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.
Highlights
Transient receptor potential (TRP) channels are six-transmembrane polypeptide subunits that assemble as tetramers to form cation-permeable pores.1 TRP subfamily melastatin, type 2 (TRPM2), is a Ca2+ permeant channel which is permeant to Na+, K+, and Cs+ ions.2 TRPM2 is unique among the known ion channels as it contains a C-terminal domain which is homologous to NUDT9 Adenosine 5′-diphosphoribose (ADPR)-hydrolase, and this has led to considerable interest
TRPM2 is unique among the known ion channels as it contains a C-terminal domain which is homologous to NUDT9 ADPR-hydrolase, and this has led to considerable interest
For the first time, we report a chemo-enzymatic approach involving chemical synthesis, coupled with use of Neurospora crassa NADase, to interrogate each major motif of the ADPR structure and Article subsequently evaluate the effect of these modifications on antagonist activity at the NUDT9 domain of the TRPM2 channel
Summary
Transient receptor potential (TRP) channels are six-transmembrane polypeptide subunits that assemble as tetramers to form cation-permeable pores. TRP subfamily melastatin, type 2 (TRPM2), is a Ca2+ permeant channel which is permeant to Na+, K+, and Cs+ ions. TRPM2 is unique among the known ion channels as it contains a C-terminal domain which is homologous to NUDT9 ADPR-hydrolase, and this has led to considerable interest. TRP subfamily melastatin, type 2 (TRPM2), is a Ca2+ permeant channel which is permeant to Na+, K+, and Cs+ ions.. TRPM2 is unique among the known ion channels as it contains a C-terminal domain which is homologous to NUDT9 ADPR-hydrolase, and this has led to considerable interest. The NUDT9-homology (NUDT9H) domain of human TRPM2 extends from residue 1236 to the Cterminus. NUDT9 was identified in an EST database screen for proteins with homology to the C-terminus of TRPM2. It is an enzyme of the Nudix family of pyrophosphatases, with adenosine 5′-diphosphoribose (ADPR, 1, Figure 1) as sole substrate. A NUDT9 crystal structure illustrated that this is a two-domain enzyme with a C-terminal ADPRase and Nterminal domain which enhances affinity for ADPR..
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