Substrate-Based Design of Cytosolic Nucleotidase IIIB Inhibitors and Structural Insights into Inhibition Mechanism

Cytosolic nucleotidases (cNs) catalyze dephosphorylation of nucleoside 5’-monophosphates and thereby contribute to the regulation of nucleotide levels in cells. cNs have also been shown to dephosphorylate several therapeutically relevant nucleotide analogues. cN-IIIB has shown in vitro a distinctive activity towards 7-mehtylguanosine monophosphate (m(7)GMP), which is one key metabolites of mRNA cap. Consequently, it has been proposed that cN-IIIB participates in mRNA cap turnover and prevents undesired accumulation and salvage of m(7)GMP. Here, we sought to develop molecular tools enabling more advanced studies on the cellular role of cN-IIIB. To that end, we performed substrate and inhibitor property profiling using a library of 41 substrate analogs. The most potent hit compounds (identified among m(7)GMP analogs) were used as a starting point for structure–activity relationship studies. As a result, we identified several 7-benzylguanosine 5’-monophosphate (Bn(7)GMP) derivatives as potent, unhydrolyzable cN-IIIB inhibitors. The mechanism of inhibition was elucidated using X-ray crystallography and molecular docking. Finally, we showed that compounds that potently inhibit recombinant cN-IIIB have the ability to inhibit m(7)GMP decay in cell lysates.