Identification and validation of calmodulin as a binding protein of an anti-proliferative small molecule 3,4-dihydroisoquinolinium salt

IHY-153 (2-(2,5-difluorobenzyl)-3,4-dihydro-5-(10-hydroxydecyl)-6- methoxy-1-undecylisoquinolinium bromide) was recently discovered as a small molecule that potently inhibits proliferation of tumor cells by inducing cell-cycle arrest at G0-G1 phase. To investigate the basis of anti-proliferative activity of IHY-153, cellular binding proteins of biotinyl-IHY-153 were screened using T7 phage displayed human cDNA libraries. Calmodulin-expressing phage specifically bound to immobilized IHY-153 in a Ca²⁺-dependent manner. The interaction between IHY-153 and Ca²⁺//CaM was validated through phage competition binding assays, surface plasmon resonance analysis, and molecular modeling. IHY-153 induced sustained phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and subsequently increased p21^WAF1 expression in colon cancer cells. These results demonstrate that IHY-153, a novel small molecule, targets Ca²⁺/CaM and indicate that this compound functions as an anti-proliferative agent by influencing Ca²⁺/CaM- dependent signal transduction.