Putative role of the adenosine A3 receptor in the antiproliferative action of N6-(2-isopentenyl)adenosine

We tested a panel of naturally occurring nucleosides for their affinity towards adenosine receptors. Both N6-(2-isopentenyl)adenosine (IPA) and racemic zeatin riboside were shown to be selective human adenosine A3 receptor (hA3R) ligands with affinities in the high nanomolar range (Ki values of 159 and 649 nM, respectively). These values were comparable to the observed Ki value of adenosine on hA3R, which was 847 nM in the same radioligand binding assay. IPA also bound with micromolar affinity to the rat A3R. In a functional assay in Chinese hamster ovary cells transfected with hA3R, IPA and zeatin riboside inhibited forskolin-induced cAMP formation at micromolar potencies. The effect of IPA could be blocked by the A3R antagonist VUF5574. Both IPA and reference A3R agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (Cl-IB-MECA) have known antitumor effects. We demonstrated strong and highly similar antiproliferative effects of IPA and Cl-IB-MECA on human and rat tumor cell lines LNCaP and N1S1. Importantly, the antiproliferative effect of low concentrations of IPA on LNCaP cells could be fully blocked by the selective A3R antagonist MRS1523. At higher concentrations, IPA appeared to inhibit cell growth by an A3R-independent mechanism, as was previously reported for other A3R agonists. We used HPLC to investigate the presence of endogenous IPA in rat muscle tissue, but we could not detect the compound. In conclusion, the antiproliferative effects of the naturally occurring nucleoside IPA are at least in part mediated by the A3R

N6-Cyclopentyl-3'-substituted-xylofuranosyladenosines : a new class of non-xanthine adenosine A1 receptor antagonists

The present study explores the C-3' site of the 3-deoxy-3-xylofuranosyl ring of nucleoside analogues with an adenine or N-6-cyclopentyladenine (CPA) base moiety and evaluates the effect on adenosine receptor affinity. Two series of sugar-modified adenosines, i.e., 3'-amido-3'-deoxyadenosines and 3'-amidated 3'-deoxyxylofuranosyladenines, were synthesized and tested for their affinity at A(1) and A(2a) receptors in rat brain cortex and rat striatum, respectively. The modest affinity found in the ''xylo series'' prompted us to synthesize the corresponding N-6-cyclopentyl derivatives, which proved to be well accommodated by the A(1) receptors with potencies in the lower nanomolar range. This represents a new perspective in the purinergic field. The absence of a GTP-induced shift, i.e., the ratio between the affinities measured in the presence and absence of 1 mM GTP indicates an antagonistic behavior of this new class of CPA analogues

Allosteric modulation, thermodynamics and binding to wild-type and mutant (T277A) adenosine A(1) receptors of LUF5831, a novel nonadenosine-like agonist

1. The interaction of a new nonribose ligand (LUF5831) with the human adenosine A(1) receptor was investigated in the present study. 2. Radioligand binding experiments were performed in the absence and presence of diverse allosteric modulators on both wild-type (wt) and mutant (T277A) adenosine A(1) receptors. Thermodynamic data were obtained by performing these assays at different temperatures. In addition, cyclic adenosine monophosphate (cAMP) assays were performed. 3. The presence of allosteric modulators had diverse effects on the affinity of LUF5831, N(6)-cyclopentyladenosine (CPA), a full agonist, and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an inverse agonist/antagonist, for the adenosine A(1) receptor. PD81,723, for example, increased the affinity of CPA, while the affinity of LUF5831 was decreased. However, the affinity of DPCPX was decreased even more. In addition, LUF5831 was shown to have an affinity for the mutant (T277A) adenosine A(1) receptor (K(i)=122±22 nM), whereas CPA's affinity was negligible. The results of temperature-dependent binding assays showed that the binding of LUF5831 was entropy driven, in between the behaviour of CPA binding to the high- and low-affinity states of the receptor, respectively. 4. The inhibition of the forskolin-induced production of cAMP through activation of the wt adenosine A(1) receptor showed that LUF5831 had a submaximal effect (37±1%) in comparison to CPA (66±5%). On the mutant receptor, however, neither CPA nor LUF5831 inhibited cAMP production. 5. This study indicates that the nonribose ligand, LUF5831, is a partial agonist for the adenosine A(1) receptor