Journal of Medicinal Chemistry permeabilized Jurkat T cells, and the results are shown in Figure 8. Because of the intrinsic fluorescence of 6-thio cIDPR, Figure 8. 6-Thio-cIDPR-induced Ca2+ release and effect of 6-thio- cIDPR on cADPR and cIDPR-induced Ca2+ release in permeabilized Jurkat T cells. Jurkat T cells were permeabilized, and [Ca2+] was measured in the presence of Fluo-3, ATP, and an ATP regenerating system as detailed in the Experimental Section. (a) Ca2+ release induced by addition of 6-thio-cIDPR. Characteristic tracings from representative experiments are shown. (b) Concentration−response curve of 6-thio-cIDPR induced Ca2+ release. Results represent Ca2+ increase over baseline (Δ values) expressed as mean ± SD (n = 4−7) of single tracings. (c) The inhibitory effect of 6-thio-cIDPR was esti- mated by previous addition of 6-SH-cIDPR and subsequent addition of either cIDPR or cADPR. Since 6-thio-cIDPR also elicited a weak agonist effect on its own (a, b), calculation of its antagonist effect on cADPR (30 μM) or cIDPR (30 μM) was carried out by subtracting the corresponding agonist data (data in part b). Concentration− response curves represent the mean ± SD (n = 3) of single tracings. A one-phase exponential decay was used to fit curves; r2 was 0.6486 for cADPR and 0.7107 for cIDPR. Fluo3 was used as a Ca2+ indicator to avoid any possible inter- ference. At low concentration, 6-thio cIDPR 3 did not stimulate Ca2+ release in T cells. Ca2+ release was, however, observed at significantly higher concentration (100 μM and above, Figure 8a and Figure 8b). In comparison with N1-cIDPR 2, weaker agonist activity was observed for 6-thio cIDPR 3; however, in the cur- rent series of experiments the permeabilized cell preparations responded also somewhat more weakly to the naturally occur- ring cADPR 1. Regarding antagonism by 6-thio cIDPR 3 a concentration-dependent effect on cADPR and N1-cIDPR 2 Article induced Ca2+ release was observed (Figure 8c). While the IC50 for cADPR induced Ca2+ release was between 30 and 100 μM, a somewhat better inhibition of Ca2+ release induced by N1- cIDPR 2 was obtained (IC50 ≈ 3 μM). We thus demonstrate here that a simple modification from a CO to CS bond at C-6 weakens agonist activity but enhances antagonist activity in Jurkat T cells. Antagonist activity was specifically strong when N1-cIDPR 2 was used to trigger Ca2+ release, indicating that the oxygen group in cIDPR can be replaced at lower 6-thio cIDPR 3 concentrations compared to the situation where Ca2+ release was induced by cADPR (Figure 8c). The most likely explanation for this differential effect of 6-thio cIDPR 3 is differ- ences in protonation pattern and hydrogen bonding interactions. The amino group in cADPR can be a hydrogen bond donor or acceptor, whereas the oxygen group in cIDPR is a good hy- drogen bond acceptor. Obviously, 6-thio cIDPR 3 competes more effectively with cIDPR for binding to the cADPR receptor. Taken together, these results show that replacement of the keto moiety by a more hydrophobic thione does interfere with the functional consequences of binding of the ligand to its receptor. There is an obvious role in the C-6 group in antago- nizing Ca2+ release, if not directly then through subtle confor- mational effects or other. It may well be that a change from a hypoxanthine to a mercaptopurine results in a different proto- nation pattern and hydrogen bonding interactions. The amino group in cADPR can be a hydrogen bond donor or acceptor, whereas the oxygen group in cIDPR is a good hydrogen bond acceptor, as opposed to the sulfur atom. A C2′-endo/syn con- formation is obviously not the sole factor that governs agonist/ antagonist activity, since all three nucleotides cADPR, cIDPR, and 6-thio cIDPR have the same global conformation, although one can suggest that a C3′-endo/anti conformation (as in N7- cIDPR and N7-cGDPR, Table 1) would most likely lead to inactive compounds. It is also well-known that the hydrophobicity of sulfur leads to differences in the associated water shell. Thus, it may be that differential arrangements of additional water mole- cules in the receptor binding site can influence the receptor machinery. Although surprising, this is not the first time that Ca2+ release has been observed at higher concentrations. Indeed, we pre- viously reported that the classical and widely used antagonists such as 8-bromo cADPR and 8-amino cADPR also show such unexpected agonist activity at high concentration.20 One of the reasons invoked to explain this earlier unobserved effect is that there may be two previously unrecognized different sites of action, such as an additional lower affinity nucleotide binding site, which causes Ca2+ mobilization distinct from the high affi- nity cADPR site. This would not have been noticed with the parent cIDPR, since it only acts as a potent agonist, but is again revealed with the structurally related 6-thio cIDPR. The C-6 position in cADPR may therefore be revealed as an attractive new site to imbue antagonistic properties in a cADPR analogue. Its proximity to the crucial N1−C1″ linkage makes it likely that structural modifications could lead to modulation of activity of cADPR binding proteins. As such, it will be of interest to develop new ways to approach the synthetic prob- lem of C-6 modification. Although the present result has been achieved somewhat unexpectedly via a chemoenzymatic route, it seems most likely that greater flexibility in this regard will be offered by total synthetic approaches in the future. 1484 dx.doi.org/10.1021/jm201127y | J. Med. Chem. 2012, 55, 1478−1489