A promising drug candidate for the treatment of glaucoma based on a P2Y6-receptor agonist.

Extracellular nucleotides can regulate the production/drainage of the aqueous humor via activation of P2 receptors, thus affecting the intraocular pressure (IOP). We evaluated 5-OMe-UDP(α-B), 1A, a potent P2Y6-receptor agonist, for reducing IOP and treating glaucoma. Cell viability in the presence of 1A was measured using [3-(4, 5-dimethyl-thiazol-2-yl) 2, 5-diphenyl-tetrazolium bromide] (MTT) assay in rabbit NPE ciliary non-pigmented and corneal epithelial cells, human retinoblastoma, and liver Huh7 cells. The effect of 1A on IOP was determined in acute glaucomatous rabbit hyaluronate model and phenol-induced chronic glaucomatous rabbit model. The origin of activity of 1A was investigated by generation of a homology model of hP2Y6-R and docking studies. 1A did not exert cytotoxic effects up to 100 mM vs. trusopt and timolol in MTT assay in ocular and liver cells. In normotensive rabbits, 100 µM 1A vs. xalatan, trusopt, and pilocarpine reduced IOP by 45 vs. 20-30%, respectively. In the phenol animal model, 1A (100 µM) showed reduction of IOP by 40 and 20%, following early and late administration, respectively. Docking results suggest that the high activity and selectivity of 1A is due to intramolecular interaction between Pα-BH3 and C5-OMe which positions 1A in a most favorable site inside the receptor. P2Y6-receptor agonist 1A effectively and safely reduces IOP in normotense, acute, and chronic glaucomatous rabbits, and hence may be suggested as a novel approach for the treatment of glaucoma.

5-OMe-uridine-5'-O-(α-boranodiphosphate), a novel nucleotide derivative highly active at the human P2Y(6) receptor protects against death-receptor mediated glial apoptosis.

P2Y receptors are activated by nucleotides and involved in numerous physiological/pathophysiological processes. However, investigations of specific P2Y receptor functions have been hampered by lack of suitable receptor agonists-antagonists. Recently, we identified the nucleotide 5-OMe-UDP as potent and selective agonist for human P2Y6 receptors. We studied a series of derivatives of this analog with a Pα-borano group substituting a non-bridging oxygen and found increased potency and receptor specificity. Rp-5-OMe-UDPαB (Rp-5-OMe-uridine 5'-O-α-boranodiphosphate) was most potent and selective in inducing intracellular calcium signaling in 1321N1 astrocytoma cells expressing the human P2Y6 receptor. Here, we investigated whether Rp-5-OMe-UDPαB evokes cell protection through human P2Y6 receptors. We tested a well-established model, tumor necrosis factor α (TNFα)-induced cell death in 1321N1 astrocytoma cells. Rp-5-OMe-UDPαB inhibited TNFα-induced cell death even stronger than UDP. These first data of a neuro-protective activity of the human P2Y6 receptor emphasize the potential of the stable, selective, and potent Rp-5-OMe-UDPαB analog for exploiting P2Y6 receptor-mediated cellular functions, like cytoprotection in human tissues, with suitability for future neuro-protective drug development.

UDP made a highly promising stable, potent, and selective P2Y6-receptor agonist upon introduction of a boranophosphate moiety.

P2Y(6) nucleotide receptor (P2Y(6)-R) plays important physiological roles, such as insulin secretion and reduction of intraocular pressure. However, this receptor is still lacking potent and selective agonists to be used as potential drugs. Here, we synthesized uracil nucleotides and dinucleotides, substituted at the C5 and/or P(α) position with methoxy and/or borano groups, 18-22. Compound 18A, R(p) isomer of 5-OMe-UDP(α-B), is the most potent and P2Y(6)-R selective agonist currently known (EC(50) 0.008 µM) being 19-fold more potent than UDP and showing no activity at uridine nucleotide receptors, P2Y(2)- and P2Y(4)-R. Analogue 18A was highly chemically stable under conditions mimicking gastric juice acidity (t(1/2) = 16.9 h). It was more stable to hydrolysis by nucleotide pyrophosphatases (NPP1,3) than UDP (15% and 28% hydrolysis by NPP1 and NPP3, respectively, vs 50% and 51% hydrolysis of UDP) and metabolically stable in blood serum (t(1/2) = 17 vs 2.4, 11.9, and 21 h for UDP, 5-OMe-UDP, and UDP(α-B), respectively). This newly discovered highly potent and physiologically stable P2Y(6)-R agonist may be of future therapeutic potential.

5-OMe-UDP is a potent and selective P2Y(6)-receptor agonist.

P2Y nucleotide receptors (P2Y-Rs) play important physiological roles. However, most of the P2Y-R subtypes are still lacking potent and selective agonists and antagonists. Based on data mining analysis of binding interactions in 44 protein-uridine nucleos(t)ides complexes, we designed uracil nucleotides, substituted at the C5/C6 position. All C6-substituted derivatives were inactive at the P2Y(2,4,6)-Rs, while out of the C5-substituted analogues, only 5-OMe-UD(T)P showed activity. To rationalize the data, the ionization and conformation of these analogues were evaluated. The pK(a) values of most analogues substituted at the C5/C6 positions were unaltered compared to UTP (pK(a) 9.42), except for 5-F-UTP nucleotide (pK(a) 7.85). C6-substituted analogues adopt the syn or high-syn conformations, which are disfavored by the receptors, while 5-OMe-UD(T)P adopt the favored anti conformation. Furthermore, 5-OMe-UDP adopts the S sugar puckering, which is the conformation preferred by the P2Y(6)-R, but not the P2Y(2)- or P2Y(4)-Rs. 5-OMe-UDP fulfills the conformational and H-bonding requirements of P2Y(6)-R, thus, making a potent P2Y(6)-R agonist (EC(50) 0.08 microM), more than UDP (EC(50) 0.14 microM).