Ecto-5'-nucleotidase (CD73) is the main enzyme that catalyzes the hydrolysis of extracellular AMP to produce anti-inflammatory, immunosuppressive adenosine. Many tumor cells over-express ectonucleotidases accumulating adenosine in the tumor microenvironment, which promotes tumor growth, metastasis, angiogenesis, and immune escape. CD73 is upregulated in inflammation, and possesses potential as a biomarker and as a novel drug target for inflammatory diseases and cancer immunotherapy. New, metabolically stable N6-disubstituted adenosine-5'-diphosphate analogs were synthesized providing a basis for the design and preparation of the CD73-selective radioligand [3H]PSB-17230 by catalytic hydrogenation of a propargyl-substituted precursor. It showed high, pico- to low nanomolar affinity for human, rat and mouse CD73, slow dissociation kinetics, negligible non-specific binding, and high selectivity, as confirmed by studies on an inactive CD73 mutant and CD73 knockout cells. A high-resolution co-crystal structure (2.35 Å) of PSB-17230 with human CD73 elucidated its binding interactions. Radioligand binding was employed to characterize competitive CD73 inhibitors and to study expression levels of the enzyme in tissues and tumor cell lines of different species. Moreover, [3H]PSB-17230 was employed in autoradiography studies to determine CD73 expression in healthy and diseased mouse and human tissues. Significant upregulation of CD73 was observed in a mouse asthma model and in kidney cancer biopsies as compared to healthy controls. [3H]PSB-17230 represents a high-affinity tracer which is anticipated to find broad application in drug screening, preclinical studies, and for diagnostic purposes in inflammation and cancer, enabling drug monitoring and targeted therapies.
