title: Establishment of a novel cell-based assay for screening small molecule antagonists of human interleukin-6 receptor.
abstract: OBJECTIVE
  Blockade of interleukin-6 (IL-6) or its receptor (IL-6R) is effective in preventing the progression of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis. In the present study, we established a novel cell-based assay for identifying small molecule IL-6R antagonists.
  
  
  METHODS
  HEK293A cells were transfected with recombinant plasmids pTaglite-SNAP-IL6R and pABhFc-IL6 to obtain membrane-bound IL-6R and recombinant human IL-6 coupled with human Fc fragment (rhIL-6), respectively. A novel screening assay based on the interaction between IL-6R and rhIL-6 was established, optimized and validated. The stability of the assay was also assessed by calculating the Z'-factor.
  
  
  RESULTS
  RhIL-6 dose-dependently bound to IL-6R expressed at HEK293A cell surface. The IC50 value of the known antagonist ab47215 was 0.38±0.08 μg/mL, which was consistent with that obtained using the traditional method (0.36±0.14 μg/mL). The value of Z'-factor was 0.68, suggesting that the novel assay was stable for high throughput screening. A total of 474 compounds were screened using the novel screening assay, and 3 compounds exhibited antagonistic activities (IC50=8.73±0.28, 32.32±9.08, 57.83±4.24 μg/mL). Furthermore, the active compounds dose-dependently inhibited IL-6-induced proliferation of 7TD1 cells, and reduced IL-6-induced STAT3 phosphorylation in U937 cells.
  
  
  CONCLUSIONS
  A novel cell-based screening assay for identifying small molecule IL-6R antagonists was established, which simplifies the procedures in traditional cellular ELISA screening and profiling and reduces the costs.
authors.affiliation: Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
authors.forename: Yang-yang
authors.initials: YY
authors.lastname: He
authors.affiliation: Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Yan
authors.affiliation: Institute of Biotechnology, Chinese Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Chang
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Drug Manufacturing Room of Pharmaceutical Department, Henan Provincial People's Hospital, Zhengzhou 450003, China.
authors.forename: Peng-yuan
authors.initials: PY
authors.lastname: Li
authors.affiliation: Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Wu
authors.affiliation: Institute of Biotechnology, Chinese Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Du
authors.affiliation: Institute of Biotechnology, Chinese Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Da-di
authors.initials: DD
authors.lastname: Zeng
authors.affiliation: Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
authors.forename: Jian-song
authors.initials: JS
authors.lastname: Fang
authors.affiliation: Institute of Biotechnology, Chinese Academy of Military Medical Sciences, Beijing 100071, China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Wang
authors.affiliation: Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
authors.forename: Guan-hua
authors.initials: GH
authors.lastname: Du
mesh_terms: D001667:Binding, Competitive; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism / Q000494:pharmacology*; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D010766:Phosphorylation; D019947:Receptors, Interleukin-6 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D020298:U937 Cells
chemical_list: C508600:IL6 protein, human; C508603:IL6R protein, human; D018836:Inflammation Mediators; D015850:Interleukin-6; D008024:Ligands; D019947:Receptors, Interleukin-6; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human