Figure 5. Drug‐screening pipeline overview.

Top: Left: MECP2‐KO neurons exhibit aberrant synaptic transcriptomics and synaptic morphology as well as decreased calcium transients and neural network activities. Right: Pharmacological compounds were selected for screening based on the expected ability of their mechanisms of action to counteract the synaptic and neurotransmitter pathologies we identified. Center: With step‐wise progression through a pipeline of assays (shown as dark‐to‐light shades of blue), drugs were eliminated either if they did not reverse the MECP2‐KO pathology identified in the assay or if they affected controls. Nefiracetam and PHA 543613 emerged as the two compounds that could reverse MECP2‐KO neurocytopathology without affecting controls. Middle: The effects of Nefiracetam and PHA 543613 were validated in two 3D human cell models, MECP2‐mosaic neurospheres (Left) and MECP2‐KO cortical organoids (Right). Left: One or both drugs reversed calcium transient frequency and amplitude as well as MEA spike frequency in MECP2‐mosaic neurospheres, but not their size. Right: One or both drugs reversed organoid size, SYN1 puncta, and MEA spike frequency in MECP2‐KO cortical organoids. Bottom: A schematic proposing a hypothetical mechanism by which Nefiracetam and PHA 543613 might influence synaptic function.