Inhibitors of BRAF dimers using an allosteric site

Abstract

BRAF kinase, a critical effector of the ERK signaling pathway, is hyperactivated in many cancers. Oncogenic BRAF^V600E signals as an active monomer in the absence of active RAS, however, in many tumors BRAF dimers mediate ERK signaling. FDA-approved RAF inhibitors poorly inhibit BRAF dimers, which leads to tumor resistance. We found that Ponatinib, an FDA-approved drug, is an effective inhibitor of BRAF monomers and dimers. Ponatinib binds the BRAF dimer and stabilizes a distinct αC-helix conformation through interaction with a previously unrevealed allosteric site. Using these structural insights, we developed PHI1, a BRAF inhibitor that fully uncovers the allosteric site. PHI1 exhibits discrete cellular selectivity for BRAF dimers, with enhanced inhibition of the second protomer when the first protomer is occupied, comprising a novel class of dimer selective inhibitors. This work shows that Ponatinib and BRAF dimer selective inhibitors will be useful in treating BRAF-dependent tumors.

Fig. 1. Ponatinib is a RAF inhibitor.

a Schematic representation of ERK signaling pathway from different BRAF species. b Schematic representation of the in-cell-western screening assay. c Screening of a library of 200 kinase inhibitors using in-cell-western assay. SKMEL239-C4 melanoma cells left untreated (regular media), treated with 0.5 μM Vemurafenib, 0.1 μM Trametinib, or with 5 μM of known RAF, MEK, and other kinase inhibitors in the presence of 0.5 μM Vemurafenib for 3 h. d SKMEL239-C4 melanoma cells left untreated (regular media), treated with 0.5 μM Vemurafenib, 0.1 μM Trametinib, Ponatinib 5 μM without or with 0.5 μM Vemurafenib for 3 h, and assayed with in-cell-western. Data are mean of n = 2 independent experiments. e Chemical structure of Ponatinib. f BRAF kinase activity assay in the absence or presence of Ponatinib or Vemurafenib was assayed by western blot with the indicated antibodies. g CRAF kinase activity assay in the absence or presence of Ponatinib or Vemurafenib was assayed by western blot with the indicated antibodies. g Kinase activity inhibition profiles of BRAF^V600E and BRAF^WT upon Ponatinib titration using SelectScreen assay. Data are mean of two technical replicates from n = 2 independent experiments. Source data are provided as a Source Data file.

Fig. 2. Structural analysis of BRAFV600E bound to Ponatinib (PON).

a Crystal structure of the BRAF^V600E/PON protomer structure. BRAF^V600E structure depicted in ribbons (gray) showing the αC-helix (green). The partially disordered activation loop (orange) is shown in a tube representation. PON is shown in sticks and molecular surface (yellow). b F_o – F_c electron density of PON in the BRAF^V600E/PON complex contoured at 3σ. c Close-up of PON binding (in ball-and-sticks) to its binding pocket (transparent surface) in BRAF^V600E. Protein sub-pockets recognizing various inhibitor moieties are colored, including the allosteric pocket (green). d A close-up view of PON binding interactions with BRAF^V600E. PON is shown in ball-and-sticks and BRAF^V600E in a cartoon model. Protein residues interacting with PON are shown in sticks. Note hydrogen bond interactions of PON with the backbone of residues H574 and I573 in the allosteric site and interaction with the D594 and F595 of the DFG motif and catalytic E501 of the αC-helix. A structural water molecule in the binding site (W1) is shown as a sphere. H-bonds are shown in dashed lines. e A superposition of BRAF type-II/αC-IN inhibitors LY3009120 (LY, green, PDB 5C9C), TAK-632 (TAK, orange, PDB 4KSP), and AZ-628 (AZ, blue, PDB 4G9R) bound in their BRAF recognition pockets with PON in BRAF^V600E/PON structure. Protein parts were omitted from clarity. f A superposition of BRAF type-Ib/αC-OUT inhibitors Vemurafenib (VEM, cyan, PDB 3OG7) and Dabrafenib (DAB, orange, PDB 4XV2) bound in their BRAF recognition pockets with PON in BRAF^V600E/PON structure. Inhibitors are depicted in sticks and proteins in a cartoon model.

Fig. 3. Ponatinib induces symmetrical BRAF dimers and promotes BRAF complexes in cells.

a The BRAF^V600E/PON symmetrical dimer structure. The dimer is viewed along its twofold axis with two protomers in silver and green ribbon representations. PON bound to each protomer is shown in orange sticks/molecular surface. The αC-helix of each protomer is illustrated in magenta. b The αC-helix of BRAF^V600E/PON complex adopts a αC-CENTRE position. Structural superposition of BRAF protomer structures in ribbon representation showing that αC-helix of BRAF-PON structure (magenta) lies between typical αC-OUT position observed in BRAF–Vemurafenib complex (cyan, PDB 3OG7) and αC-IN position in MEK-bound BRAF (gold, PDB 4MNE). Protein parts were omitted for clarity. c Various cell lines expressing BRAF^V600E or RAS^MUT/BRAF^WT were left untreated or treated with 1 or 5 μM Ponatinib for 1 h. Cells were then collected, assayed for CRAF by immunoprecipitation and immunoblot with the indicated antibodies for monitoring BRAF/CRAF heterodimerization and activation of ERK signaling. d Similar analysis to c assayed for MEK by immunoprecipitation and immunoblot with the indicated antibodies for monitoring BRAF/MEK complex formation. Data are representative of n = 3 independent experiments. Source data are provided as a Source Data file.

Fig. 4. Ponatinib inhibits different BRAF species and ERK signaling in cells.

a Melanoma A375, SKMEL239-C4, SKMEL-30, SKMEL-2, and lung cancer CALU6 cell lines expressing BRAF^V600E or RAS^MUT/BRAF^WT left untreated or treated with increasing concentration of Ponatinib or Vemurafenib for 1 h, then assayed for western blot and immunoblot with the indicated antibodies to probe ERK-signaling inhibition. Data are representative of three independent experiments. b Breast cancer SKBR3 cells, which are RTK-dependent HER2 amplified and melanoma A375 (BRAF^V600E monomer) cells were left untreated or treated with increasing concentration of Ponatinib and Lapatinib, an EGFR/HER2 inhibitor, for 1 h, then assayed by immunoblot with the indicated antibodies. Data are representative of n = 3 independent experiments. Source data are provided as a Source Data file.

Fig. 5. Targeting the allosteric site in BRAF with PHI1.

Comparison of binding modes of PON (a) and PHI1 (b) with BRAF^V600E in their corresponding crystal structures, illustrating interactions of PHI1 with the αC-allosteric site. Protein residues lining the base of αC-helix and HRD motif are shown in surface representation. Protein–ligand interface contact area (within 3.6 Å of ligand atoms) is colored in orange (PON) and magenta (PHI1), respectively. H-bonds are shown with dashed white lines. Protein parts are omitted for clarity. c Major PHI1-induced structural rearrangement of αC-helix (green) in BRAF^V600E/PHI1 complex compared to BRAF^V600E/PON (orange). The αC-allosteric site is shown in surface representation as in b and PHI1 atoms as van der Waals spheres. d Cartoon map showing molecular interactions between PHI1 and BRAF^V600E. e Superposition of BRAF^V600E/PHI1 (green) and BRAF^V600E/PON (orange) complexes, demonstrates that PHI1 binding defines a new back pocket (BP-VI, red circle), according to KLIFS database nomenclature, which corresponds to the aC-allosteric site shown in b. PON is classified as a BP-IV binder. The position of BP-V is also shown. f KinomeEDGE^® of PON and PHI1 at 1 μM. Interaction maps of human kinases, including mutated, pathogen and lipid kinases, illustrating levels of PON and PHI1 binding (radii of red circles, see inset) giving better than 90% displacement of control binding (see “Methods” section). g Inhibition of kinase activity of BRAF^V600E, BRAF^WT, and other tyrosine kinases targets, identified by KinomeEDGE^®, using SelectScreen (Invitrogen) in the presence of 100 μM ATP. Half-maximal inhibition values (IC₅₀ ± SD) of two technical replicates from n = 2 independent experiments are tabulated. Source data are provided as a Source Data file.

Fig. 6. PHI1 is a selective BRAF dimer inhibitor that displays positive co-operativity.

a Melanoma A375 and SKMEL239-C4 cell lines were treated with increasing. concentrations of PON or PHI1 for 1 h. Whole-cell lysate were assayed by western blot with the indicated antibodies to assess ERK-pathway inhibition. Representative blots from n = 3 independent experiments are shown. b Quantitation of p-ERK inhibition; normalized values (mean ± SEM, n = 3 independent experiments) of p-ERK levels obtained by densitometry with corresponding fitted curves (see “Methods” section). c SKMEL239-C4 cells without Encorafenib (−Enco) treatment and after Encorafenib (+Enco) treatment for 1 h, followed by exchange with fresh medium for another hour, were treated with increasing concentrations of c PHI1, e LY3009120, g AZ-628 and i TAK-632 for 1 h and cell lysates were assayed by western blot with the indicated antibodies to assess ERK-pathway inhibition. A representative blot from n = 3 (c, d) and n = 2 (e–j) independent experiments is shown. Normalized values and non-linear regression fits of p-ERK activity for different compounds in c, e, g, and i is shown respectively. Error bars represent mean ± SEM, n = 3 (d) and mean of two replicates from n = 2 independent experiments (f, h, j). k Table summarizing p-ERK inhibition results from all inhibitors in Encorafenib-free and Encorafenib-treated SKMEL239-C4 cells. Source data are provided as a Source Data file.

Fig. 7. Positive co-operativity in inhibition of non-BRAFV600E dimers by PHI1.

Lung cancer H1666 (BRAF^G466V), H2087 (BRAF^L597V), and melanoma SKMEL-30 (BRAF^WT/NRAS^Q61R), SKMEL-2 (BRAF^WT/NRAS^Q61K) cells without Encorafenib (−Enco) treatment and after Encorafenib (+Enco) treatment for 1 h, followed by exchange with fresh medium for another hour, were treated with increasing concentrations of a PHI1, c TAK-632, and d Vemurafenib for 1 h and cell lysates were assayed by western blot with the indicated antibodies to assess the ERK-pathway inhibition. A representative blot from n = 3 (a, b) and n = 2 (c, e) independent experiments is shown. Normalized values and non-linear regression fits of p-ERK activity for each treatment is shown in b, d, and f, respectively. Error bars represent mean ± SEM, n = 3 (b) and mean of two replicates from n = 2 independent experiments (d, f). Notably, Encorafenib pre-treatment promotes paradoxical activation in these cells. Source data are provided as a Source Data file.

Fig. 8. Mechanisms of RAF inhibitors action.

Scheme of four distinct mechanisms of RAF inhibitors action and representative RAF inhibitors for each mechanism. This work revealed that PHI1 displays a distinct structural, inhibitory, and therapeutic mode of action compared to previously characterized αC-IN and αC-OUT RAF inhibitors. Moreover, Ponatinib, an FDA-approved inhibitor, is an effective inhibitor of BRAF monomers and RAF dimers with a distinct structural binding mode.