title: Development of a scintillation proximity binding assay for high-throughput screening of hematopoietic prostaglandin D2 synthase.
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.
authors.affiliation: Discovery Technologies, Amgen Inc., South San Francisco, CA 94080, USA.
authors.forename: Cesar
authors.initials: C
authors.lastname: Meleza
authors.affiliation: Inflammation, Amgen Inc., Seattle, WA 98119, USA.
authors.forename: Bobbie
authors.initials: B
authors.lastname: Thomasson
authors.affiliation: Inflammation, Amgen Inc., Seattle, WA 98119, USA.
authors.forename: Chidambaram
authors.initials: C
authors.lastname: Ramachandran
authors.affiliation: Biologic Optimization, Amgen Inc., Seattle, WA 98119, USA.
authors.forename: Jason W
authors.initials: JW
authors.lastname: O'Neill
authors.affiliation: Discovery Attribute Sciences, Amgen Inc., Cambridge, MA 02141, USA.
authors.forename: Klaus
authors.initials: K
authors.lastname: Michelsen
authors.affiliation: Discovery Technologies, Amgen Inc., South San Francisco, CA 94080, USA. Electronic address: meil@amgen.com.
authors.forename: Mei-Chu
authors.initials: MC
authors.lastname: Lo
mesh_terms: D002460:Cell Line; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054834:Lipocalins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015230:Prostaglandin D2 / Q000096:biosynthesis / Q000097:blood; D044262:Prostaglandin H2 / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D054834:Lipocalins; D011994:Recombinant Proteins; D044262:Prostaglandin H2; D019746:Intramolecular Oxidoreductases; C022466:prostaglandin R2 D-isomerase; D015230:Prostaglandin D2
keywords: H-PGDS; High-throughput screening; Inhibitor; SPA