Synthesis and Biological Evaluation of Novel Membrane-Permeant Cyclic ADP-Ribose Mimics:  N-[(5‘ ‘-O-Phosphorylethoxy)methyl]-5‘-O-phosphorylinosine 5‘,5‘ ‘-Cyclicpyrophosphate (cIDPRE) and 8-Substituted Derivatives

Abstract

N1-[(5' '-O-Phosphorylethoxy)methyl]-5'-O-phosphorylinosine 5',5''-cyclicpyrophosphate (cIDPRE 2a) and the 8-substituted derivatives 8-bromo-, 8-azido-, 8-amino-, and 8-Cl-cIDPRE (2b-e) were synthesized from N1-[(5''-acetoxyethoxy)methyl]-2',3'-O-isopropylideneinosine (5) in good yields. The pharmacological activities of cIDPRE and the 8-substituted derivatives (2a-e) were analyzed in intact and permeabilized human Jurkat T-lymphocytes. The results indicate that cIDPRE permeates the plasma membrane, releases Ca2+ from an intracellular, cADPR-sensitive Ca2+ store, and subsequently initiates Ca2+ release-activated Ca2+ entry. The Ca(2+)-releasing activity of cIDPRE was confirmed directly in permeabilized cells. Using time-resolved confocal Ca2+ imaging at the single cell level, the development of global Ca2+ signals starting from local small Ca2+ signals evoked by cIDPRE was observed. 8-N3-cIDPRE 2c and 8-NH2-cIDPRE 2d were similarly effective in their agonistic activity as compared to cIDPRE 2a, showing almost indistinguishable concentration-response curves for 2a, 2c, and 2d and very similar kinetics of Ca2+ signaling. In contrast, the halogenated derivatives 8-Br- and 8-Cl-cIDPRE (2b and 2e) did not significantly elevate [Ca2+]i. Therefore, cIDPRE 2a, 8-N3-cIDPRE 2c, and 8-NH2-cIDPRE 2d are novel membrane permeant cADPR mimic and may provide important novel tools to study cADPR-mediated Ca2+ signaling in intact cells.

A Minimal Structural Analogue of Cyclic ADP-ribose

“…The biological activity of cIDP-DE in Jurkat T cells was quite similar to that of cIDPRE in the following ways: (i) Ca 2ϩ release activity in permeabilized cells; (ii) induction of both Ca 2ϩ release and Ca 2ϩ entry in the Ca 2ϩ -free/Ca 2ϩ reintroduction protocol; and (iii) stimulation of Ca 2ϩ signaling (11). This similarity of cIDP-DE and cIDPRE, especially in the induction of Ca 2ϩ signaling in intact T cells, indicates that the further replacement of the southern ribose by the second ether bridge in cIDP-DE did not significantly influence its biological activity.…”

“…Biochemistry and Pharmacology-Because cIDPRE and some of its analogues were recently identified as membranepermeant Ca 2ϩ -mobilizing agonists of the cADPR/Ca 2ϩ signaling system (11), we intended to also analyze the biological activities of cIDP-DE. However, due to the unexpected fluorescence properties of cIDP-DE, any interference with the Ca 2ϩ indicator fura2 had to be ruled out first.…”

“…A series of N 1 -glycosyl-substituted cIDPR derivatives with different configurations at the N 1 -glycosyl moiety also retained agonistic Ca 2ϩ -releasing activities (10). More interestingly, a simplified molecule in which the northern ribose was replaced by an ether strand, the N 1 -ethoxymethyl-cIDPR (cI-DPRE) analogue, exhibited a strong potency for inducing Ca 2ϩ release in intact and permeabilized human Jurkat T lymphocytes (11). The 8-substituted cIDPRE analogues 8-N 3 -cIDPRE and 8-NH 2 -cIDPRE showed similar biological activities as compared with cIDPRE, whereas the halogenated derivatives 8-BrcIDPRE and 8-Cl-cIDPRE did not significantly elevate [Ca 2ϩ ] i (11).…”

“…More interestingly, a simplified molecule in which the northern ribose was replaced by an ether strand, the N 1 -ethoxymethyl-cIDPR (cI-DPRE) analogue, exhibited a strong potency for inducing Ca 2ϩ release in intact and permeabilized human Jurkat T lymphocytes (11). The 8-substituted cIDPRE analogues 8-N 3 -cIDPRE and 8-NH 2 -cIDPRE showed similar biological activities as compared with cIDPRE, whereas the halogenated derivatives 8-BrcIDPRE and 8-Cl-cIDPRE did not significantly elevate [Ca 2ϩ ] i (11). Thus, the N 1 -ribosyl moiety (northern ribose) and the 6-amino group in cADPR are not, per se, critical structural factors for biological activity, whereas different substituents at the 8-position of the base moiety obviously influence strongly the overall electron affinity and sterical conformation of the analogue, thereby greatly influencing its biological activity.…”

Cyclic ADP-Carbocyclic-Ribose and -4-Thioribose, as Stable Mimics of Cyclic ADP-Ribose, a Ca²⁺-Mobilizing Second Messenger

“…The chemical synthetic method is now generally employed for synthesizing these types of biologically important cyclic nucleotides particularly those chemically modified in the N-1-linked ribose moiety, which are not expected to be accessible by an enzymatic route. [55][56][57][58][59][60][61][62][63][64][65][66] …”