Structure–Activity Relationship of Adenosine 5′-diphosphoribose at the Transient Receptor Potential Melastatin 2 (TRPM2) Channel: Rational Design of Antagonists

Abstract

Adenosine 5′-diphosphoribose (ADPR) activates TRPM2, a Ca2+, Na+, and K+ permeable cation channel. Activation is induced by ADPR binding to the cytosolic C-terminal NudT9-homology domain. To generate the first structure–activity relationship, systematically modified ADPR analogues were designed, synthesized, and evaluated as antagonists using patch-clamp experiments in HEK293 cells overexpressing human TRPM2. Compounds with a purine C8 substituent show antagonist activity, and an 8-phenyl substitution (8-Ph-ADPR, 5) is very effective. Modification of the terminal ribose results in a weak antagonist, whereas its removal abolishes activity. An antagonist based upon a hybrid structure, 8-phenyl-2′-deoxy-ADPR (86, IC50 = 3 μM), is more potent than 8-Ph-ADPR (5). Initial bioisosteric replacement of the pyrophosphate linkage abolishes activity, but replacement of the pyrophosphate and the terminal ribose by a sulfamate-based group leads to a weak antagonist, a lead to more drug-like analogues. 8-Ph-ADPR (5) inhibits Ca2+ signalling and chemotaxis in human neutrophils, illustrating the potential for pharmacological intervention at TRPM2.

Selective inhibition of TRPM2 channel by two novel synthesized ADPR analogues

“…To begin with adenosine modification, ADPR analogues ( 7h , 7g , 10h , 10g ) with 6‐methylamino or 6‐dimethylamino substitution showed no inhibitory activity, indicating the necessity of 6‐amino for inhibitory activity. This is in accordance with Moreau's report, in which 6‐oxygen substituted ADPR (IDPR) shows no inhibitory activity 28. Substitutions at C‐2 position of purine base showed different influences.…”

“…As to the terminal ribose, it has been shown that a five‐membered ring but the hydroxyl groups of the terminal ribose plays a critical role in filling the binding site 28. Here, compounds 7i and 8a (both terminal ribose hydroxyl protected) showed inhibitory activity, while their corresponding deprotected products 10i and 10a lost inhibitory activity.…”

“…ADPR analogues 13 and 14 were synthesized with similar procedures to route 1 (Scheme 3), in which 2′‐ O ‐methyl‐5′‐ O ‐tosyl adenosine 11 was synthesized from reagent 1a , treated with methyl p ‐toluenesulphonate and tosyl chloride. In Moreau's report,28 ADPR analogues are synthesized using NADase as an important catalyst, and quite a number of individual synthetic routes were applied. Taken together, in this study, a more efficient way for synthesis of ADPR analogues was developed.…”

“…In contrast, inactive compound 7o reserves oxygen‐linked pyrophosphate. Previous report considered pyrophosphate as an activity‐keeping group and none of the sulphonamide, squarate or triazole substituted ADPR analogues showed any activity 28. Therefore, appropriate modifications on the pyrophosphate can be tolerable.…”

“…In addition, several nucleotide analogues such as adenosine monophosphate (AMP), 8‐bromoadenosine 5′‐diphosphoribose (8‐Br‐ADPR) have also been reported to be TRPM2 inhibitors (Figure 1). 26, 27 Recently, a series of ADPR analogues mainly modified at C‐8 position of purine such as 8‐phenyl‐2′‐ deoxy ‐ADPR (Figure 1) have been shown to potently inhibit TRPM2 current (IC 50  = 3 μ m ) without interfering Ca 2+ release induced by cADPR, NAADP or IP 3 28. Despite the efforts in searching for TRPM2 inhibitors, no TRP‐subtype selective TRPM2 inhibitor has yet been identified.…”