Discovery of rafoxanide as a dual CDK4/6 inhibitor for the treatment of skin cancer

Abstract

Since cyclin‑dependent kinases 4/6 (CDK4/6) play pivotal roles in cell cycle regulation and are overexpressed in human skin cancers, CDK4/6 inhibitors are potentially effective drugs for skin cancer. In the present study, we present a mixed computational and experimental study attempting to repurpose approved small‑molecule drugs as dual CDK4/6 inhibitors for skin cancer treatment. We performed structure‑based virtual screening using the docking software idock, targeting an ensemble of CDK4/6 structures. We identified and selected nine compounds with significant predicted scores, and evaluated their cytotoxic effects in vitro in A375 and A431 human skin cancer cell lines. Rafoxanide was found to exhibit the highest cytotoxic effects (IC50: 1.09 µM for A375 and 1.31 µM for A431 cells). Consistent with the expected properties of CDK4/6 inhibitors, rafoxanide significantly increased the G1 phase population. Notably, we revealed that rafoxanide specifically decreased the expression of CDK4/6, cyclin D, retinoblastoma protein (Rb) and the phosphorylation of CDK4/6 and Rb. Furthermore, the anticancer effect of rafoxanide was demonstrated in vivo in BALB/C nude mice subcutaneously xenografted with human skin cancer A375 cells. Rafoxanide (40 mg/kg, i.p.) exhibited significant antitumor activity, comparable to that of oxaliplatin (5 mg/kg, i.p.). The combined administration of rafoxanide and oxaliplatin produced a synergistic therapeutic effect. To the best of our knowledge, the present study is the first to indicate that rafoxanide inhibits CDK4/6 activity and is a potential candidate drug for the treatment of human skin cancer.

Discovery of Vanoxerine Dihydrochloride as a CDK2/4/6 Triple-Inhibitor for the Treatment of Human Hepatocellular Carcinoma

“…cells. As positive controls, three siRNAs targeting each of the CDK2/4/6were designed as described previously [22], and used to inhibit the expressions of each of the CDK2/4/6proteins. *p<0.05, signi cantly different from the control PBS treatment group.…”

“…The cell cycle pro le was determined by Flow cytometry analysis, as described previously [20][21][22].Brie y, Western blot analysis.…”

Discovery of Vanoxerine Dihydrochloride as a CDK2/4/6 Triple-Inhibitor for the Treatment of Human Hepatocellular Carcinoma

“…The X-ray crystallographic protein structures used in the studies were obtained from the Protein Data Bank (PDB) [23,24]. We selected 5 structures of CDK4 and 8 structures of CDK6, and rst screened for CDK4/6 inhibitors as described in our previous studies [21,22].…”

“…MTT assay were conducted as described in previous studies [20][21][22]. QGY7703 and Huh7 cells were plated at an initial density of 9x10 3 cells/well in 96-well plates, incubated with MTT (Sigma) reagents and the absorbance measured at 570 nm with a microplate reader (Multiskan Spectrum, Thermo Scienti c Microplate Reader, USA).…”

“…As positive controls, three siRNAs targeting each of the CDK2/4/6were designed as described previously [22], and used to inhibit the expressions of each of the CDK2/4/6proteins in QGY7703 and Huh7 cells. The proteins were measured using enhanced chemiluminescence detection system (Thermo Fisher scienti c, USA).…”