Figure 4.

Identification of hits from the CC_NCI test library. (a) Cartoon representations of the primary assay and counter-screening assays used to identify false positives. Top: example of a compound that binds the Nav1.6 C-tail, preventing FGF14 binding and resulting in reduced complementation of luciferase fragments. Alternative mechanisms (not shown) include direct FGF14 binding or modulation of signaling pathways that regulate FGF14 or Nav1.6 through phosphorylation. Middle: example of a compound that reduces luminescence through direct inhibition of the luciferase enzyme, which could lead to false positives in the LCA. Bottom: example of a cytotoxic compound, leading to decreased NADH production and resulting in reduced fluorescence in the CTB cell viability assay. (b) Scatter plot of all compounds tested from the CC_NCI library showing % maximal luminescence or fluorescence (normalized to DMSO). Top: LCA results with preliminary hits highlighted as green (50 inhibitors; Z ≤ −4 and % max luminescence ≤ 50.7%) or red (15 enhancers; Z ≥ 3 and % max luminescence ≥ 137.0%). Middle: full-length luciferase assay in-cells used to identify false positives (Z ≥ ±3, equivalent to % max luminescence cut-offs of 76.6% and 123.4%, respectively); 1 enhancer and 36 inhibitors were identified, 22 of which were in the initial set of hits. Bottom: cell viability assay (CellTiter-Blue) identified 7 toxic compounds, 5 of which were in the initial set of hits (Z ≥ ±3, equivalent to % fluorescence cut-offs of 78.54% or 121.46%). Note: 2 of these toxic compounds were also inhibitors of the full-length luciferase. (c) Heat map representation of LCA, luciferase, and cell viability assay results. (d,e) Final hits were selected following exclusion of false positives and toxic compounds, resulting in a final set of hits including 14 enhancers and 26 inhibitors. (d) Scatter plot from (a) with final hits highlighted as green (inhibitors) or red (enhancers). (e) Heat map representation of final hits.