Biological evaluation of a novel sorafenib analogue, t-CUPM

Abstract

Sorafenib (Nexavar®) is currently the only FDA-approved small molecule targeted therapy for advanced hepatocellular carcinoma. The use of structural analogues and derivatives of sorafenib has enabled the elucidation of critical targets and mechanism(s) of cell death for human cancer lines. We previously performed a structure-activity relationship study on a series of sorafenib analogues designed to investigate the inhibition overlap between the major targets of sorafenib Raf-1 kinase and VEGFR-2, and an enzyme shown to be a potent off-target of sorafenib, soluble epoxide hydrolase. In the current work, we present the biological data on our lead sorafenib analogue, t-CUPM, demonstrating that this analogue retains cytotoxicity similar to sorafenib in various human cancer cell lines and strongly inhibits growth in the NCI-60 cell line panel. Co-treatment with the pan-caspase inhibitor, Z-VAD-FMK, failed to rescue the cell viability responses of both sorafenib and t-CUPM, and immunofluorescence microscopy shows similar mitochondrial depolarization and apoptosis-inducing factor release for both compounds. These data suggest that both compounds induce a similar mechanism of caspase-independent apoptosis in hepatoma cells. In addition, t-CUPM displays anti-proliferative effects comparable to sorafenib as seen by a halt in G0/G1 in cell cycle progression. The structural difference between sorafenib and t-CUPM significantly reduces inhibitory spectrum of kinases by this analogue, and pharmacokinetic characterization demonstrates a 20-fold better oral bioavailability of t-CUPM than sorafenib in mice. Thus, t-CUPM may have the potential to reduce the adverse events observed from the multikinase inhibitory properties and the large dosing regimens of sorafenib.

Fig. 1

NCI-60 human tumor cell line screen data from t-CUPM treatment. 51 cell lines from various cancer types were screened according to the NCI-60 cell line screening procedure. Comparison of the NCI-60 data for t-CUPM to known FDA-approved targeted therapies is presented in Table 2

Fig. 2

Comparison of the kinase inhibitory profile of sorafenib and t-CUPM. a Selected kinase targets of sorafenib were screened for inhibition at 10 μM test concentrations. Data were collected from a 2-point kinase screen, limiting statistical analysis calculations. However, a change in inhibition of >40 % is deemed significant for these data. b Comparison of sorafenib and t-CUPM on the suppression of the RAF/MEK/ERK and JAKs/STAT3 signaling pathways using western blot analysis. HepG2 cells were exposed to compounds for 24 h at indicated concentrations. Note that the apparent suppression of total ERK by sorafenib is considered an artifact

Fig. 3

t-CUPM exhibits similar caspase-dependent apoptosis to sorafenib. Dose response effects of sorafenib and t-CUPM on HepG2 (a) and Huh-7 (b) cell viability and caspase 3/7 induction. Data for cell viability (MTT assay) and caspase 3/7 induction (luminescence) were determined after a 72-h incubation period for each compound

Fig. 4

t-CUPM exhibits similar caspase-independent apoptosis. a HepG2 cells were pre-incubated for 60 min with the pan-caspase inhibitor, Z-VAD-FMK (20 μM), prior to 24-h incubation with 30 μM of test compound. *P value < 0.05 as compared to Z-VAD-FMK addition. b HepG2 cells were incubated at 30 μM of each compound for 6 h. MitoTracker^® Red CMXRos and DAPI were used to stain mitochondria and nuclei, respectively. AIF primary antibody was stained using Alexa Fluor^® 488 conjugated secondary antibody. Arrows show areas of AIF nuclear accumulation after mitochondrial depolarization

Fig. 5

t-CUPM exhibits similar anti-proliferative responses to sorafenib. Effects of sorafenib and t-CUPM on hepatoma cell cycle distribution. HepG2 cells were exposed at concentrations of 30 μM for 24 h and fluorescence detection of incorporated EdU, and 7AAD was analyzed by flow cytometry. *P value < 0.05 (n = 3) as compared to DMSO control (+EdU)

Fig. 6

Comparison of the pharmacokinetic profiles of sorafenib and t-CUPM. PK study was performed by cassette oral administration at a dose of 1 mg/kg (n = 3) in mice. R² is the square of the correlation coefficient between predict and observed value; T_max the time of maximum concentration, C_max the maximum blood concentration, t_1/2 half-life, and AUC_t area under the concentration–time curve to terminal time. *P value < 0.05 as compared to the AUC_t of sorafenib