Abstract
Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system caused by binding of anti-aquaporin-4 (AQP4) autoantibodies (NMO-IgG) to AQP4 on astrocytes. A screen was developed to identify inhibitors of NMO-IgG-dependent, complement-dependent cytotoxicity. Screening of 50,000 synthetic small molecules was done using CHO cells expressing human AQP4 and a human NMO recombinant monoclonal antibody (rAb-53). The screen yielded pyrano[2,3-c]pyrazoles that blocked rAb-53 binding to AQP4 and prevented cytotoxicity in cell culture and spinal cord slice models of NMO. Structure-activity analysis of 82 analogs yielded a blocker with IC(50) ∼ 6 μm. Analysis of the blocker mechanism indicated idiotype specificity, as (i) pyrano[2,3-c]pyrazoles did not prevent AQP4 binding or cytotoxicity of other NMO-IgGs, and (ii) surface plasmon resonance showed specific rAb-53 binding. Antibody structure modeling and docking suggested a putative binding site near the complementarity-determining regions. Small molecules with idiotype-specific antibody targeting may be useful as research tools and therapeutics.
Highlights
A high-throughput cytotoxicity-based screen was developed to identify small-molecule inhibitors of Neuromyelitis optica (NMO)-IgGAQP4 binding
We previously reported smallmolecule and antibody blockers of NMO-IgG binding to AQP4 that targeted the extracellular surface of AQP4 and blocked the binding of polyclonal NMO-IgG to AQP4 and downstream cytotoxicity [12, 13]
We identified a class of small molecules that exclusively blocked AQP4 binding of the NMO monoclonal antibody used for screening
Summary
A high-throughput cytotoxicity-based screen was developed to identify small-molecule inhibitors of NMO-IgGAQP4 binding. Results: Pyrano[2,3-c]pyrazoles were identified as inhibitors and were shown to bind directly to the NMO monoclonal antibody used for screening but did not interfere with other NMO-IgGs that bound to AQP4. Screening of 50,000 synthetic small molecules was done using CHO cells expressing human AQP4 and a human NMO recombinant monoclonal antibody (rAb-53).
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