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Anal Biochem. 2016 Oct 15;511:17-23. doi: 10.1016/j.ab.2016.07.028. Epub 2016 Jul 30.

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

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Discovery Technologies, Amgen Inc., South San Francisco, CA 94080, USA.
Inflammation, Amgen Inc., Seattle, WA 98119, USA.
Biologic Optimization, Amgen Inc., Seattle, WA 98119, USA.
Discovery Attribute Sciences, Amgen Inc., Cambridge, MA 02141, USA.
Discovery Technologies, Amgen Inc., South San Francisco, CA 94080, USA. Electronic address:


Prostaglandin D2 synthase (PGDS) catalyzes the isomerization of prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2). PGD2 produced by hematopoietic prostaglandin D2 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 PGH2, 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 IC50s ranging from 6 to 807 nM in SPA and 82 nM to 10 μM in the LAD2 cell assay.


H-PGDS; High-throughput screening; Inhibitor; SPA

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