Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity

Schlessinger et al. 10.1073/pnas.0711741105.

Supporting Information

Files in this Data Supplement:

SI Figure 5
SI Figure 6
SI Methods
SI Scheme 1
SI Scheme 2
SI Table 3

Fig. 5. Effects of PLX4720 on activated ERK in melanocytes. Primary human melanocytes (FOM180) were isolated from fetal foreskins and established in culture. Melanocytes were treated at the indicated concentrations for 1 h before protein lysates were collected and immunoblots were performed. Actin served as a loading control.

Fig. 6. Effects of PLX4720 on cell-cycle profiles of melanoma cells. B-Raf V600E+ (1205Lu) and B-Raf wild-type (C8161) melanoma cells were treated with PLX4720 for 24 h at the indicated concentrations, and DNA content was assessed via propidium iodide staining through flow cytometry. Percentages of each stage of the cell cycle are depicted in the upper right portion of each histogram.

SI Methods

Synthesis and Characterization of PLX4720, PLX3203, Compound 1, and Compound 2. Propane-1-sulfonic acid [3-(5-chloro-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluorophenyl]amide, PLX4720, was synthesized from commercially available 2,4-difluoro-aniline as described in SI Scheme 1.

Step 1: Synthesis of propane-1-sulfonic acid (2,4-difluorophenyl)amide, 4. Propane 1-sulfonylchloride chloride was added dropwise (185 ml, 1.64 mol) to a solution of 2,4-difluoroaniline (3, 200g, 1.55 mol), pyridine (133 ml, 1.64 mol), and DMAP (7.0g, 62 mmol) in anhydrous dichloromethane (2.0 L) at room temperature. Upon completion of the addition, the reaction mixture was refluxed overnight and evaporated in vacuo. The crude residue was dissolved in ethyl acetate (4L), washed with water (2 ´ 2.0 L) and brine (2.0 L), dried over sodium sulfate and evaporated in vacuo to afford 4 as a tan solid in quantitative yield.

Step 2: Synthesis of propane-1-sulfonic acid (2,4-difluoro-3-formylphenyl)amide, 5. n-BuLi in hexane (2.475 liters of 1.6 M, 3.96 mol) was added to a solution of diisopropylamine (554.6 ml, 3.96 mol) in anhydrous THF (6.3 liters) at -78°C. The reaction was then stirred for 1 h at 0°C. This lithium diisopropylamide solution was cooled to -78°C, and a solution of 3 (423.2g, 1.8 mol) in anhydrous THF (2.7 liters) was added. The reaction mixture was stirred 4 h at -78°C, and N-formylmorpholine (216 ml, 2.16 mol) was added. The resulting mixture was allowed to warm to ambient temperature overnight, quenched with 1 N hydrochloric acid (8 liters), and extracted with ethyl acetate (3 ´ 7 liters). The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give a crude solid that was triturated with 1:3 ethylacetate:heptane (1:3) to yield 4 (240 g, 51%) as a light brown solid.

Step 3: Synthesis of propane-1-sulfonic acid {3-[(5-chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)hydroxymethyl]-2,4-difluorophenyl}amide, 7. Potassium carbonate (253.1 g, 1.83 mol) was added to a solution of 5 (72 g, 273.5 mmol) and 5-chloro-7-azaindole (Compound 6, 44.2 g, 289.9 mmol) in methanol/water (1:1, 1.37 liters). After stirring the reaction mixture for 48 h at room temperature, the pH was adjusted to 7 with 4 N hydrochloric acid. The volatiles were removed, and the crude mixture was extracted with ethyl acetate (3 ´ 1.2 liters). The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give a crude solid that was purified on a silica gel column with 3:7 to 3:1 ethyl acetate/heptane as eluent to give 7 (99.7 g, 88%) as a light brown solid.

Step 4: Synthesis of propane-1-sulfonic acid [3-(5-chloro-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluorophenyl]amide (PLX4720). 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (81.6g, 359.5 mmol) and then 39 ml of water were added to a solution of 7 (115g, 276.5 mmol) in 1,4-dioxane (775 ml) at room temperature. The reaction mixture was stirred for 2 h at room temperature and then quenched with saturated sodium bicarbonate (800 ml). The volatiles were removed under reduced pressure. The residue was diluted with water and extracted with THF/ethyl acetate (1:1, 3 ´ 2 liters). The combined organic layers were dried over sodium sulfate and evaporated in vacuo to give a crude solid that was purified on a silica gel column with ethyl acetate:heptane (1:1) as eluent to yield PLX4720 (103 g, 90%) as a light brown solid. MS(API-ES) [M+H⁺]⁺ = 414 and 416 (3:1); ¹H-NMR (300 MHz, DMSO-d₆): d 13.13 p.p.m. (brs, 1H), 9.78 (brs, 1H), 8.45 (d, J = 2.3 Hz, 1H), 8.31 (s, 1H), 7.60 (m, 1H), 7.29 (m, 1H), 3.33 (m, 2H), 1.75 (m, 1H), 0.97 (m, 3H); IR (KBr Pellet): 3,232 cm^-1, 1,649 cm^-1.

N-[2,4-difluoro-3-(5-pyridin-3-yl-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-phenyl]-ethanesulfonamide, PLX3203, was synthesized from commercially available 2,4-difluoroaniline as described in SI Scheme 2.

Step 1 - Preparation of dibenzyl-(2,4-difluoro-phenyl)-amine, 8. Potassium carbonate (32.1 g, 0.23 mol) and benzyl bromide (21.2 ml, 0.18 mol) were added to 2,4-difluoroaniline (3, 10.0 g, 77.4 mmol) in N,N-dimethylformamide (130 ml). The reaction was stirred at room temperature overnight, poured into water, and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated and purified by silica gel column chromatography eluting with 10% ethyl acetate in hexane. The appropriate fractions were combined and concentrated to provide product 8 (12.0 g, 50%). MS(ESI) [M+H⁺]⁺ = 310.2.

Step 2 - Preparation of 3-dibenzylamino-2,6-difluoro-benzaldehyde, 9. n-Butyllithium (2.50 M in hexane, 6.1 ml, 15.3 mmol) was added slowly to dibenzyl-(2,4-difluoro-phenyl)-amine (8, 4.30 g, 13.9 mol) in tetrahydrofuran (60 ml), under an atmosphere of nitrogen and cooled in a -78°C acetone/dry ice bath. The reaction was stirred for 1 h. Then, N,N-Dimethylformamide (1.2 ml, 15.3 mol) was added, and the reaction was allowed to warm to room temperature for 1 h. The reaction was poured into water, and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated and purified by silica gel column chromatography eluting with 10% ethyl acetate in hexane to provide product (9, 4.0 g, 85%). MS(ESI) [M+H⁺]⁺ = 337.2.

Step 3 - Preparation of (3-dibenzylamino-2,6-difluoro-phenyl)-(5-pyridin-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-methanol, 10. 5-Pyridin-3-yl-1H-pyrrolo[2,3-b]pyridine (0.40 g, 2.1 mmol) and potassium hydroxide (0.50 g, 8.9 mol) were added to 3-dibenzylamino-2,6-difluoro-benzaldehyde (9, 0.76 g, 2.3 mmol) in methanol (50 ml) under an atmosphere of nitrogen. The reaction was stirred overnight at room temperature, poured into water, and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated and purified by silica gel column chromatography eluting with 5% methanol in methylene chloride to provide product (10, 0.60 g, 50%). MS(ESI) [M+H⁺]⁺ = 533.2.

Step 4 - Preparation of (3-dibenzylamino-2,6-difluoro-phenyl)-(5-pyridin-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-methanone, 11. Dess-Martin periodinane (0.97 g, 2.3 mmol) was added to (3-dibenzylamino-2,6-difluoro-phenyl)-(5-pyridin-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-methanol (10, 0.90g, 1.7 mmol) in methylene chloride (20 ml) under an atmosphere of nitrogen. The reaction was stirred at room temperature for 15 min, poured into a solution of sodium bicarbonate and sodium thiosulfate, and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated and purified by silica gel column chromatography eluting with 5% methanol in methylene chloride to provide product (11, 0.70 g, 78%). MS(ESI) [M+H⁺]⁺ = 531.2.

Step 5 - Preparation of (3-dibenzylamino-2,6-difluoro-phenyl)-(5-pyridin-3-yl-1-triisopropylsilanyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-methanone, 12. Sodium hydride (210.0 mg, 60% in mineral oil, 5.3 mmol) was added to (3-dibenzylamino-2,6-difluoro-phenyl)-(5-pyridin-3-yl-1H-pyrrolo[2,3-b]pyridin-3-yl)-methanone (11, 0.84 g, 1.6 mmol) in tetrahydrofuran (150 ml) under an atmosphere of nitrogen. The reaction was stirred for 5 min. Then, triisopropylsilyl chloride (0.80 ml, 3.8 mmol) was added and the reaction was stirred at room temperature for 3 h. The reaction mixture was poured into water, and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by silica gel column chromatography eluting with 10% ethyl acetate in hexane to provide product (12, 420 mg, 39%). MS(ESI) [M+H⁺]⁺ = 687.4.

Step 6 - Preparation of (3-amino-2,6-difluoro-phenyl)-(5-pyridin-3-yl-1-triisopropylsilanyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-methanone, 13. Twenty percent palladium hydroxide on carbon (20 mg) was added to (3-dibenzylamino-2,6-difluoro-phenyl)-(5-pyridin-3-yl-1-triisopropylsilanyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-methanone (12, 55.0 mg, 0.080 mmol) in methanol (15 ml). The reaction was stirred under an atmosphere of hydrogen overnight. The reaction was filtered to remove the catalyst, and then concentrated to give crude product that was used in the next step.

Step 7 - Preparation of Ethanesulfonic acid [3-(1-ethanesulfonyl-5-pyridin-3-yl-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluoro-phenyl]-amide, 14. Methanesulfonyl chloride (0.30 ml, 3.9 mmol) and triethylamine (0.40 ml, 2.9 mmol) was added to (3-amino-2,6-difluoro-phenyl)-(5-pyridin-3-yl-1-triisopropylsilanyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-methanone (13, 35.0 mg, 0.069 mmol) in methylene chloride (6 ml). The reaction was stirred at room temperature overnight, poured into water, and extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and concentrated to give crude product that was used in the next step.

Step 8 - Preparation of N-[2,4-difluoro-3-(5-pyridin-3-yl-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-phenyl]-ethanesulfonamide, PLX3203. Tetra-n-butylammonium fluoride (20 mg, 0.075 mol) was added to N-[2,4-difluoro-3-(5-pyridin-3-yl-1-triisopropylsilanyl-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-phenyl]-methanesulfonamide (14, 15.0 mg, 0.028 mmol) in tetrahydrofuran (10 ml). The reaction was stirred at room temperature for 1 h, then poured into water and extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated and purified by silica gel column chromatography eluting with 5% methanol in methylene chloride to provide product (15, 6.0 mg, 48%). MS(ESI) [M+H⁺]⁺ = 443.1; ¹H NMR d (CD₃OD) 8.95 (d, 1H, J = 1.6 Hz), 8.88 (s, 1H), 8.74 (d, 1H, J = 1.6 Hz), 8.63 (d, 1H, J = 4.0 Hz), 8.26 (d, 1H, J = 8.4 Hz), 8.07 (s, 1H), 7.72 (m, 1H), 7.64 (m, 1H), 7.20 (m, 1H), 3.19 (q, 2H, J = 7.2 Hz), 1.40 (s, 3H, J = 7.2 Hz)

Synthesis of Compounds 1 and 2. Compounds 1 and 2 were prepared from commercially available 3-bromo-7-azaindole and 3-formyl-7-azaindole respectively. These two compounds were synthesized and purified by reverse phase HPLC as a part of a focused library synthesis.

Cloning, expression, purification and crystallization of B-Raf.To enable crystallization, the BRAF cDNAs (both V600 and E600) fragment encoding amino acid residues 448-723 with 16 mutations to improve expression (I543A, I544S, I551K, Q562R, L588N, K630S, F667E, Y673S, A688R, L706S, Q709R, S713E, L716E, S720E, P722S, K723G) were cloned into a pET29a vector (Invitrogen), in frame with a N-terminal histidine tag for bacterial expression. The protein was expressed in E. coli cells with an overall yield of 5-10 mg/liter.

B-Raf kinase was produced using 30 liters of Bioreactor cultures of E. coli strain BL21(DE3) RIL (Stratagene) grown in Terrific Broth, with 18 h induction at 18°C by using 1 mM IPTG. Frozen cell pastes were suspended with 40 ml of lysis buffer [100 mM Hepes (pH 7.0), 250 mM NaCl, 0.75% Nonidet P-40, 5% glycerol, 25 mM imidazole, 5 mM MgCl₂, and 2 mM PMSF] per liter of cells. Cell suspensions were lysed by using a microfluidizer (M-110H; Microfluidics) at 18,000 psi, and clarified by centrifugation at 25,000 ´g at 4°C for 1 h. Supernatants were fractionated using Ni-Chelating Sepharose FF (GE Healthcare), washing with lysis buffer containing 50 mM imidazole, and by 10 column volumes of 20 mM Hepes (pH 7.5), 200 mM NaCl, 25 mM imidazole, 5 mM MgCl₂, followed by elution with 500 mM imidazole in the same buffer. Eluted protein was fractionated further by using a 20-ml SP FF column (GE Health Care) in 20 mM Hepes (pH 7.5), 5% glycerol, protein eluted from Ni-chelating column was diluted 3-fold into SP binding buffer and passed over SP column. B-Raf protein was eluted by using linear NaCl gradient (200-750 mM) over 20 column volumes. Eluted protein was concentrated and fractionated by using a Superdex200 26/60 SEC (GE HealthCare) column in 20 mM Hepes (pH 7.5), 200 mM NaCl, 5% glycerol, 10 mM DTT. B-Raf protein behaved as monomers and was concentrated to 20 mg/ml and stored at -80°C.

The isolated protein was characterized by N-terminal sequencing, mass spectrometry and a biochemical assay for the enzyme activity. The protein was crystallized by a sitting drop vapor diffusion experiment in which equal volumes of protein (at 10 mg/ml concentration) and reservoir solution (100 mM BisTris at pH 6.0, 12.5% 2,5-hexanediol, and 12% PEG3350) were mixed and allowed to equilibrate against the reservoir at 4°C. To obtain co-crystals of complexes of the protein with ligands, the protein solution was initially mixed with the compound (dissolved in DMSO) at a final compound concentration of 1 mM, and then set up for co-crystallization using the method described above. For enzymatic activity, B-Raf (residues D448 through K723) with or without the V600E mutation and fused at the N terminus with 6 residue HIS-tag, was co-expressed with CDC37 in insect cells using a baculovirus vector. For comparative enzymatic determinations, c-Raf-1 (residues Q307 through F648, with Y340D and Y341D mutations, fused at N terminus with GST) from baculovirus-infected insect cells was purchased from UBI. Protein expression, purification and crystallization of Pim-1 (1) and FGFR1 (2) have been previously described.

Structure determination. The B-Raf, Pim-1, and FGFR1 crystallographic data were collected at the Advanced Light Source (Lawrence Berkeley National Laboratory, Berkeley, CA), with. ADSC Quantum 210 CCD detectors. All data were processed and reduced with Mosflm and scaled with Scala of the CCP4 suite of programs (3) using the software ELVES (4). The space group crystals was determined to be P2₁2₁2₁for B-Raf crystals, P65 for Pim1crystals and C2 for FGFR1 crystals with the cell axes shown in table 3. Initial phases are obtained by molecular replacement method with the program MOLREP (5) using a previously published B-Raf crystal structure (PDB ID: 1UWJ), or the Pim-1 structure (PDB ID: 1YXX) or the FGFR1 structure (PDB ID: 1FGK) as the search model. Initial electron density map indicated regions of conformational changes. The model was then rebuilt using the program O (6). This model was finally refined against data up to the maximum diffraction resolution using CNX (CNS (7) licensed to industrial users by Accelerys) and refmac5 (8) using a maximum-likelihood target function and with cross-validation throughout. Crystallography statistics are shown in the following Table.

Protein kinase activity measurements. The in vitro kinase activities of wild type and mutants were determined by measuring phosphorylation of biotinylated-MEK protein using Perkin-Elmer's AlphaScreen Technology. For each enzyme (0.1 ng), 20-ml reactions were carried out in 20 mM Hepes (pH 7.0), 10 mM MgCl₂, 1 mM DTT, 0.01% Tween-20, 100 nM biotin-MEK protein, and various ATP concentrations at room temperature. Reactions were stopped at 2, 5, 8, 10, 20, and 30 min with 5 ml of a solution containing 20 mM Hepes (pH 7.0), 200 mM NaCl, 80 mM EDTA, 0.3% BSA. The stop solution also included phospho-MEK Antibody (Cell Signaling Technology), Streptavidin-coated Donor beads and Protein A Acceptor beads from the AlphaScreen Protein A Detection Kit (PerkinElmer Life Sciences). The antibody and beads were preincubated in stop solution in the dark at room temperature for 30 min. The final dilution of antibody was 1/2,000, and the final concentration of each bead was 10 mg/ml. The assay plates were incubated at room temperature for one hour then were read on a PerkinElmer AlphaQuest reader.

Fifty-eight selected kinases were profiled for inhibition by PLX4720 using the Z´-LYTE biochemical assay format (SelectScreen; Invitrogen) according to the manufacturer's instructions. All reactions were done in the presence of 10 mM ATP except for CLK1, KIT, MEK1, MKK6, MLK1, P38a, PDK1, TAO1, and TRKA, which were carried out in the presence of 100 mM ATP. Assays for AKT1,2,3 with eNOS substrate peptide, JNK1,2,3 with substrate ATF2, and the tyrosine kinases FGFR and Pyk2 with substrate peptide E4Y were performed by using the Alpha screen technology (Perkin-Elmer) as described for the Raf assays.

Phosphorylated ERK immunoassay. Cells were serum-starved overnight, treated with compound for 1 h at 37°C, and fixed (4% formaldehyde in PBS, for 30 min at ambient temperature). After washing in PBS, 0.1% Tween-20 (PBST), the endogenous peroxidase was quenched for 30 min with 0.6% H₂O₂ in PBST followed by further washes. After blocking with 10% FBS in PBST for 1 h at ambient temperature and washing, plates were incubated with anti-phospho-ERK monoclonal antibody (1:2000; Cell Signaling Technology) in PBST containing 5% BSA for 1 h at ambient temperature followed by further washes. Plates were then incubated with goat-anti-mouse-linked horseradish peroxidase (1:1000; Pierce) in PBST containing 5% BSA for 1 h at ambient temperature. Signal was developed with SuperSignal (Pierce) ELISA substrate, and light units were detected with a luminometer (Safire, Tecan).

Cell proliferation assays. Cells were plated at 5 ´ 10³ cells/well in 96-well plates (Corning #3603) and treated with compound the next day. After 72 h, proliferation was measured by using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions.

Melanoma Cell Culture

After stable establishment, melanoma cell lines were maintained in melanoma 2% media containing a 4:1 ratio of MCDB153 and L15 supplemented with 2 mmol/L Ca⁺², 2% (vol/vol) heat-inactivated FBS (FBS), and 5 mg/ml insulin in 5% CO₂ at a constant temperature (37°C) and humidity. Human melanocytes were isolated from foreskin and maintained in medium 254CF supplemented with human melanocyte growth supplement (HMGS) from Cascade Biologics.

Melanoma Cell Proliferation Assay

Cells were plated into a 96-well plate at a density of between 1.8 ´10⁴ and 2.5 ´ 10⁴ per ml and allowed to adhere overnight. The following day, cells were treated with increasing concentrations of PLX4720 in quadruplicate. Cells were left to grow for 72 h before being treated with 10% (vol/vol) 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent for 3 h. Medium containing MTT was quickly removed, and the crystallized precipitate was solubilized by using DMSO. Absorbance was subsequently read in a plate reader at 540 nm. Relative growth was calculated as the absorbance of cells in the absence of PLX4720 (UA_F) divided by the absorbance of cells in a given concentration of PLX4720 (TA_F) after the initial absorbance (IA) was subtracted from both.

TA_F - IA

Relative Growth = ----

UA_F-IA

Data show the mean of at least three independent experiments ± SE.

Cell Cycle Analysis

Melanoma cells were seeded onto 100-mm dishes at 60% confluence and allowed to adhere overnight. Increasing concentrations of PLX4720 were administered for 24 h before supernatant and cells were collected, pelleted, and fixed with 70% ethanol. Before staining with propidium iodide (10 mg/ml), cells were incubated for 1 h at 37°C in 0.5 mg/ml RNase I to rid samples of residual RNA contamination. Samples were then analyzed by using the EPICS XL (Beckman-Coulter) apparatus.

Apoptosis Assay

Melanoma cells were plated into 100-mm dishes at 60% confluence and subsequently treated with PLX4720 for 24-hour intervals. Media and cells were harvested and pelleted before staining with annexin-FITC and propidium iodide. Samples were subsequently analyzed by using the EPICS XL apparatus.

Three-Dimensional Spheroid Growth

Melanoma spheroids were prepared by using the liquid overlay method. Briefly, 2,000 melanoma cells in 200 ml (10,000 cells per ml) were added to a 96-well plate coated with 1.5% agar. Plates were incubated for 72 h, by which time cells had organized into three-dimensional spheroids. Spheroids were then harvested by using a P1000 pipetteman. The medium was removed, and the spheroids were implanted into a gel of bovine collagen I containing EMEM, L-glutamine, and 2% FBS. Normal 2% melanoma medium was overlaid on top of the solidified collagen. Spheroids were treated with increasing concentrations of PLX4720 and allowed to grow for 72 h. Spheroids were then washed twice in PBS before being treated with calcein-AM and ethidium bromide for 1 h at 37°C according to the manufacturer's directions. Photographs of the invading spheroids were then taken by using a Nikon-300 inverted fluorescence microscope.

Construction of Synthetic Skin

Reconstructions of normal human skin were prepared as previously reported. Three milliliters of fibroblast-containing bovine type I collagen (7.5 ´ 104 cells per ml) plus Matrigel (4:1 ratio) was added to each insert of tissue culture trays and allowed to constrict in DMEM plus 10% FBS for 4 days at 37°C. For epidermal reconstruction, keratinocytes were mixed with melanoma cells at a ratio of 10:1 in keratinocyte serum-free medium containing 2% dialyzed FCS, 60 mg/ml bovine pituitary extract, 4.5 ng/ml bFGF, 100 nM human endothelin-3, and 10 ng/ml human SCF. A total of 5 ´ 10⁶ cells were seeded on each contracted collagen gel. Cultures were kept submerged in medium containing 1 ng/ml EGF for 2 days, 0.2 ng/ml EGF for another 2 days, and were raised to the air-liquid interface via feeding from below with high calcium (2.4 mM) medium in EGF-free medium. After 15 days, skin reconstructs were treated with either 1 mM PLX4720 or vehicle control for 72 h and subsequently embedded in paraffin for subsequent sectioning and staining.

Immunohistochemistry

Seven-micrometer paraffin sections were processed by deparaffinization and rehydration followed by endogenous peroxidase blocking (1% H₂O₂ in methanol for 20 min) and antigen retrieval (boiled in 10 mM citrate buffer for 10 min). Tissue sections were blocked with 2% goat or horse serum (Vector Laboratories) and incubated with rabbit polyclonal antibody against phospho-MAPK/ERK or rabbit monoclonal antibody control for 30 min at 37°C followed by overnight exposure at 4°C. Samples were subsequently incubated with biotinylated secondary antibodies (Vector Laboratories). Immunoreactivity was detected by using the ABC Elite kit (Vector Laboratories). AEC was used as a final chromogen and hematoxylin as the nuclear counterstain. Negative controls for all antibodies were made by replacing the primary antibody with nonimmunogenic IgG.

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SI Scheme 1

Scheme 1. Synthesis of PLX4720 (Compound 1).

SI Scheme 2

Scheme 2. Synthesis of PLX3203 (Compound 2).

Table 3. Statistics of crystallographic data and refinement

*R_sym = ∑ | I_avg - I_j | / ∑ I_j.

^†R_cryst = ∑ | F_o - F_c | / ∑ F_o , where F_o and F_c are observed and calculated structure factors, respectively, R_free was calculated from a randomly chosen 5% of reflections excluded from the refinement, and R_cryst was calculated from the remaining 95% of reflections.

^‡rmsd is the root-mean-square deviation from ideal geometry.

^§Numbers in parentheses are for the highest resolution shell.

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