The RAS-RAF-MEK-ERK signaling pathway controls cellular growth and proliferation, and mutational activation of this pathway is a frequent cause of cancer. Most prominently, the V600E mutation in BRAF causes malignant melanoma, papillary thyroid cancer and other malignancies. Rare but recurrent activating mutations in the other two RAF isoforms, ARAF and CRAF, have also been identified in diverse cancers. Distinct classes of RAF inhibitors have been developed, particularly for BRAFV600E, but their potencies against the three RAF isoforms have not been systematically compared. Here we biochemically characterize monomeric and dimeric preparations of ARAF, BRAF, and CRAF and measure the potencies of a panel of thirteen type I, type I.5, and type II RAF inhibitors against each active RAF preparation. Type I inhibitor SB590885 is roughly equipotent across RAF isoforms and, as expected, type I.5 inhibitors are typically most potent against BRAFV600E. Despite their reputation as pan-RAF inhibitors, type II inhibitors as a class are potent inhibitors of CRAF but exhibit relative sparing of ARAF and intermediate potencies against BRAF. Type II compounds inhibit BRAF and CRAF with marked positive cooperativity, and their apparent potencies are insensitive to ATP-concentrations. Crystal structures of CRAF in complex with type I.5 inhibitor PLX4720 reveal an asymmetric CRAF dimer with one CRAF subunit bound in the inactive state and the second bound in an aC-helix-in, active conformation with an altered inhibitor pose. Our findings have important implications for understanding the pharmacology of current RAF inhibitors and will inform development of new agents with distinct isoform selectivity.
Tkacik et al. (Sun,) studied this question.