title: Development of a high-throughput cell-based assay for identification of IL-17 inhibitors.
abstract: Human interleukin 17 (IL-17) is a proinflammatory cytokine derived mainly from activated T cells. Extensive evidence points to a significant role of IL-17 in many autoimmune and infectious diseases, as well as tumorigenesis and transplant rejection, and suggests that targeting IL-17 could be a promising therapeutic strategy. Robust cell-based assays would thus be essential for lead identification and the optimization of therapeutic candidates. Herein, we report a well-characterized two-step assay, consisting of (a) in vitro activation and stimulation of CD4(+) T lymphocytes by a defined complex of antibodies and cytokines, leading to T helper 17 (Th17) cell differentiation and IL-17 production, and (b) IL-17 quantification in cell supernatants using a homogeneous time-resolved fluorescence (HTRF) assay. The system was optimized for and shown to be reliable in high-throughput compatible 96- and 384-well plate formats. The assay is robust (Z' > 0.5) and simple to perform, yields a stable response, and allows for sufficient discrimination of positive (IL-17-producing cells) and negative controls (uninduced cells). The assay was validated by performing dose-response testing of rapamycin and cyclosporine A, which had previously been reported to inhibit IL-17, and determining, for the first time, their in vitro potencies (IC(50)s of 80 ± 23 pM and 223 ± 52 nM, respectively). Also, IKK 16, a selective small-molecule inhibitor of IκB kinase, was found to inhibit IL-17 production, with an IC(50) of 315 ± 79 nM.
authors.affiliation: AMRI, Bothell Research Center, Bothell, WA 98021, USA. svetlana.dobritsa@amriglobal.com
authors.forename: Svetlana V
authors.initials: SV
authors.lastname: Dobritsa
authors.forename: Iok Teng
authors.initials: IT
authors.lastname: Kuok
authors.forename: Hai
authors.initials: H
authors.lastname: Nguyen
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Webster
authors.forename: Andrew Mearns
authors.initials: AM
authors.lastname: Spragg
authors.forename: Timothy
authors.initials: T
authors.lastname: Morley
authors.forename: Grant J
authors.initials: GJ
authors.lastname: Carr
mesh_terms: D000328:Adult; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D020381:Interleukin-17 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D012015:Reference Standards; D015203:Reproducibility of Results; D020123:Sirolimus / Q000494:pharmacology; D012997:Solvents / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D058504:Th17 Cells / Q000187:drug effects* / Q000378:metabolism
chemical_list: D020381:Interleukin-17; D012997:Solvents; D016572:Cyclosporine; D020123:Sirolimus; D004121:Dimethyl Sulfoxide