[3H]HEMADO— a novel tritiated agonist selective for the human adenosine A3 receptor

doi:10.1016/j.ejphar.2006.10.048

European Journal of Pharmacology

Volume 556, Issues 1–3, 5 February 2007, Pages 14-18

https://doi.org/10.1016/j.ejphar.2006.10.048 Get rights and content

Abstract

Adenosine A₃ receptors are promising drug targets for a number of conditions like inflammatory diseases including asthma, ischemic injury or certain types of cancer. Consequently, intense efforts are dedicated to the development of selective A₃ agonists and antagonists. The only tritiated agonist that is available for radioligand binding is the nonselective [³H]5′-N-ethylcarboxamidoadenosine ([³H]NECA). Based on a recently characterized series of 2-substituted adenosine receptor agonists we developed a tritiated selective A₃ radioligand with high affinity. From this series 2-hexyn-1-yl-N⁶-methyladenosine (HEMADO) with a K_i-value of 1.1 nM at the human A₃ subtype was chosen. HEMADO is 300-fold selective versus the A₁ subtype, and 1100-fold and more than 25,000-fold selective compared to the adenosine A_2A and A_2B receptors, respectively. The tritiated derivative [³H]HEMADO exhibited the same affinity as the unlabeled precursor. In concentrations up to 10 nM no specific binding to adenosine A₁, A_2A or A_2B receptors was observed confirming the high selectivity for adenosine A₃ receptors. Characterization of [³H]HEMADO in radioligand binding studies revealed reversible binding to the human adenosine A₃ subtype. In saturation binding studies for the A₃ subtype a K_D-value of 1.1 nM was determined. Nonspecific binding at a radioligand concentration of 1 nM amounted to 1–2% of total binding. Competition binding with a panel of adenosine receptor ligands clearly confirmed the correct A₃ pharmacology of the binding site labeled by [³H]HEMADO. With [³H]HEMADO we present a tritiated agonist with high affinity and A₃-selectivity and very low nonspecific binding. [³H]HEMADO is a useful tool for specific screening for A₃ receptor agonists and antagonists in improved radioligand binding assays with the human subtype.

Introduction

The metabolite adenosine mediates numerous physiological effects via four subtypes of G protein-coupled receptors known as adenosine A₁, A_2A, A_2B and A₃ receptors (Fredholm et al., 2001). The adenosine A₃ receptor is the most recent member of this family and has been identified in many tissues, usually in relatively low amounts. Higher receptor levels are found in eosinophils (Walker et al., 1997), activated T-cells (Gessi et al., 2004a) and in certain tumors (Madi et al., 2004, Gessi et al., 2004b). Like the A₁ subtype it mediates an inhibition of adenylyl cyclase and may elicit a Ca²⁺ signal (Fredholm et al., 2001, Englert et al., 2002). The two inhibitory subtypes may be distinguished in binding and functional experiments by their distinct pharmacological profiles of agonists and antagonists. Several agonist radioligands have been used for binding studies at adenosine A₃ receptors. One of the first radioligands used was the nonselective N⁶-4-aminobenzyl-3-[¹²⁵I]-iodo-adenosine ([¹²⁵I]-ABA) with a K_D-value of 10 nM at the human A₃ receptor (Salvatore et al., 1993). N⁶-4-aminobenzyl-3-[¹²⁵I]-iodo-5′-N-methylcarboxamidoadenosine ([¹²⁵I]-AB-MECA) is a high-affinity agonist allowing for the detection of low receptor levels. However, it is only about 10-fold selective versus the A₁ subtype (Fredholm et al., 2001) and, thus, cross-reaction may occur. In many studies utilizing cellular models with stably transfected receptors, tritiated ligands with long half-lives are more useful than radioiodinated ligands. However, in the past no tritiated high-affinity agonist was available for A₃ receptor studies. The only tritiated agonist in use for such studies was the nonselective adenosine receptor agonist [³H]5′-N-ethylcarboxamidoadenosine ([³H]NECA) (Klotz et al., 1998).

Based on a series of A₃-selective agonists (Volpini et al., 2002) we developed a novel tritiated ligand with high affinity and A₃-selectivity. The compound 2-(hexyn-1-yl)-6-methyladenosine (HEMADO) with an A₃ K_D of 1.1 nM and a 300- and 1100-fold selectivity versus A₁ and A_2A, respectively, was chosen for tritiation. Here we present a characterization of this new radioligand, [³H]HEMADO. All characteristics of [³H]HEMADO including A₃ affinity, selectivity and nonspecific binding are superior to the nonselective [³H]NECA. Therefore, it allows for improved binding assays which are widely used in the development of adenosine receptor ligands.

Section snippets

Materials

The adenosine receptor agonists 2-phenylethyn-1-yladenosine-5′-N-ethyluronamide (PENECA), 2-(3-hydroxy-3-phenyl)propyn-1-yladenosine-5′-N-ethyluronamide (PHPNECA) and HEMADO (structure see Fig. 1) were synthesized as described earlier (Cristalli et al., 1992, Cristalli et al., 1994, Cristalli et al., 1995, Camaioni et al., 1997, Volpini et al., 2002). All other adenosine receptor ligands and guanine nucleotides were from Sigma-RBI, Taufkirchen, Germany. [³H]HEMADO was synthesized (Tocris,

Results

As expected from the pharmacological characteristics of unlabeled HEMADO (Fig. 1) (Volpini et al., 2002) the tritiated ligand binds with high affinity to human adenosine A₃ receptors. Saturation binding reveals a K_D-value of 1.1 nM (Table 1, Fig. 2) which is in good agreement with the previously reported affinity for HEMADO (Volpini et al., 2002). The saturation experiment in Fig. 2 also demonstrates that [³H]HEMADO shows very low nonspecific binding amounting to about 1–2% of total binding at

Discussion

Adenosine A₃ receptors are promising targets for the treatment of a number of diseases including asthma, ischemic conditions or cancer (Livingston et al., 2004, Auchampach and Bolli, 1999, Fishman and Bar-Yehuda, 2003, Merighi et al., 2003). Consequently, a large number of agonists and antagonists with selectivity for this subtype were developed over the past decade or so. Nevertheless, there is still a significant interest in the development of potent and selective ligands for the A₃ and other

Acknowledgements

The excellent technical assistance of Helge Joa is gratefully acknowledged. This work was supported by grants from the Italian Ministry of Research: FIRB 2003 and PRIN 2004, PRIN 2005.

References (28)

E. Camaioni et al.
Adenosine receptor agonists: synthesis and biological evaluation of the diastereoisomers of 2-(3-hydroxy-3-phenyl-1-propyn-1-yl)NECA
Bioorg. Med. Chem.
(1997)
Z.-G. Gao et al.
N⁶-Substituted adenosine derivatives: selectivity, efficacy, and species differences at A₃ adenosine recepotrs
Biochem. Pharmacol.
(2003)
S. Merighi et al.
A glance at adenosine receptors: novel target for antitumor therapy
Pharmacol. Ther.
(2003)
C.E. Müller et al.
[³H]8-Ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]-purin-5-one ([³H]PSB-11), a novel high-affinity antagonist radioligand for human A₃ adenosine receptors
Bioorg. Med. Chem. Lett.
(2002)
J.A. Auchampach et al.
Adenosine receptor subtypes in the heart: therapeutic opportunities and challenges
Am. J. Physiol.
(1999)
P.G. Baraldi et al.
Recent developments in the field of A₃ adenosine receptor antagonists
Drug Dev. Res.
(2003)
R.F. Bruns et al.
Characterization of the A₂ adenosine receptor labeled by [³H]NECA in rat striatal membranes
Mol. Pharmacol.
(1986)
G. Cristalli et al.
2-Alkynyl derivatives of adenosine and adenosine-5′-N-ethyluronamides as selective agonists at A₂ adenosine receptors
J. Med. Chem.
(1992)
G. Cristalli et al.
2-Alkynyl derivatives of adenosine-5′-N-ethyluronamide: selective A₂ adenosine receptor agonists with potent inhibitory activity on platelet aggregation
J. Med. Chem.
(1994)
G. Cristalli et al.
2-Aralkynyl and 2-heteroalkynyl derivatives of adenosine-5′-N-ethyluronamide as selective A_2a adenosine receptor agonists
J. Med. Chem.
(1995)

A. De Lean et al.

Validation and statistical analysis of a computer modeling method for quantitative analysis of radioligand binding data for mixtures of pharmacological receptor subtypes

Mol. Pharmacol.

(1982)

M. Englert et al.

Effector coupling of stably transfected human A₃ adenosine receptors in CHO cells

Biochem. Pharmacol.

(2002)

P. Fishman et al.

Pharmacology and therapeutic applications of A₃ receptor subtype

Curr. Top. Med. Chem.

(2003)

B.B. Fredholm et al.

International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors

Pharmacol. Rev.

(2001)

Cited by (62)

Synthesis, biological evaluation and molecular modelling studies of 1,3,7,8-tetrasubstituted xanthines as potent and selective A <inf>2A</inf> AR ligands with in vivo efficacy against animal model of Parkinson's disease
2019, Bioorganic Chemistry
Citation Excerpt :
Newly synthesized xanthines were evaluated using in vitro radioligand binding assays at cloned adenosine receptors. In this study all human AR subtypes were stably transfected into Chinese Hamster Ovary (CHO) cells to study their pharmacological profile in an identical cellular background utilizing radioligand binding studies (A1, A2A, A3) or adenylyl cyclase activity assays (A2B) [27,28]. Binding affinities and selectivity profile of various newly synthesized xanthine derivatives for adenosine A1, A2A, and A3 receptors have been summarized in Table 1.
In the present study, an attempt has been made to develop a new series of 1,3,7,8-tetrasubstituted xanthine based potent and selective AR ligands for the treatment of Parkinson's disease. Antagonistic interactions between dopamine and A_2A adenosine receptors serve as the basis for the development of AR antagonists as potential drug candidates for PD. All the synthesized compounds have been evaluated for their affinity toward AR subtypes using in vitro radioligand binding assays. 1,3-Dipropylxanthine 7a with a methyl substituent at N-7 position represents the most potent compound of the series and displayed highest affinity (A_2A, K_i = 0.108 µM), however incorporation of a propargyl group at 7-positon of the xanthine nucleus seems to be the most appropriate substitution to improve selectivity towards the A_2A subtype along with reasonable potency. Antiparkinsonian activity has been evaluated using perphenazine induced catatonia in rats. Most of the synthesized xanthines significantly lowered the catatonic score as compared to control and displayed antiparkinsonian effects comparable to standard drug. All the synthesized compounds were subjected to grid-based molecular docking studies to understand the key structural requirements for the development of new molecules well-endowed with intrinsic efficacy and selectivity as adenosine receptor ligands. In silico studies carried out on newly synthesized xanthines provided further support to the pharmacological results.
[1,2,4]Triazolo[1,5-c]pyrimidines as adenosine receptor antagonists: Modifications at the 8 position to reach selectivity towards A<inf>3</inf> adenosine receptor subtype
2018, European Journal of Medicinal Chemistry
[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 K_i = 1.21 nM). Docking studies performed on the synthesized compounds inside models of human A₁, A_2A and A₃ adenosine receptors showed similar binding modes, comparable with the typical crystallographic binding mode of the inverse agonist ZM-241,385.
Anticancer activity study of A<inf>3</inf> adenosine receptor agonists
2018, Life Sciences
Citation Excerpt :
Binding assay of compounds 4–6 was performed in the present study, while, the binding affinity of compounds 1–3 are already reported in literatures 17–19. For competition binding the following radioligands and binding buffers were used: 1 nM [3H]2-chloro-N6-cyclopentyladenosine ([3H]CCPA) for A1 receptor (50 mM TRIS/HCl, pH 7.4), 10 nM [3H]N-ethylcarboxamidoadenosine ([3H]NECA) for A2A receptor (50 mM Tris, pH 7.4, 10 mM MgCl2), 1 nM [3H]2-hexynyladenosine ([3H]HEMADO) for A3AR mM TRIS/HCl, pH 8.25, 10 mM MgCl2, 1 mM EDTA) [19,22]. Nonspecific binding of [3H]CCPA was determined in the presence of 1 mM theophylline, 100 μM R-PIA was used in the case of [3H]NECA and [3H]HEMADO binding.
A₃ adenosine receptor (A₃AR) signalling activation seems to mediate anticancer effect, and it has been targeted for drug development. The identification of potent and selective A₃AR agonists could be crucial for cancer drug development.
In the present study was determined the in vitro activity of known 1–3 and newly 4–6 synthesized compounds with high A₃AR affinity and selectivity (K_i in the low nanomolar range) in binding studies. Effect of known and novel A₃AR agonists on human prostate cancer (PC3), hepatocellular carcinoma (Hep G2), and epithelial colorectal carcinoma (Caco-2) cells were analysed by cytotoxicity assay, dose and time dependent inhibitor assay, migration, apoptosis, autophagy and reactive oxygen species (ROS) assays.
Results show that the anticancer effect is not due to A₃AR activation alone. In fact, the more active and selective agonist versus A₃AR, compound 1, results inactive on cancer cells such as compounds 2–4. Moreover, results show that the novel compound 5, at micromolar concentration range (IC₅₀ = 28.0 μM), inhibits the growth of PC3, Hep G2, and Caco-2 cells and their migration in time- and dose- dependent manner. The mechanism involved in cell death is attributable to apoptosis. At the same time compound 5 promotes autophagy, which induce apoptosis producing autophagic cell death. Further investigation revealed that compound 5 elevates the level of ROS in all cancer cells tested, suggesting the involvement of ROS in cell death.
These results show that the new compound 5 exerts inhibitory effect on cancer cells through differential effect and may serve as a potential anticancer agent.
A binding kinetics study of human adenosine A <inf>3</inf> receptor agonists
2018, Biochemical Pharmacology
Citation Excerpt :
Interestingly, slow dissociation kinetics of agonists can be maintained by introducing an extended linear side chain at the C2 position and removing the bulky substituent at the N6 position (i.e. N6-Me analogue MRS7294 in RT: 125 min). The kinetic behavior of another known reference agonist, 2-(1-hexynyl)-N-methyladenosine (HEMADO), also confirmed this observation from comparison with other research [45,46]. More impressive kinetics were observed in the methanocarba series.
The human adenosine A₃ (hA₃) receptor has been suggested as a viable drug target in inflammatory diseases and in cancer. So far, a number of selective hA₃ receptor agonists (e.g. IB-MECA and 2-Cl-IB-MECA) inducing anti-inflammatory or anticancer effects are under clinical investigation. Drug-target binding kinetics is increasingly recognized as another pharmacological parameter, next to affinity, for compound triage in the early phases of drug discovery. However, such a kinetics-driven analysis has not yet been performed for the hA₃ receptor. In this study, we first validated a competition association assay for adenosine A₃ receptor agonists to determine the target interaction kinetics. Affinities and Kinetic Rate Index (KRI) values of 11 ribofurano and 10 methanocarba nucleosides were determined in radioligand binding assays. Afterwards, 15 analogues were further selected (KRI <0.70 or KRI >1.35) for full kinetics characterization. The structure-kinetics relationships (SKR) were derived and longer residence times were associated with methanocarba and enlarged adenine N⁶ and C2 substitutions. In addition, from a k_on-k_off-K_D kinetic map we divided the agonists into three subgroups. A residence time “cliff” was observed, which might be relevant to (N)-methanocarba derivatives’ rigid C2-arylalkynyl substitutions. Our findings provide substantial evidence that, next to affinity, additional knowledge of binding kinetics is useful for developing and selecting new hA₃R agonists in the early phase of the drug discovery process.
Medicinal chemistry of adenosine, P2Y and P2X receptors
2016, Neuropharmacology
Pharmacological tool compounds are now available to define action at the adenosine (ARs), P2Y and P2X receptors. We present a selection of the most commonly used agents to study purines in the nervous system. Some of these compounds, including A₁ and A₃ AR agonists, P2Y₁R and P2Y₁₂R antagonists, and P2X3, P2X4 and P2X7 antagonists, are potentially of clinical use in treatment of disorders of the nervous system, such as chronic pain, neurodegeneration and brain injury. Agonists of the A_2AAR and P2Y₂R are already used clinically, P2Y₁₂R antagonists are widely used antithrombotics and an antagonist of the A_2AAR is approved in Japan for treating Parkinson's disease. The selectivity defined for some of the previously introduced compounds has been revised with updated pharmacological characterization, for example, various AR agonists and antagonists were deemed A₁AR or A₃AR selective based on human data, but species differences indicated a reduction in selectivity ratios in other species. Also, many of the P2R ligands still lack bioavailability due to charged groups or hydrolytic (either enzymatic or chemical) instability. X-ray crystallographic structures of AR and P2YRs have shifted the mode of ligand discovery to structure-based approaches rather than previous empirical approaches. The X-ray structures can be utilized either for in silico screening of chemically diverse libraries for the discovery of novel ligands or for enhancement of the properties of known ligands by chemical modification. Although X-ray structures of the zebrafish P2X4R have been reported, there is scant structural information about ligand recognition in these trimeric ion channels. In summary, there are definitive, selective agonists and antagonists for all of the ARs and some of the P2YRs; while the pharmacochemistry of P2XRs is still in nascent stages. The therapeutic potential of selectively modulating these receptors is continuing to gain interest in such fields as cancer, inflammation, pain, diabetes, ischemic protection and many other conditions.
This article is part of the Special Issue entitled ‘Purines in Neurodegeneration and Neuroregeneration’.
[1,2,4]Triazolo[1,5-c]pyrimidines as Tools to Investigate A<inf>3</inf> Adenosine Receptors in Cancer Cell Lines
2023, ChemMedChem

View all citing articles on Scopus

View full text

[3H]HEMADO— a novel tritiated agonist selective for the human adenosine A3 receptor

Abstract

Introduction

Section snippets

Materials

Results

Discussion

Acknowledgements

Bioorg. Med. Chem.

Biochem. Pharmacol.

Pharmacol. Ther.

Bioorg. Med. Chem. Lett.

Adenosine receptor subtypes in the heart: therapeutic opportunities and challenges

Am. J. Physiol.

Recent developments in the field of A3 adenosine receptor antagonists

Drug Dev. Res.

Characterization of the A2 adenosine receptor labeled by [3H]NECA in rat striatal membranes

Mol. Pharmacol.

2-Alkynyl derivatives of adenosine and adenosine-5′-N-ethyluronamides as selective agonists at A2 adenosine receptors

J. Med. Chem.

2-Alkynyl derivatives of adenosine-5′-N-ethyluronamide: selective A2 adenosine receptor agonists with potent inhibitory activity on platelet aggregation

J. Med. Chem.

2-Aralkynyl and 2-heteroalkynyl derivatives of adenosine-5′-N-ethyluronamide as selective A2a adenosine receptor agonists

J. Med. Chem.

Validation and statistical analysis of a computer modeling method for quantitative analysis of radioligand binding data for mixtures of pharmacological receptor subtypes

Mol. Pharmacol.

Effector coupling of stably transfected human A3 adenosine receptors in CHO cells

Biochem. Pharmacol.

Pharmacology and therapeutic applications of A3 receptor subtype

Curr. Top. Med. Chem.

International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors

Pharmacol. Rev.

[³H]HEMADO— a novel tritiated agonist selective for the human adenosine A₃ receptor

Recent developments in the field of A₃ adenosine receptor antagonists

Characterization of the A₂ adenosine receptor labeled by [³H]NECA in rat striatal membranes

2-Alkynyl derivatives of adenosine and adenosine-5′-N-ethyluronamides as selective agonists at A₂ adenosine receptors

2-Alkynyl derivatives of adenosine-5′-N-ethyluronamide: selective A₂ adenosine receptor agonists with potent inhibitory activity on platelet aggregation

2-Aralkynyl and 2-heteroalkynyl derivatives of adenosine-5′-N-ethyluronamide as selective A_2a adenosine receptor agonists

Effector coupling of stably transfected human A₃ adenosine receptors in CHO cells

Pharmacology and therapeutic applications of A₃ receptor subtype