two residues: one in the EL2, E168 in human A2A/E172 in human A1, that is occupied by the hydrophobic residue V169 in the human A3 receptor; and the other in TM6, occupied by H250/H251 in human A2A and A1 receptors and by a less bulky S247 in the A3 receptor. In the end, these findings could help to design new subtype-selective adenosine receptor ligands

The Influence of the 1-(3-Trifluoromethyl-Benzyl)-1H-Pyrazole-4-yl Moiety on the Adenosine Receptors Affinity Profile of Pyrazolo[4,3-e][1,2,4]Triazolo[1,5-c]Pyrimidine Derivatives

A new series of pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (PTP) derivatives has been developed in order to explore their affinity and selectivity profile at the four adenosine receptor subtypes. In particular, the PTP scaffold was conjugated at the C2 position with the 1-(3-trifluoromethyl-benzyl)-1H-pyrazole, a group believed to confer potency and selectivity toward the human (h) A$_{2B}$ adenosine receptor (AR) to the xanthine ligand 8-(1-(3-(trifluoromethyl) benzyl)-1H-pyrazol-4-yl)-1,3-dimethyl-1H-purine-2,6(3H, 7H)-dione (CVT 6975). Interestingly, the synthesized compounds turned out to be inactive at the hA$_{2B}$ AR but they displayed affinity at the hA$_3$ AR in the nanomolar range. The best compound of the series (6) shows both high affinity (hA$_3$ AR K$_i$ = 11 nM) and selectivity (A$_1$/A$_3$ and A$_{2A}$/A$_3$ > 9090; A$_{2B}$/A$_3$ > 909) at the hA$_3$ AR. To better rationalize these results, a molecular docking study on the four AR subtypes was performed for all the synthesized compounds. In addition, CTV 6975 and two close analogues have been subjected to the same molecular docking protocol to investigate the role of the 1-(3-trifluoromethyl-benzyl)-1H-pyrazole on the binding at the four ARs