Ecto‐5′‐nucleotidase (CD73, EC 3.1.3.5) catalyzes the extracellular hydrolysis of AMP yielding adenosine, which induces immunosuppression, angiogenesis, metastasis, and proliferation of cancer cells. CD73 inhibition is therefore proposed as a novel strategy for cancer (immuno)therapy, and CD73 antibodies are currently undergoing clinical trials. Despite considerable efforts, the development of small molecule CD73 inhibitors has met with limited success. To develop a suitable drug candidate, a high resolution (2.05 Å) co‐crystal structure of the CD73 inhibitor PSB‐12379, a nucleotide analogue, in complex with human CD73 is determined. This allows the rational design and development of a novel inhibitor (PSB‐12489) with subnanomolar inhibitory potency toward human and rat CD73, high selectivity, as well as high metabolic stability. A co‐crystal structure of PSB‐12489 with CD73 (1.85 Å) reveals the interactions responsible for increased potency. PSB‐12489 is the most potent CD73 inhibitor to date representing a powerful tool compound and novel lead structure.

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 laserinduced 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 K i 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 structureactivity relationships, and provides a basis for future optimization towards selective CD39 and dual CD39/CD73 inhibitors.

Ecto-5′-nucleotidase (CD73) catalyzes the hydrolysis of AMP to anti-inflammatory, immunosuppressive adenosine. It is expressed on vascular endothelial, epithelial, and also numerous cancer cells where it strongly contributes to an immunosuppressive microenvironment. In the present study we designed and synthesized fluorescent-labeled CD73 inhibitors with low nanomolar affinity and high selectivity based on N 6 -benzyl-α,βmethylene-ADP (PSB-12379) as a lead structure. Fluorescein was attached to the benzyl residue via different linkers resulting in PSB-19416 (14b, K i 12.6 nM) and PSB-18332 (14a, K i 2.98 nM) as fluorescent high-affinity probes for CD73. These compounds are anticipated to become useful tools for biological studies, drug screening, and diagnostic applications.

ATP and adenosine have emerged as important signaling molecules involved in vascular remodeling, retinal functioning and neurovascular coupling in the mammalian eye. However, little is known about the regulatory mechanisms of purinergic signaling in the eye. Here, we used three-dimensional multiplexed imaging, in situ enzyme histochemistry, flow cytometric analysis, and single cell transcriptomics to characterize the whole pattern of purine metabolism in mouse and human eyes. This study identified ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2, and ecto-5′-nucleotidase/CD73 as major ocular ecto-nucleotidases, which are selectively expressed in the photoreceptor layer (CD73), optic nerve head, retinal vasculature and microglia (CD39), as well as in neuronal processes and cornea (CD39, NTPDase2). Specifically, microglial cells can create a spatially arranged network in the retinal parenchyma by extending and retracting their branched CD39high/CD73low processes and forming local “purinergic junctions” with CD39low/CD73− neuronal cell bodies and CD39high/CD73− retinal blood vessels. The relevance of the CD73–adenosine pathway was confirmed by flash electroretinography showing that pharmacological inhibition of adenosine production by injection of highly selective CD73 inhibitor PSB-12489 in the vitreous cavity of dark-adapted mouse eyes rendered the animals hypersensitive to prolonged bright light, manifested as decreased a-wave and b-wave amplitudes. The impaired electrical responses of retinal cells in PSB-12489-treated mice were not accompanied by decrease in total thickness of the retina or death of photoreceptors and retinal ganglion cells. Our study thus defines ocular adenosine metabolism as a complex and spatially integrated network and further characterizes the critical role of CD73 in maintaining the functional activity of retinal cells.

Ocular ATP and adenosine have emerged as important signalling molecules involved in vascular remodeling, retinal functioning and neurovascular coupling in the mammalian eye. However, little is known about the regulatory mechanisms of purinergic signaling in the eye. Here, we used three-dimensional multiplexed imaging in combination with in situ enzyme histochemistry, flow cytometric analysis and single cell transcriptomics to characterize the pattern of purine metabolism in the mouse and human eyes. This study identified NTPDase1/CD39 and ecto-5’-nucleotidase/CD73 as major ecto-nucleotidases, which are selectively expressed in the optic nerve head, vascular endothelial and perivascular cells, outer segments of photoreceptors, retinal microglia, and cornea and coordinately control ATP and adenosine levels. The relevance of the CD73-adenosine axis was confirmed by flash electroretinography showing that pharmacological inhibition of CD73 in dark-adapted mouse eyes rendered the animals hypersensitive to prolonged bright light, manifested as decreased a-wave and b-wave amplitudes and a loss of retinal ganglion cells. Our study thus defines ocular adenosine metabolism as a complex and spatially integrated network and characterizes its critical role in protecting the retina from light-induced phototoxicity.