Compartmentalization of adenosine metabolism in cancer cells and its modulation during acute hypoxia.

Extracellular adenosine mediates diverse anti-inflammatory, angiogenic and vasoactive effects and becomes an important therapeutic target for cancer, which has been translated into clinical trials. This study was designed to comprehensively assess adenosine metabolism in prostate and breast cancer cells. We identified cellular adenosine turnover as a complex cascade, comprised of (a) the ectoenzymatic breakdown of ATP via sequential nucleotide pyrophosphatase/phosphodiesterase-1, ecto-5'-nucleotidase/CD73 and adenosine deaminase reactions, and ATP re-synthesis through counteracting adenylate kinase and nucleoside diphosphokinase; (b) the uptake of nucleotide-derived adenosine via equilibrative nucleoside transporters; and (c) the intracellular adenosine phosphorylation into ATP by adenosine kinase and other nucleotide kinases. The exposure of cancer cells to 1% O2 for 24 hours triggered ∼2-fold up-regulation of CD73, without affecting nucleoside transporters, adenosine kinase activity and cellular ATP content. The ability of adenosine to inhibit the tumor-initiating potential of breast cancer cells via receptor-independent mechanism was confirmed in vivo using a xenograft mouse model. The existence of redundant pathways controlling extracellular and intracellular adenosine provides a sufficient justification for reexamination of the current concepts of cellular purine homeostasis and signaling in cancer

Soluble and membrane-bound adenylate kinase and nucleotidases augment ATP-mediated inflammation in diabetic retinopathy eyes with vitreous hemorrhage

ATP and adenosine are important signaling molecules involved in vascular remodeling, retinal function, and neurovascular coupling in the eye. Current knowledge on enzymatic pathways governing the duration and magnitude of ocular purinergic signaling is incompletely understood. By employing sensitive analytical assays, this study dissected ocular purine homeostasis as a complex and coordinated network. Along with previously characterized ecto-5-nucleotidase/CD73 and adenylate kinase activities, other enzymes have been identified in vitreous fluids, including nucleoside triphosphate diphosphohydrolase (NTPDase), adenosine deaminase, and alkaline phosphatase. Strikingly, activities of soluble adenylate kinase, adenosine deaminase, ecto-5-nucleotidase/CD73, and alkaline phosphatase, as well as intravitreal concentrations of ATP and ADP, were concurrently upregulated in patients suffering from diabetic retinopathy (DR) with non-clearing vitreous hemorrhage (VH), when compared to DR eyes without VH and control eyes operated due to macular hole or pucker. Additional histochemical analysis revealed selective distribution of key ecto-nucleotidases (NTPDase1/CD39, NTPDase2, ecto-5-nucleotidase/CD73, and alkaline phosphatase) in the human sensory neuroretina and optic nerve head, and also in pathological neofibrovascular tissues surgically excised from patients with advanced proliferative DR. Collectively, these data provide evidence for specific hemorrhage-related shifts in purine homeostasis in DR eyes from the generation of anti-inflammatory adenosine towards a pro-inflammatory and pro-angiogenic ATP-regenerating phenotype. In the future, identifying the exact mechanisms by which a broad spectrum of soluble and membrane-bound enzymes coordinately regulates ocular purine levels and the further translation of purine-converting enzymes as potential therapeutic targets in the treatment of proliferative DR and other vitreoretinal diseases will be an area of intense interest.Key messagesNTPDase, alkaline phosphatase, and adenosine deaminase circulate in human vitreous.Purinergic enzymes are up-regulated in diabetic eyes with vitreous hemorrhage.Soluble adenylate kinase maintains high ATP levels in diabetic retinopathy eyes.Ecto-nucleotidases are co-expressed in the human retina and optic nerve head.Alkaline phosphatase is expressed on neovascular tissues excised from diabetic eyes.Peer reviewe

Soluble and membrane-bound adenylate kinase and nucleotidases augment ATP-mediated inflammation in diabetic retinopathy eyes with vitreous hemorrhage

ATP and adenosine are important signaling molecules involved in vascular remodeling, retinal function, and neurovascular coupling in the eye. Current knowledge on enzymatic pathways governing the duration and magnitude of ocular purinergic signaling is incompletely understood. By employing sensitive analytical assays, this study dissected ocular purine homeostasis as a complex and coordinated network. Along with previously characterized ecto-5′-nucleotidase/CD73 and adenylate kinase activities, other enzymes have been identified in vitreous fluids, including nucleoside triphosphate diphosphohydrolase (NTPDase), adenosine deaminase, and alkaline phosphatase. Strikingly, activities of soluble adenylate kinase, adenosine deaminase, ecto-5′-nucleotidase/CD73, and alkaline phosphatase, as well as intravitreal concentrations of ATP and ADP, were concurrently upregulated in patients suffering from diabetic retinopathy (DR) with non-clearing vitreous hemorrhage (VH), when compared to DR eyes without VH and control eyes operated due to macular hole or pucker. Additional histochemical analysis revealed selective distribution of key ecto-nucleotidases (NTPDase1/CD39, NTPDase2, ecto-5′-nucleotidase/CD73, and alkaline phosphatase) in the human sensory neuroretina and optic nerve head, and also in pathological neofibrovascular tissues surgically excised from patients with advanced proliferative DR. Collectively, these data provide evidence for specific hemorrhage-related shifts in purine homeostasis in DR eyes from the generation of anti-inflammatory adenosine towards a pro-inflammatory and pro-angiogenic ATP-regenerating phenotype. In the future, identifying the exact mechanisms by which a broad spectrum of soluble and membrane-bound enzymes coordinately regulates ocular purine levels and the further translation of purine-converting enzymes as potential therapeutic targets in the treatment of proliferative DR and other vitreoretinal diseases will be an area of intense interest.</p

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 CD39(high)/CD73(low) processes and forming local "purinergic junctions" with CD39(low)/CD73(-) neuronal cell bodies and CD39(high)/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.</p

Structure-Activity Relationship of 3-Methylcytidine-5'-α,β-methylenediphosphates as CD73 Inhibitors

We recently reported N-4-substituted 3-methylcytidine-5'-alpha,beta-methylenediphosphates as CD73 inhibitors, potentially useful in cancer immunotherapy. We now expand the structure-activity relationship of pyrimidine nucleotides as human CD73 inhibitors. 4-Chloro (MRS4598 16; K-i = 0.673 nM) and 4-iodo (MRS4620 18; K-i = 0.436 nM) substitution of the N-4-benzyloxy group decreased K-i by similar to 20-fold. Primary alkylamine derivatives coupled through a p-amido group with a varying methylene chain length (24 and 25) were functionalized congeners, for subsequent conjugation to carrier or reporter moieties. X-ray structures of hCD73 with two inhibitors indicated a ribose ring conformational adaptation, and the benzyloxyimino group (E configuration) binds to the same region (between the C-terminal and N-terminal domains) as N-4-benzyl groups in adenine inhibitors. Molecular dynamics identified stabilizing interactions and predicted conformational diversity. Thus, by N-4-benzyloxy substitution, we have greatly enhanced the inhibitory potency and added functionality enabling molecular probes. Their potential as anticancer drugs was confirmed by blocking CD73 activity in tumor tissues in situ