CD73 controls ocular adenosine levels and protects retina from light-induced phototoxicity.

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.

The CD73 is induced by TGF-ß1 triggered by nutrient deprivation and highly expressed in dedifferentiated human melanoma.

CD73 is the key enzyme in the generation of extracellular adenosine, a mediator involved in tumor progression, tumor immune escape and resistance to anti-cancer therapeutics. Microenvironmental conditions influence the expression of CD73 in tumor cells. However how CD73 expression and activity is regulated in a stress condition of lower nutrient availability are largely unknown. Our results indicate that serum starvation leads to a marked up-regulation of CD73 expression on A375 melanoma cells in a time-dependent manner. The cell-surface expression of CD73 is associated with an increased release of TGF-ß1 by starved cells. Blockade of TGF-ß1 receptors or TGFß/SMAD3 signaling pathway significantly reduce the expression of CD73 induced by starvation. Treatment of cells with rTGF-ß1 up-regulates the expression of CD73 in a concentration-dependent manner, confirming the role of this pathway in regulating CD73 in melanoma A375 cells. The increased expression of CD73 is associated with enhanced AMPase activity, which is selectively reduced by inhibitors of CD73 activity, APCP and PSB-12489. Pharmacological blockade of CD73 significantly inhibits invasion of melanoma cells in a transwell system. Furthermore, using multiplex immunofluorescence imaging we found that, within human melanoma metastases, tumor cells at the dedifferentiated stage show the highest CD73 protein expression. In summary, our data provide new insights into the mechanism regulating the expression/activity of CD73 in melanoma cells in a condition of lower availability of nutrients, which is a common feature of the tumor microenvironment. Within human metastatic melanoma tissues elevated protein expression of CD73 is associated with an invasive-like phenotype.

2-Substituted α,ß-Methylene-ADP Derivatives: Potent Competitive Ecto-5'-nucleotidase (CD73) Inhibitors with Variable Binding Modes.

CD73 inhibitors are promising drugs for the (immuno)therapy of cancer. Here, we present the synthesis, structure-activity relationships, and cocrystal structures of novel derivatives of the competitive CD73 inhibitor α,ß-methylene-ADP (AOPCP) substituted in the 2-position. Small polar or lipophilic residues increased potency, 2-iodo- and 2-chloro-adenosine-5'-O-[(phosphonomethyl)phosphonic acid] (15, 16) being the most potent inhibitors with Ki values toward human CD73 of 3-6 nM. Subject to the size and nature of the 2-substituent, variable binding modes were observed by X-ray crystallography. Depending on the binding mode, large species differences were found, e.g., 2-piperazinyl-AOPCP (21) was >12-fold less potent against rat CD73 compared to human CD73. This study shows that high CD73 inhibitory potency can be achieved by simply introducing a small substituent into the 2-position of AOPCP without the necessity of additional bulky N6-substituents. Moreover, it provides valuable insights into the binding modes of competitive CD73 inhibitors, representing an excellent basis for drug development.

Fluorescent Probes for Ecto-5'-nucleotidase (CD73).

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,ß-methylene-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.