Nucleoside triphosphate diphosphohydrolase1 (
NTPDase1, CD39) inhibitors have potential as novel drugs for the (immuno)
therapy of
cancer. They increase the extracellular concentration of immunostimulatory
ATP and reduce the formation of
AMP, which can be further hydrolyzed by
ecto-5'-nucleotidase (CD73) to immunosuppressive,
cancer-promoting
adenosine. In the present study, we synthesized analogs and derivatives of the standard CD39 inhibitor
ARL67156, a
nucleotide analog which displays a competitive mechanism of inhibition. Structure-activity relationships were analyzed at the human
enzyme with respect to substituents in the N 6- and C8-position of the
adenine core, and modifications of the triphosph(on)ate chain. Capillary electrophoresis coupled to
laser-induced fluorescence detection employing a fluorescent-labeled
ATP derivative was employed to determine the compounds' potency. Selected inhibitors were additionally evaluated in an orthogonal,
malachite green assay versus the natural substrate
ATP. The most potent CD39 inhibitors of the present series were
ARL67156 and its derivatives 31 and 33 with Ki values of around 1 µM. Selectivity studies showed that all three
nucleotide analogs additionally blocked CD73 acting as dual-target inhibitors. Docking studies provided plausible binding modes to both targets. The present study provides a full characterization of the frequently applied CD39 inhibitor
ARL67156, presents structure-activity relationships, and provides a basis for future optimization towards selective CD39 and dual CD39/CD73 inhibitors.