Synthesis of Terminal Ribose Analogues of Adenosine 5'-Diphosphate Ribose as Probes for the Transient Receptor Potential Cation Channel TRPM2.

Ondřej Baszczyňski,Monika D Rozewitz,Barry V L Potter,Andreas H Guse,Ralf Fliegert,Joanna M Watt

doi:10.1021/acs.joc.9b00338

Abstract

TRPM2 (transient receptor potential cation channel, subfamily M, member 2) is a nonselective cation channel involved in the response to oxidative stress and in inflammation. Its role in autoimmune and neurodegenerative diseases makes it an attractive pharmacological target. Binding of the nucleotide adenosine 5′-diphosphate ribose (ADPR) to the cytosolic NUDT9 homology (NUDT9H) domain activates the channel. A detailed understanding of how ADPR interacts with the TRPM2 ligand binding domain is lacking, hampering the rational design of modulators, but the terminal ribose of ADPR is known to be essential for activation. To study its role in more detail, we designed synthetic routes to novel analogues of ADPR and 2′-deoxy-ADPR that were modified only by removal of a single hydroxyl group from the terminal ribose. The ADPR analogues were obtained by coupling nucleoside phosphorimidazolides to deoxysugar phosphates. The corresponding C2″-based analogues proved to be unstable. The C1″- and C3″-ADPR analogues were evaluated electrophysiologically by patch-clamp in TRPM2-expressing HEK293 cells. In addition, a compound with all hydroxyl groups of the terminal ribose blocked as its 1″-β-O-methyl-2″,3″-O-isopropylidene derivative was evaluated. Removal of either C1″ or C3″ hydroxyl groups from ADPR resulted in loss of agonist activity. Both these modifications and blocking all three hydroxyl groups resulted in TRPM2 antagonists. Our results demonstrate the critical role of these hydroxyl groups in channel activation.

Highlights

The nonselective cation channel TRPM2 is activated in a Ca2+-dependent manner after binding of the nucleotide adenosine 5′-diphosphate (ADP)-ribose (ADPR) to its cytosolic C-terminal NUDT9 homology (NUDT9H) domain that shares homology with a mitochondrial nucleotide pyrophosphatase NUDT9.1 While earlier studies indicated that the NUDT9H domain has a low pyrophosphatase activity, hydrolyzing adenosine 5′-diphosphate ribose (ADPR) to adenosine 5′-monophosphate (AMP) and ribose 5-phosphate (R5P),[2] a recent study showed that the Nudix box motif in TRPM2 does not support catalysis and the production of AMP might have been due to spontaneous hydrolysis of ADPR at alkaline pH.[3]
The terminal ribose of ADPR is essential for the ligand-driven activation of the cation channel TRPM2.29 To investigate the structure−activity relationship (SAR) of this part of ADPR in more detail, we focused upon the synthesis of all three possible 1′′-deoxy, 2′′-deoxy, and 3′′-deoxy terminal ribose ADPR derivatives 2, 3, and 4 by selective deletion of the appropriate hydroxyl group (Figure 2)
ADPR analogues, such as those reported here, are at the cutting edge of progress to unravel the mechanism of Building on our previous study that showed the essential role of the terminal ribose of ADPR in gating of the Ca2+

Summary

■ INTRODUCTION

The nonselective cation channel TRPM2 (transient receptor potential cation channel, subfamily M, member 2) is activated in a Ca2+-dependent manner after binding of the nucleotide ADP-ribose (ADPR) to its cytosolic C-terminal NUDT9H domain that shares homology with a mitochondrial nucleotide pyrophosphatase NUDT9.1 While earlier studies indicated that the NUDT9H domain has a low pyrophosphatase activity, hydrolyzing ADPR to AMP and ribose 5-phosphate (R5P),[2] a recent study showed that the Nudix box motif in TRPM2 does not support catalysis and the production of AMP might have been due to spontaneous hydrolysis of ADPR at alkaline pH.[3]. Reactive oxygen species (ROS) and genotoxic stress can result in release of ADPR from the nucleus due to the activation of the poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase pathways.[4,5] The ADPR so generated can activate TRPM2, resulting in prolonged Ca2+-entry, mitochondrial Ca2+-overload, and apoptosis,[6] thereby contributing to cell damage in post ischemic reperfusion injury during myocardial infarction[7] and stroke.[8]. A recently reported improved procedure for the preparation of NDP sugars from nucleoside phosphorimidazolides[36] uses 2−4 equiv of magnesium chloride to achieve high reaction yields and short reactions times for nucleoside 5-phosphorimidazolide coupling to sugar phosphates

■ RESULTS AND DISCUSSION

■ CONCLUSIONS

■ ACKNOWLEDGMENTS

■ REFERENCES