Development of a scintillation proximity binding assay for high-throughput screening of hematopoietic prostaglandin D2 synthase

Abstract ProstaglandinD 2 synthase (PGDS) catalyzes the isomerization of prostaglandinH 2 (PGH 2 ) to prostaglandinD 2 (PGD 2 ). PGD 2 produced by hematopoietic prostaglandinD 2 synthase (H-PGDS) in mast cells and Th2 cells is proposed to be a mediator of allergic and inflammatory responses. Consequently, inhibitors of H-PGDS represent potential therapeutic agents for the treatment of inflammatory diseases such as asthma. Due to the instability of the PGDS substrate PGH 2 , an in-vitro enzymatic assayis not feasible for large-scale screening of H-PGDS inhibitors. Herein, we report the development of a competition binding assayamenable to high-throughput screening (HTS) in a scintillation proximity assay(SPA) format. This assaywas used to screen an in-house compound library of approximately 280,000 compounds for novel H-PGDS inhibitors. The hit rateof the H-PGDS primary screen was found to be 4%. This high hit ratesuggests that the active site of H-PGDS can accommodate a large diversity of chemical scaffolds. For hit prioritization, these initial hits were rescreened at a lower concentration in SPA and tested in the LAD2 cell assay. 116 compounds were active in both assayswith IC 50 s ranging from 6 to 807 nM in SPA and 82 nM to 10 μM in the LAD2 cell assay.