Identification of novel selective P2Y^sub 6^ receptor antagonists by high-throughput screening assay

Aims The P2Y6 nucleotide receptor is widely involved in inflammatory responses, and is a promising molecular target for the treatment of inflammatory diseases. Although several P2Y6 receptor antagonists have been developed and evaluated thus far, none has successfully been developed into a therapeutic drug. In this study, we explored new promising compounds that inhibit the human P2Y6 receptor. Main methods High-throughput screening (HTS) was used to study the effects of various compounds on human P2Y6 receptors expressed in 1321N1 human astrocytoma cells by monitoring intracellular Ca2 + concentration ([Ca2 +]i) levels using an FDSS7000 real-time fluorescence detector. IL-8 concentration was measured by enzyme-linked immunosorbent assay. Key findings Among structurally diverse chemical libraries totalling 141,700 compounds, 43 compounds with an inhibitory activity against the P2Y6 receptor were identified. Further studies using a dose-response assay, receptor selectivity assay, and chemokine measurement assay revealed the selective P2Y6 receptor inhibitor TIM-38, which inhibited UDP-induced [Ca2 +]i elevation in a dose-dependent manner. TIM-38 had an IC50 value of 4.3 µM and inhibited P2Y6 without affecting the response induced by four other human P2Y or muscarinic receptors. In addition, TIM-38 inhibited UDP-induced interleukin-8 release in a dose-dependent manner without affecting releases caused by other stimulus such as interleukin-1β or tumour necrosis factor-α. Analyses of TIM-38 derivatives revealed that the nitro moiety is vital to P2Y6 receptor inhibition. Significance TIM-38 acts as a novel structural antagonist of P2Y6 receptor and may be a good lead compound for developing a P2Y6 receptor-targeted anti-inflammatory drug.