TLN-4601, a novel anticancer agent, inhibits Ras signaling post Ras prenylation and before MEK activation.

TLN-4601 is a structurally novel farnesylated dibenzodiazepinone discovered through DECIPHER, Thallion's proprietary drug discovery platform. The compound was shown to have a broad cytotoxic activity (low micromol/l) when tested in the NCI 60 tumor cell line panel and has shown in-vivo antitumor activity in several xenograft models. Related to its farnesylated moiety, the effect of TLN-4601 on Ras mitogen-activated protein kinase signaling was assessed. Downstream Ras signaling events, Raf-1, MEK, and ERK1/2 phosphorylation in MCF7 cells were evaluated by western blot analysis. TLN-4601 prevented epidermal growth factor-induced phosphorylation of Raf-1, MEK, and ERK1/2. This effect was time-dependent and dose-dependent with complete inhibition of protein phosphorylation within 4-6 h at 10 micromol/l. The inhibition of Ras signaling was not mediated by the inhibition of protein prenylation, documented by the lack of effect TLN-4601 on the prenylation of HDJ2 (specific substrate of farnesyltransferase), RAP1A (specific substrate of geranylgeranyl transferase-1), or Ras. As TLN-4601 did not inhibit EGFR, Raf-1, MEK or ERK1/2 kinase activities, the inhibitory effect of TLN-4601 on Ras signaling is not mediated by direct kinase inhibition. Using an Elk-1 trans-activation reporter assay, we found that TLN-4601 inhibits the MEK/ERK pathway at the level of Raf-1. Interestingly, TLN-4601 induces Raf-1 proteasomal-dependent degradation. These data indicate that TLN-4601 may inhibit the Ras-mitogen-activated protein kinase-signaling pathway by depleting the Raf-1 protein.