Apilimod inhibits not only PIKfyve-catalyzed synthesis of PtdIns(3,5)P₂ but also that of PtdIns5P.

Lysates, derived from HEK293 cells transduced with recombinant adenovirus expressing HA-PIKfyve^WT and GFP from separate promoters, were immunoprecipitated with a polyclonal anti-PIKfyve (α-PIK) or preimmune (Pre) sera. Washed immunoprecipitates (IPs) were pretreated with various apilimod concentrations or with vehicle alone (0.1% DMSO) for 15 min at 37°C along with PI substrate and then subjected to a lipid kinase assay with 15 μM ATP and [γ-³²P]ATP (30 μCi) in a 50-μl final volume. Lipid products were resolved by TLC n-propanol/2 M acetic acid solvent system (65:35 v:v). (A): Shown are autoradiograms from representative TLCs out of 6 independent experiments demonstrating that both PIKfyve lipid products, i.e., PtdIns5P and PtdIns(3,5)P₂ (denoted by arrowheads) are inhibited significantly at low nanomolar concentrations of apilimod. (B): Quantification of the autoradiograms from six experiments using variable slope non-linear regression curve fitting option of ImageJ software (mean ± SEM). Note that the two lipids are inhibited with a similar efficiency.