| In Vitro | In vitro activity: RAF709 may have very slow dissociation kinetics (T1/2 > 6.5 h) using the rapid dilution method to measure its dissociation rate constant. In cellular assays, the dose–response of pMEK and pERK are measured in Calu-6 cells with EC50 of 0.02 and 0.1 μM with minimal paradoxical activation and inhibition of proliferation with EC50 of 0.95 μM. RAF709 stabilizes BRAF–CRAF dimers with an EC50 of 0.8 μM. Of the 456 kinases tested, RAF709 shows a high level of selectivity, demonstrating greater than 99% on-target binding to BRAF, BRAFV600E, and CRAF at 1 μM and very few off-targets with DDR1 (>99%), DDR2 (86%), FRK (92%), and PDGFRb (96%), the only kinases with binding>80% at 1 μM. RAF709 shows equal activity against both RAF monomers and dimers. In in vitro biochemical assays, RAF709 exhibits potent inhibitory activity targeting BRAF, BRAFV600E, and CRAF with IC50 values ranging between 0.3 to 1.5 nmol/L. RAF709 treatment leads to a dose-dependent induction of B/CRAF heterodimerization in HCT116, but inhibits MEK and ERK phosphorylation, in line with the ability of RAF709 to effectively inhibit the RAF dimers. RAF709 selectively inhibits oncogenic signaling and proliferation in tumor cells with BRAF, NRAS, or KRAS mutations with minimal paradoxical activation.
Kinase Assay: Enzymatic activities of purified B/CRAF proteins were measured using inactive MEK1 protein as a substrate. Substrate phosphorylation was detected using the anti-pMEK1/2 (S217/S221) antibody, AlphaScreen Protein A coated acceptor beads and streptavidin coated donor beads, and read in an EnVision reader. To measure the inhibitory activity of RAF709, compound was added to the enzyme assay plates with the final concentration from 25 to 1.74E-6 μmol/L. Kinase selectivity of RAF709 was determined using the KINOMEscan screening platform (DiscoverX) that quantitatively measures interactions between RAF709 and 456 human kinases.
Cell Assay: Cells were seeded in 15-cm dishes and incubated with the indicated concentrations of compound for 1 hour. Cell lysates were prepared in immunoprecipitation buffer supplemented with 1× protease and 1× phosphatase inhibitor cocktails. Cleared lysates were normalized for protein concentration and incubated with specific antibody overnight at 4°C. Protein A Ultra Link Resin (Thermo Scientific) was then added to each sample and incubated for 2 hours at 4°C. Resin was washed with immunoprecipitation lysis buffer before bound proteins were eluted in SDS sample buffer. |
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| In Vivo | RAF709 treatment led to 83% inhibition of DUSP6 at 4 hours postdose compared with vehicle control; however, this inhibition was not durable as demonstrated by the increased levels of DUSP6 at 16 and 24 hours postdose. Similarly, trametinib treatment led to a partial and transient inhibition of DUSP6. In contrast, the combination of RAF709 and trametinib led to a more sustained DUSP6 inhibition, showing greater than 80% of inhibition even at 16 hours postdose. We next evaluated antitumor efficacy of the different treatments in the same tumor xenograft model. Tumor-bearing animals were dosed with vehicle, RAF709 at 100 mg/kg once daily, trametinib at 0.3 mg/kg once daily, or a combination of both for 10 days. In line with DUSP6 inhibition, the combination of RAF709 and trametinib treatment resulted in greater antitumor activity than either of the single agents alone, resulting in 33% regression as compared with 40% T/C or 54% T/C by RAF709 or trametinib, respectively. |
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