Figure 2. Apilimod binds to and inhibits PIKfyve kinase activity.

(A) Structure of apilimod and its analogs and their IC₅₀ for IFNγ (50 ng/ml)/LPS (1 µg/ml) induced-IL12p40 secretion in THP-1 cells. (B) Scatter plot depicting proteins identified in a quantitative chemical proteomics experiment. Proteins are plotted as a function of the percentage competition with apilimod relative to DMSO, on the y-axis, versus the interaction specificity (e-value) on the x-axis. (see also Table S1 for protein list for apilimod quantitative chemical proteomics) (C) U2OS cells lysates were pre-incubated with DMSO or indicated competitor compound for 30 mins before adding beads with immobilized APA10. The PIKfyve captured on the beads was detected by Western Blot. (D) The effect of apilimod on PIKfyve kinase activity was measured in vitro, by quantifying the ratio of synthesized PI(3,5)P₂ to an internal standard. (E) Lipid kinase inhibition profiling for apilimod. (see also Table S2 for protein kinase inhibition profiling for apilimod) (F) The binding of apilimod with indicated PIKfyve truncants was assessed using a APA10-based Sepharose HP affinity resin. (G) The K_D of Cy5-apilimod to PIKfyve kinase domain was determined. (H) The IC₅₀ of apilimod for the interaction between Cy5-apilimod and PIKfyve kinase domain was determined.