Cellular effects and metabolic stability of N1-cyclic inosine diphosphoribose and its derivatives

Abstract

Background and purpose: Recently, a number of mimics of the second messenger cyclic ADP-ribose (cADPR) with replacement of adenosine by inosine were introduced. In addition, various alterations in the molecule ranging from substitutions at C8 of the base up to full replacement of the ribose moieties still retained biological activity. However, nothing is known about the metabolic stability and cellular effects of these novel analogues.

Experimental approach: cADPR and the inosine-based analogues were incubated with CD38, ADP-ribosyl cyclase and NAD-glycohydrolase and metabolism was analysed by RP-HPLC. Furthermore, the effect of the analogues on cytokine expression and proliferation was investigated in primary T-lymphocytes and T-lymphoma cells.

Key results: Incubation of cADPR with CD38 resulted in degradation to adenosine diphosphoribose. ADP-ribosyl cyclase weakly catabolised cADPR whereas NAD-glycohydrolase showed no such activity. In contrast, N1-cyclic inosine 5'-diphosphoribose (N1-cIDPR) was not hydrolyzed by CD38. Three additional N1-cIDPR analogues showed a similar stability. Proliferation of Jurkat T-lymphoma cells was inhibited by N1-cIDPR, N1-[(phosphoryl-O-ethoxy)-methyl]-N9-[(phosphoryl-O-ethoxy)-methyl]-hypoxanthine-cyclic pyrophosphate (N1-cIDP-DE) and N1-ethoxymethyl-cIDPR (N1-cIDPRE). In contrast, in primary T cells neither proliferation nor cytokine expression was affected by these compounds.

Conclusions and implications: The metabolic stability of N1-cIDPR and its analogues provides an advantage for the development of novel pharmaceutical compounds interfering with cADPR mediated Ca2+ signalling pathways. The differential effects of N1-cIDPR and N1-cIDPRE on proliferation and cytokine expression in primary T cells versus T-lymphoma cells may constitute a starting point for novel anti-tumor drugs.

Figure 1

Metabolism of cADPR by CD38, ADP-ribosyl cyclase and NADase. cADPR (50 μM) was incubated with Jurkat T-lymphocytes (1 × 10⁷ ml⁻¹) (a, b, g), ADP-ribosyl cyclase (100 ng ml⁻¹) (c, d, h) and NADase (180 μg ml⁻¹) (e, f, i) for 120 min at RT (n=4–5 each condition). Aliquots were taken at 0 or 120 min and were analysed by RP-HPLC. Characteristic chromatograms are shown. Data in panels g, h and i are mean±s.d. (n=4–5).

Figure 2

Metabolism of cADPR and N1-cIDPR by CD38. Vehicle (no substrate), cADPR (50 μM) or N1-cIDPR (50 μM) were incubated at RT either with 1 × 10⁷ ml⁻¹ Jurkat T-lymphocytes for the times indicated (a–i, j), with recombinant soluble mouse CD38 (0.75 μg ml⁻¹) (k) or with increasing numbers of Jurkat T-lymphocytes for 6 h (l). Aliquots were taken at the time points indicated and were analysed by RP-HPLC. The detection was performed at 270 nm for cADPR and at 250 nm for N1-cIDPR. Data are presented as mean±s.d. (n=3). Note that at some time points s.d. values are smaller than symbols and thus cannot be seen properly.

Figure 3

Metabolism of 8-Br-N1-cIDPR, N1-cIDPRE or N1-cIDP-DE by CD38. Jurkat T-lymphocytes (1 × 10⁷ ml⁻¹) were incubated with each 50 μM 8-Br-N1-cIDPR (a, b), N1-cIDPRE (c, d) or N1-cIDP-DE (e, f) for 18 h at RT (n=2). Aliquots were taken at 0 min and 18 h and were analysed by RP-HPLC (detection at 250 nm).

Figure 4

Proliferation of Jurkat T-lymphocytes in the presence of cADPR, N1-cIDPR, 8-Br-N1-cIDPR, N1-cIDPRE or N1-cIDP-DE. Jurkat T-lymphocytes (1 × 10⁵ ml⁻¹) were incubated with increasing concentrations of cADPR (c), 8-Br-N1-cIDPR (c), N1-cIDPR (a, d), N1-cIDPRE (d) or N1-cIDP-DE (b, d) at 37°C/5% CO₂. Cell density was determined after 48, 72 and 96 h. (a, b) Cell density plotted against incubation time and concentration of N1-cIDPR (a) and N1-cIDP-DE (b); data are presented as mean±s.d. (n=3–9). (c, d) Concentration–response relationship; data are presented as mean±s.e. (n=2–3).