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

Prostaglandin D-2 synthase (PGDS) catalyzes the isomerization of prostaglandin H-2 (PGH(2)) to prostaglandin D-2 (PGD(2)). PGD(2) produced by hematopoietic prostaglandin D-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 assay is not feasible for large-scale screening of H-PGDS inhibitors. Herein, we report the development of a competition binding assay amenable to high-throughput screening (HTS) in a scintillation proximity assay (SPA) format. This assay was used to screen an in-house compound library of approximately 280,000 compounds for novel H-PGDS inhibitors. The hit rate of the H-PGDS primary screen was found to be 4%. This high hit rate suggests 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 assays with IC(50)s ranging from 6 to 807 nM in SPA and 82 nM to 10 mu M in the LAD2 cell assay. (C) 2016 Elsevier Inc. All rights reserved.