10.6084/m9.figshare.5129602.v1 Sun M. Sun M. Wang J. Wang J. Zhou Y. Zhou Y. Wang Z. Wang Z. Jiang Y. Jiang Y. Li M. Li M. Supplementary Material for: Isotetrandrine Reduces Astrocyte Cytotoxicity in Neuromyelitis Optica by Blocking the Binding of NMO-IgG to Aquaporin 4 Karger Publishers 2016 High-throughput screening Traditional Chinese herbs NMO-IgG binding Aquaporin 4 Neuromyelitis optica Isotetrandrine 2016-04-12 00:00:00 Dataset https://karger.figshare.com/articles/dataset/Supplementary_Material_for_Isotetrandrine_Reduces_Astrocyte_Cytotoxicity_in_Neuromyelitis_Optica_by_Blocking_the_Binding_of_NMO-IgG_to_Aquaporin_4/5129602 <b><i>Objective:</i></b> 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. <b><i>Methods:</i></b> 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. <b><i>Results:</i></b> 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 <i>Mahonia japonica </i>(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 IC<sub>50</sub> at ∼3 μM. <b><i>Conclusions:</i></b> 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.