Nucleotide-Derived Competitive Inhibitors of Ectonucleotidase CD39─A Promising Extracellular Target for Immunotherapy...

Ectonucleotidases catalyze the hydrolysis of extracellular nucleotides, maintaining the balance between proinflammatory ATP and immunosuppressive adenosine. In the present study, we developed potent competitive inhibitors of the main ATP-hydrolyzing ectoenzyme nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39) based on 8-butylthio-AMP as a lead structure. Altogether, 88 purine nucleotides and analogs with broad structural modifications were synthesized, 78 of which are new compounds. 8-Substitution of the purine scaffold with bulky residues is essential for high potency and confers metabolic stability. 8-(1-Naphthylthio)-N⁶-(4-phenylbutyl)-AMP (42b, PSB-24379) is the most potent CD39 inhibitor of the series (K_i 77.4 nM), showing ancillary CD73 inhibition (K_i 240 nM). Docking into a human CD39 homology model rationalized key interactions. PSB-24379 reduced ATP hydrolysis in melanoma and breast cancer cell membranes and partially reverted ATP-mediated effects on T cell activation and proliferation in an ATP-rich environment. These CD39 inhibitors represent high-quality tool compounds with potential as drugs for immunotherapy of cancer.