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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.

Author information

1
Discovery Technologies, Amgen Inc., South San Francisco, CA 94080, USA.
2
Inflammation, Amgen Inc., Seattle, WA 98119, USA.
3
Biologic Optimization, Amgen Inc., Seattle, WA 98119, USA.
4
Discovery Attribute Sciences, Amgen Inc., Cambridge, MA 02141, USA.
5
Discovery Technologies, Amgen Inc., South San Francisco, CA 94080, USA. Electronic address: meil@amgen.com.

Abstract

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.

KEYWORDS:

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

PMID:
27485270
DOI:
10.1016/j.ab.2016.07.028
[Indexed for MEDLINE]

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