Cell experiment:

Cells (2×103) are seeded in a 96-well plate and cultured in DMEM containing 2% (vol/vol) FBS in the presence of one of the compounds. Viable cell numbers are measured by formazan formation using a Cell Counting Kit 8. Apoptotic cells are detected by a standard TUNEL assay using an In Situ Cell Detection kit.

Animal experiment:

Cells (5×106) are implanted into the right flanks of female athymic nude mice (6-8 wk old). After tumor sizes reached appr 50 mm3 on average, compounds suspended in Cremophor:ethanol:water (1:1:6) are administered orally for five consecutive days per week for 17 d. Tumor volumes (V) are calculated. Dissected tumors after 17-d administration of the 80 mg/kg compounds are fixed in 4% (wt/vol) paraformaldehyde and embedded in paraffin. Their sections are subjected to immunohistochemistry with an anti-ERK1/2 antibody or an anti-CD31 antibody using a HISTMOUSE-PLUS kit. Apoptotic cells are detected by a TUNEL assay. Statistical significance for groups of three or more is determined by one-way ANOVA with Tukey’s test for post hoc analysis.

Kobe0065 is a small-molecule inhibitor of Ras with Ki value of 46 μM for the binding of H-Ras.GTP to c-Raf-1 [1].

Kobe0065 is a compound screened out by an in silico screen method as a potent inhibitor of the Ras–Raf interaction. It showed favorite efficacy to inhibit the binding of M-Ras.GTP and H-Ras.GTP to the Ras-binding domain of c-Raf-1. Kobe0065 also dose-dependently inhibited the binding of H-RasG12V to c-Raf-1 in NIH 3T3 cells with a rough IC50 value of 10 μM. Besides that, 20 μM of Kobe0065 effectively suppressed the phosphorylation of down-stream kinases of Raf, including MEK and ERK. In NIH 3T3 cells transfected with H-rasG12V, Kobe0065 inhibited the colony formation with IC50 value of 0.5 μM. Kobe0065 also showed effect on other cancer cells carrying activated ras oncogenes, such as PANC-1(K-rasG12V), HT1080 (N-rasQ61L) and HCT116 (H-rasG13D). Moreover, in mice bearing SW480 xenografts, administration of Kobe0065 resulted in 40-50% inhibition of the tumor growth at dose of 80 mg/kg. This effect turned to be more distinct when the dose was up to 160 mg/kg [1].

References:
[1] Shima F, Yoshikawa Y, Ye M, et al. In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras–effector interaction. Proceedings of the National Academy of Sciences, 2013, 110(20): 8182-8187.