title: Isotetrandrine Reduces Astrocyte Cytotoxicity in Neuromyelitis Optica by Blocking the Binding of NMO-IgG to Aquaporin 4.
abstract: Neuromyelitis optica (NMO) is a severe neurological demyelinating autoimmune disease that affects the optic nerves and spinal cord with no cure and no FDA-approved therapy. Research over the last decade revealed that the binding of NMO-IgG to the water channel protein astrocyte aquaporin 4 (AQP4) might be the primary cause of NMO pathogenesis. The purpose of this study was to identify potential blockers of NMO-IgG and AQP4 binding.
  
  
  
  We developed a two-step screening platform consisting of a reporter cell-based high-throughput screen assay and a cell viability-based assay. Purified NMO-IgG from NMO patient serum and transfected Chinese hamster lung fibroblast V79 cells stably expressing human M23-AQP4 were used for primary screening of 40,000 small molecule fractions from 500 traditional Chinese herbs.
  
  
  
  Thirty-six positive fractions were identified, of which 3 active fractions (at 50 μg/ml) were found to be from the same Chinese traditional herb Mahonia japonica (Thunb.). A bioactivity-guided method based on a primary screening assay for blocking activity led to the isolation of an active single natural compound, isotetrandrine, from the 3 fractions. Our immunofluorescence staining results showed that isotetrandrine can block NMO-IgG binding to AQP4 without affecting the expression and function of AQP4. It can also inhibit NMO-IgG binding to astrocyte AQP4 in NMO patient sera and block NMO-IgG-dependent complement-mediated cytotoxicity with the IC50 at ∼3 μM.
  
  
  
  The present study developed a cell-based high-throughput screen to identify small molecule inhibitors for NMO-IgG and AQP4 binding, and suggests a potential therapeutic value of isotetrandrine in NMO.
authors.affiliation: Department of Laboratory Medicine, Jilin Medical University, Changchun, PR China.
authors.forename: Meiyan
authors.initials: M
authors.lastname: Sun
authors.forename: Jie
authors.initials: J
authors.lastname: Wang
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhou
authors.forename: Zijian
authors.initials: Z
authors.lastname: Wang
authors.forename: Yong
authors.initials: Y
authors.lastname: Jiang
authors.forename: Miao
authors.initials: M
authors.lastname: Li
mesh_terms: D000818:Animals; D051401:Aquaporin 4 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D044182:Benzylisoquinolines / Q000494:pharmacology* / Q000627:therapeutic use; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004365:Drugs, Chinese Herbal / Q000494:pharmacology* / Q000627:therapeutic use; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D007166:Immunosuppressive Agents / Q000494:pharmacology / Q000627:therapeutic use; D051379:Mice; D018345:Mice, Knockout; D009471:Neuromyelitis Optica / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology
chemical_list: C496030:AQP4 protein, human; D051401:Aquaporin 4; D044182:Benzylisoquinolines; D004365:Drugs, Chinese Herbal; D007074:Immunoglobulin G; D007166:Immunosuppressive Agents; C009438:tetrandrine