The
phenylpiperazine derivative
naftopidil is an α1-adrenoceptor (AR) antagonist that has been used clinically to treat
benign prostatic hyperplasia. In our
drug repositioning research,
naftopidil shows the unique growth-inhibitory effects.
Naftopidil inhibits cell cycle progression not only in
cancer cells, but also in fibroblasts and vascular endothelial cells.
Naftopidil-inhibited cell cycle progression is independent of α1-AR expression in cells. Therefore, the antiproliferative effects of
naftopidil may be due to the off-target effects of the
drug. In this study, we attempted to identify the off-target molecules of
naftopidil using the magnetic nanobeads,
ferrite glycidyl metharcrylate (FG) beads. Similar to
naftopidil, its derivatives TG09-01 and TG09-02, which were introduced with amino groups for immobilizing to FG beads, inhibited cell growth in human HT29
colon adenocarcinoma cells. Both derivatives were associated with inhibition of cell cycle progression in HT29 cells. This observation is consistent with that seen with
naftopidil. Using TG09-02-immobilized FG beads, α- and β-tubulins were identified as the specific
binding proteins of
naftopidil. The
tubulin polymerization assay clearly indicated that
naftopidil bound directly to
tubulin and inhibited the polymerization of
tubulin. Other
phenylpiperazine derivatives, such as
RS100329, BMY7378, and
KN-62, also inhibited the polymerization of
tubulin. These results suggest that
phenylpiperazine derivatives including
naftopidil may have broad spectrum of cellular cytotoxicity in various types of cells. In addition, the
tubulin polymerization-inhibiting activity of
phenylpiperazine derivatives may be a specific feature of the
phenylpiperazine-based structure. These findings can allow us to design and synthesize new
tubulin-binding drugs derived from
naftopidil as a lead compound.