Structure activity and molecular modeling analyses of ribose- and base-modified uridine 5′-triphosphate analogues at the human P2Y2 and P2Y4 receptors

Abstract

With the long-term goal of developing receptor subtype-selective high affinity agonists for the uracil nucleotide-activated P2Y receptors we have carried out a series of structure activity and molecular modeling studies of the human P2Y2 and P2Y4 receptors. UTP analogues with substitutions in the 2'-position of the ribose moiety retained capacity to activate both P2Y2 and P2Y4 receptors. Certain of these analogues were equieffective for activation of both receptors whereas 2'-amino-2'-deoxy-UTP exhibited higher potency for the P2Y2 receptor and 2'-azido-UTP exhibited higher potency for the P2Y4 receptor. 4-Thio substitution of the uracil base resulted in a UTP analogue with increased potency relative to UTP for activation of both the P2Y2 and P2Y4 receptors. In contrast, 2-thio substitution and halo- or alkyl substitution in the 5-position of the uracil base resulted in molecules that were 3-30-fold more potent at the P2Y2 receptor than P2Y4 receptor. 6-Aza-UTP was a P2Y2 receptor agonist that exhibited no activity at the P2Y4 receptor. Stereoisomers of UTPalphaS and 2'-deoxy-UTPalphaS were more potent at the P2Y2 than P2Y4 receptor, and the R-configuration was favored at both receptors. Molecular docking studies revealed that the binding mode of UTP is similar for both the P2Y2 and P2Y4 receptor binding pockets with the most prominent dissimilarities of the two receptors located in the second transmembrane domain (V90 in the P2Y2 receptor and I92 in the P2Y4 receptor) and the second extracellular loop (T182 in the P2Y2 receptor and L184 in the P2Y4 receptor). In summary, this work reveals substitutions in UTP that differentially affect agonist activity at P2Y2 versus P2Y4 receptors and in combination with molecular modeling studies should lead to chemical synthesis of new receptor subtype-selective drugs.

Expression and function of rat urothelial P2Y receptors

“…Based on current pharmacological profiling, this rank order is not consistent with that reported for P2Y 1 (ADP Ͼ UTP), P2Y 6 (UDP Ͼ UTP), P2Y 11 (ATP Ͼ Ͼ UTP), P2Y 12 (ADP Ͼ UTP), P2Y 13 (2-MeSADP ϭ ADP Ͼ UTP, ATP) or P2Y 14 (UDP-glucose Ͼ Ͼ UTP, ATP, ADP) (1,14,41). Only the rat P2Y 2 and P2Y 4 receptors have been reported to be activated preferentially and equipotently by UTP and ATP (14,26). Closer analysis revealed that UTP-and UTP␥S-evoked increases in cytosolic calcium in rat urothelial cells were mediated by the release of calcium from intracellular stores and via PLC-linked mechanisms, consistent with the mode of action of putative P2Y 2/4 receptors (3,12).…”

Defining the nucleotide binding sites of P2Y receptors using rhodopsin-based homology modeling

“…This difference in the size of the pocket seemed to be critical for the selective recognition of 5-substituted UTP derivatives. For example, 5-methyl UTP is 9.8-fold less potent than UTP at the P2Y 2 receptor, but 53-fold less potent than UTP at the P2Y 4 receptor [16].…”

“…2C). For example, molecular modeling combined with SAR analysis was recently applied to study the binding modes of UTP and its analogues in the P2Y 2 and P2Y 4 receptors [16]. A conformational analysis of UTP in the binding pockets of the P2Y 2 and P2Y 4 receptors was performed by means of the mixed MCMM/LMCS sampling method as implemented in MacroModel 9.0 [22] The search was performed on the ligand and the residues located within 5 Å of the ligand, while the remaining residues were conformationally frozen.…”

“…Detailed homology models have been constructed for other members of the P2Y receptor family [2,[15][16][17][18][19]. The models were built using the program Modeler [20] implemented in InsightII [21].…”

UDP Is a Competitive Antagonist at the Human P2Y14Receptor

“…We have identified and developed novel, selective ligands for several P2Y recep-tors that have proven useful for pharmacological resolution of molecularly defined P2Y-R in cells and tissues (Boyer et al, 1996;Houston et al, 2006Houston et al, , 2008Jacobson et al, 2006). Accordingly, we are interested in identifying a selective antagonist for the P2Y 14 -R. Ault and Broach (2006) recently used a yeast model system in which various nucleotides and nucleotide sugars were examined for their ability to stimulate growth of mutant P2Y 14 -R-expressing yeast cells in studies focused on the identification of mutant P2Y 14 receptors with differential agonist sensitivities.…”