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).

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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-BH 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.