Structure and RAF family kinase isoform selectivity of type II RAF inhibitors tovorafenib and naporafenib

Abstract

Upon activation by RAS, RAF family kinases initiate signaling through the MAP kinase cascade to control cell growth, proliferation, and differentiation. Among RAF isoforms (ARAF, BRAF, and CRAF), oncogenic mutations are by far most frequent in BRAF. The BRAF^V600E mutation drives more than half of all malignant melanoma and is also found in many other cancers. Selective inhibitors of BRAF^V600E (vemurafenib, dabrafenib, encorafenib) are used clinically for these indications, but they are not effective inhibitors in the context of oncogenic RAS, which drives dimerization and activation of RAF, nor for malignancies driven by aberrantly dimerized truncation/fusion variants of BRAF. By contrast, a number of "type II" RAF inhibitors have been developed as potent inhibitors of RAF dimers. Here, we compare potency of type II inhibitors tovorafenib (TAK-580) and naporafenib (LHX254) in biochemical assays against the three RAF isoforms and describe crystal structures of both compounds in complex with BRAF. We find that tovorafenib and naporafenib are most potent against CRAF but markedly less potent against ARAF. Crystal structures of both compounds with BRAF^V600E or WT BRAF reveal the details of their molecular interactions, including the expected type II-binding mode, with full occupancy of both subunits of the BRAF dimer. Our findings have important clinical ramifications. Type II RAF inhibitors are generally regarded as pan-RAF inhibitors, but our studies of these two agents, together with recent work with type II inhibitors belvarafenib and naporafenib, indicate that relative sparing of ARAF may be a property of multiple drugs of this class.

Keywords: RAF kinase; cooperativity; drug action; fluorescence resonance energy transfer (FRET); inhibitor mechanism; x-ray crystallography.

Figure 1

RAF protein constructs and their activity.A, schematic of RAF complexes studied here. For simplicity, we refer to these preparations as ARAF^SSDD, BRAF^WT, BRAF^V600E CRAF^WT, and CRAF^SSDD, as indicated. B, coomassie-stained SDS-PAGE gel of purified RAF preparations. C, activity of purified RAF complexes as assayed by TR-FRET. Ratio of emission at 665/620 nm is plotted for increasing concentrations of each RAF preparation. TR-FRET, time-resolved FRET.

Figure 2

Representative concentration-response curves of tovorafenib and naporafenib generated with the purified RAF complexes described inFigure 1. ARAF^SSDD dimer and BRAF^V600E monomer dose-response curves have a standard Hill slopes (−1.0) but BRAF^WT, CRAF^WT, and CRAF^SSDD dimer curves have nonstandard Hill slopes less than −1.0, indicating that these dimer RAF complexes are inhibited with positive cooperativity. Data are plotted as mean ± SD from one independent experiment performed in triplicate (n = 3).

Figure 3

Crystal structures of BRAF with tovorafenib and naporafenib.A, chemical structures of tovorafenib and naporafenib. B, the asymmetric unit of the BRAF^V600E structure contains a back-to-back BRAF^V600E dimer bound to tovorafenib, with the inhibitor bound in each protomer of the dimer. C, detailed view of tovorafenib in complex with BRAF^V600E. Hydrogen bonds are depicted as dashed lines. The DFG-motif is colored orange and the αC-helix is shown in green. Tovorafenib spans the nucleotide-binding site, and the kinase adopts a DFG-out, αC-helix-in conformation. D, detailed view of naporafenib in complex with BRAF^WT, colored as in panel C. E and F, simulated annealing composite omit electron density maps for tovorafenib (E) and naporafenib (F) in complex with BRAF^V600E and BRAF^WT, respectively. Maps represent 2F_o-F_c density contoured at 1σ.