Supporting Information

Zhang et al. 10.1073/pnas.1219457110 SI Experimental Procedures with brine (2.5 L), dried over anhydrous sodium sulfate, and filtered. Synthesis and Characterization of PLX647. PLX647, 5-(1H-pyrrolo The solvents were removed at reduced pressure to provide ∼560 g [2,3-b]pyridin-3-ylmethyl)-N-[[4-(trifluoromethyl)phenyl]methyl] of crude product. This material was triturated with methyl tert-butyl pyridin-2-amine, was synthesized from commercially available ether:hexanes (1:1.3) to yield compound 4 (360 g, 79.0%) as a yel- 1 5-bromopyridin-2-amine, 4-(trifluoromethyl)benzaldehyde and low solid. HNMRδ (CDCl3) 9.84 (s, 1H), 8.57 (s, 1H), 7.95 (dd, 1H-pyrrolo[2,3-b]pyridine following the synthetic route depicted 1H, J = 2.0, 8.8 Hz), 7.65 (d, 2H, J = 8.4Hz),7.50(d,2H,J= 8.4 Hz), in Scheme S1. 6.50 (d, 1H, J = 8.8 Hz), 5.72 (s, 1H), 4.77 (d, 2H, J = 5.2 Hz).

Step 3 Step 1 Step 2

1 2 3 4

Step 4 Step 5

7a R = H 5 6 PLX647 7b R = CH3

Scheme S1.

Step 1. Synthesis of 5-bromo-N-[[4-(trifluoromethyl)phenyl]methyl]pyridin- Step 3. Synthesis of tert-butyl N-(5-formyl-2-pyridyl)-N-[[4-(trifluoromethyl) 2-amine (compound 3). To a suspension of 5-bromopyridin-2-amine phenyl]methyl]carbamate (compound 5). To a solution of 6-[[4-(tri- (compound 1, 446.8 g, 2.58 mol) in acetonitrile (7.2 L) under an fluoromethyl)phenyl]methylamino]pyridine-3-carbaldehyde (com- atmosphere of nitrogen at room temperature, 4-(trifluoromethyl) pound 4, 485.5 g, 1.73 mol) in anhydrous THF (3.7 L), under benzaldehyde (compound 2,452.0g,2.60mol),triethylsilane nitrogen, di-tert-butyl dicarbonate (589.0 g, 2.70 mol), 4-dimethy- (1,250 mL, 7.82 mol), and trifluoroacetic acid (600 mL, 7.84 mol) laminopyridine (11.8 g, 0.097 mol), and N,N-diisopropylethyl- were added sequentially via addition funnel. The reaction was amine (550.0 mL, 3.16 mol) were added sequentially via additional refluxed (∼73 °C) for 16 h. The reaction mixture was cooled, funnel over 30 min. The reaction was stirred at room temperature concentrated under reduced pressure, and poured into a mixture for 1 h, poured into water (3 L), and extracted twice with ethyl of water (5 L) and hexane (3 L). The organic layer was separated acetate ( 3L and 1 L). The organic layers were combined, washed and discarded. Saturated potassium carbonate solution was added with brine (1.5 L), dried over anhydrous sodium sulfate, filtered, to the aqueous layer with stirring until a pH ∼9 and was extracted and concentrated to give crude product 5 (709 g, 107.6%), which twice with ethyl acetate (3 L each). The organic layers were com- was used in the next step without further purification. bined, washed with brine (2 L), dried over anhydrous sodium sul- Step 4. Synthesis of tert-butyl N-[5-[hydroxy(1H-pyrrolo[2,3-b]pyridin-3- fate, and filtered. Solvents were removed under reduced pressure yl)methyl]-2-pyridyl]-N-[[4-(trifluoromethyl)phenyl]methyl] carbamate to provide ∼995 g of crude product, which upon recrystallization (compound 7). To a solution 1H-pyrrolo[2,3-b]pyridine (compound from methyl tert-butyl ether/hexane (2:3) yielded 656.2 g of com- 6, 142.9 g, 1.21 mol) in methanol (4.5 L), under nitrogen, tert-butyl 1 fl pound 3 (76.6% yield) as white needles. HNMRδ (CDCl3) 8.16 N-(5-formyl-2-pyridyl)-N-[[4-(tri uoromethyl)phenyl]methyl] (d, 1H, J = 2.4 Hz), 7.62 (d, 2H, J = 8.0 Hz), 7.52 (dd, 1H, J = 2.4, carbamate (compound 5, 478.1 g, 1.26 mol) and sodium hydroxide 8.8Hz),7.48(d,2H,J= 8.0Hz),6.33(d,1H,J= 8.8 Hz), 5.12 (145 g, 3.63 mol) were added. The reaction was stirred at room (s, 1H), and 4.61 (d, 2H, J = 6.0 Hz). temperature for 15 h, poured into water (12 L), and the product Step 2. Synthesis of 6-[[4-(trifluoromethyl)phenyl]methylamino]pyridine-3- was extracted twice with ethyl acetate (9 L and 4 L). The com- carbaldehyde (compound 4). To a cooled (dry ice–acetone bath) bined organic layers were washed twice with brine (2.5 L each), solution of 5-bromo-N-[[4-(trifluoromethyl)phenyl]methyl]pyridin- dried over sodium sulfate, filtered, and concentrated under re- 2-amine (compound 3, 541.7 g, 1.63 mol) in anhydrous THF (6.5 L) duced pressure to provide a crude mixture of the hydroxy 7a and under nitrogen was added n-butyllithium (2.5 M in hexane, 1,970 mL, methoxy 7b (682 g, 117%) which was used in the next step without + + 4.93 mol) via additional funnel maintaining the internal temperature further purification. MS (electrospray ionization) [M+H ] = below –57 °C. The reaction mixture was stirred for 1.5 h at ≤–65 °C 499.4 (7a). before the addition of anhydrous dimethylformamide (265 mL, Step 5. Synthesis of 5-(1H-pyrrolo[2,3-b]pyridin-3-ylmethyl)-N-[[4- 3.42 mol) over a period of 30 min. The reaction mixture was al- (trifluoromethyl)phenyl]methyl]pyridin-2-amine (PLX647). To a solution lowed to warm up to about 0 °C in 1 h. Saturated NH4Cl aqueous of tert-butyl N-[5-[hydroxy(1H-pyrrolo[2,3-b]pyridin-3-yl)methyl]- solution (3 L) was added to quench the reaction and the products 2-pyridyl]-N-[[4-(trifluoromethyl)phenyl] methyl] carbamate and were extracted with ethyl acetate (8 L). The organic layer was washed tert-butyl N-[5-[methoxy(1H-pyrrolo[2,3-b]pyridin-3-yl)methyl]-

Zhang et al. www.pnas.org/cgi/content/short/1219457110 1of10 2-pyridyl]-N-[[4-(trifluoromethyl)phenyl]methyl]carbamate (7a were grown in ESF921 media (Expression Systems) at 2.0 × 106 and 7b, 682 g, 1.37 mol) in anhydrous acetonitrile (4.1 L), under cells/mL and were infected with a multiplicity of infection (MOI) = 5. nitrogen, triethylsilane (700 mL, 4.38 mol) and trifluoroacetic Cells were harvested 72 h postinfection and lysed using 10 mM acid (500 mL, 6.54 mol) were added via additional funnel over K-phosphate (pH 8.0), 25 mM NaCl, 0.5 mM PMSF, and pro- a period of 25 min. The reaction mixture was refluxed (∼66 °C) tease inhibitor mixture (EMD Millipore). Following clarification for 22 h, cooled, concentrated under reduced pressure, and by centrifugation the supernatant was adjusted to 40 mM K- poured into water (4 L) and hexane (4 L). The organic layer was phosphate (pH 8.0), 200 mM NaCl, and 30 mM imidazole. FMS separated and discarded. The pH of the aqueous layer was ad- were purified through a combination of IMAC and further justed by the addition of a saturated aqueous solution of po- purified by ion-exchange and size-exclusion chromatography. The tassium carbonate with stirring until the pH solution was at ∼9 clarified extract was bound to an IMAC column (HiTrap, and the product was extracted twice with ethyl acetate (3 L each). nickel-charged chelating Sepharose Fast Flow; GE Healthcare) The organic layers were combined, washed with water (2 L) and then washed using 40 mM phosphate (pH 8.0), 200 mM NaCl, and brine (2 L), dried over anhydrous sodium sulfate, filtered, and 30 mM imidazole and eluted using 25 mM Hepes (pH 7.0), 150 mM concentrated to give crude product (546 g). The solid was tritu- NaCl, and 300 mM imidazole. Following elution, 10 mM DTT was rated with methanol and dried to give PLX647 (262.8 g, 58.9%) as added to the eluent. Pooled fractions were further diluted with + + a yellow solid. MS (electrospray ionization) [M+H ] = 383.1. 1H 20 mM Hepes (pH 7.0) before loading over a cation exchange NMR δ (DMSO-d6) 11.36 (s, 1H), 8.15 (s, 1H), 7.92 (s, 1H), 7.81 column (HiTrap SP Fast Flow; GE Healthcare), proteins separated/ (1H, d, J = 7.2 Hz), 7.62 (d, 2H, J = 7.6 Hz), 7.48 (d, 2H, J = eluted using a NaCl gradient. Final purity was achieved by further 7.2 Hz), 7.27 (d, 1H, J = 7.6 Hz), 7.23 (s, 1H), 7.00 (t, 1H,, J = chromatography using a Superdex S200 gel filtration column (26/ 4.0 Hz), 6.95 (d, 1H, J = 7.2 Hz), 6.44 (d, 1H, J = 8.0 Hz), 4.51 60; GE Healthcare) in 20 mM Hepes (pH 7.0), 150 mM NaCl, and 13 (d, 2H, J = 4.0 Hz), 3.81 (s, 2H). C NMR δ (DMSO-d6) 157.50, 10 mM DTT. 149.32, 147.23, 146.65, 142.97, 137.84, 128.24, 127.66 (q, JCF = Crystallization and structure determination. The KIT , at 31.7 Hz), 127.20, 125.56 (q, JCF = 5.6 Hz), 125.01, 124.98 (q, JCF = a concentration of ∼5 mg/mL, was incubated with 1 mM PLX647 217 Hz), 123.80, 119.60, 115.38, 113.76, 108.61, 44.40, 27.94. before setting up the 24-well crystallization trays using the sitting drop method at 20 °C. The protein was diluted 1:1 with mother Protein Purification, Crystallization, and Structure Determination. liquor consisting of 1.6 M ammonium sulfate, 2.0 M sodium Purification of KIT proteins. To enable crystallization, the v-kit Hardy- chloride, and 0.1 M Bis-Tris (pH6.0). The cocrystal of FMS with Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) PLX647-OMe was obtained using a similar setup as the KIT- cDNA fragment encoding amino acid residues P551–H934, PLX647 cocrystallization experiment, but at 4 °C. The reservoir – containing a deletion between (Q694 T753) and the introduction contains 20% (wt/vol) polyethylene glycol 8000, 0.2 M MgCl2, of 16 surface mutations were expressed using a bacterial ex- and 0.1M Tris (pH 7.5). Both KIT and FMS cocrystals were then pression system. In addition to the insert domain (KID) soaked in a solution containing the mother liquor, plus 20% (vol/ deletion, the 16 surface mutations were introduced to improve vol) glycerol, followed by flash-freezing with liquid nitrogen. X- protein solubility and stability. These mutations included I563S, ray diffraction data were collected at beamline ALS 8.3.1 at the V569S, Y609Q, L631S, M651E, I662H, I690H, C691S, K693D, Advanced Light Source (Lawrence Berkeley Laboratory, Ber- I756S, L762N, V825D, C844S, L890S, L912D, and L923D. KIT keley, CA). Data were processed and scaled using MOSFLM (1) cDNAs were cloned into a custom pET-vector (Invitrogen), in and SCALA in the CCP4 package (2) (Table S4). frame with a noncleavable N-terminal 6xHistidine tag. Both KIT and FMS costructures were solved by molecular Proteins were expressed in Escherichia coli [ArcticExpress replacement with the program MOLREP (3) and using the (DE3) RIL; Stratagene] and protein induction was carried out at structure of apo-KIT ( ID code 1T45) (4) and 10 °C with 0.1 mM isopropylthio-β-galactoside. Following 18 h of an inhibitor-bound FMS (5) as a starting model, respectively. The induction cells were harvested. KIT proteins were purified through structures were refined with the program PHENIX (6) (Table S4). a combination of immobilized metal affinity chromatography Cycles of manual rebuilding withCOOT(7)andstructurerefine- (IMAC) and further purified by gel filtration and ion exchange ment were continued until there was no further improvement, chromatography. Cell pellets were lysed in lysis buffer containing indicated by the convergence of the Rfree factor (8). 100 mM potassium phosphate buffer (pH 8.0), 10% (vol/vol) glycerol, 0.75% Nonidet P-40, and 1 mM PMSF using a cell dis- Biochemical Assays. The in vitro kinase activities were determined ruptor (MicroFluidics). The clarified cell extract was initially by measuring of a biotinylated substrate peptide purified using IMAC (nickel-charged Chelating Sepharose Fast as described previously (9). Flow; GE Healthcare) and bound protein was further washed with 50 mM Hepes (pH 8.0), 200 mM NaCl, and 15 mM im- Cell Proliferation Assays. To evaluate the ability of PLX647 to idazole and eluted with 50 mM Hepes (pH 8.0), 200 mM NaCl, inhibit FMS, KIT, fms-related 3 (FLT3), and and 100 mM EDTA. The eluted protein was concentrated and kinase insert domain receptor (KDR) catalytic activity in cells, separated on a Superdex S200 gel filtration column (26/60; GE growth assays were established with cell lines that are dependent Healthcare) in 20 mM Tris (pH 8.0), 250 mM NaCl, and 10 mM upon FMS, KIT, FLT3, or KDR activity for proliferation. Eight β-mercaptoethanol. Final purity was achieved by further chro- cell lines were evaluated, including the engineered Ba/F3 cell matography on an anion exchange column (HiTrap Q Fast Flow; lines expressing breakpoint cluster region (BCR)–FMS, BCR– GE Healthcare) using a 0–0.5 M NaCl gradient in 20 mM Tris KIT, and BCR–KDR. In addition, the colony stimulating factor (pH 8.0). 1 (CSF-1)-ligand-dependent M-NFS-60 cell line that expresses Purification of FMS proteins. To enable crystallization, the McDo- endogenous FMS, the (SCF)-dependent M-07e nough feline sarcoma viral (v-fms) oncogene homolog (FMS) cell line that expresses endogenous KIT, and the vascular en- cDNA fragment encoding amino acid residues Q542–R919, also dothelial growth factor (VEGF)-dependent HUVEC cell line containing a deletion between (G696–D741), and additional mu- that expresses endogenous KDR were tested. In addition, the tations (C667T, C830S, and C907T) to improve expression were MV4-11 cell line that expresses mutationally activated receptor cloned into a pFastbac vector (Invitrogen), in frame with a non- (FLT3–ITD) and the FLT3-ligand dependent OCI-AML5 cell cleavable N-terminal histidine tag for insect cell expression. line that expresses wild-type FLT3 were used. Proteins were expressed using the baculovirus system in Sf9 The BCR–FMS, BCR–KIT, and BCR–-KDR Ba/F3 cell lines insect cells using 20/50 wave bioreactors (GE Healthcare). Cells were created by introduction of FMS, KIT, and KDR fusion

Zhang et al. www.pnas.org/cgi/content/short/1219457110 2of10 constructs that render the cells dependent on the introduced MERTK_(cMER), NEK5, BMX, MAPK3_(ERK1), SNF1LK_ kinase for growth. DNA constructs were created that encode (SIK), MAPK4_(ERK4), PLK4, and STK36. the BCR protein fused to the intracellular domain of FMS, KIT, with <10% inhibition at 1μM. RPS6KB1_(p70S6K), LYN_A, or KDR. The resulting BCR–FMS, BCR–KIT and BCR–KDR CSNK1A1L, ACVRL1, MARK4, PAK7_(KIAA1264), ROCK1, proteins exhibit constitutively active catalytic activity. Stable DAPK3_(ZIPK), STK38_(NDR1), SBK1, PHKG1, PRKCZ_ transfection of these constructs into the IL-3–dependent Ba/F3 (PKC_zeta), LATS1, PDPK1, CDK3, YANK3, MAP3K12_(DLK), cell line renders the cells IL-3–independent, and growth of the ERBB4_(HER4), DAPK2, MAP3K13_(LZK), RAF1_(cRAF), cells becomes dependent upon the catalytic activity of FMS, AMPK_A2/B1/G1, HCK, MAP3K8_(COT), IRAK3, CDC42_ KIT, or KDR. BPB_(MRCKB), MYLK2_(skMLCK), CHEK2_(CHK2), For the growth assay, cells were incubated with either DMSO STK22B_(TSSK2), MAPK12_(p38_gamma), CDK9/cyclinK, (0.2% final concentration) or PLX647 for 3 d and were subse- FRK_(PTK5), AKT1_(PKBa), DNA-PK, GRK1_(RHOK), quently evaluated for viability by quantifying the amount of ATP AMPK_A1/B1/G1, JAK1, FYN, YES1, MARK3, FGR, EPHA7, present in the cell culture. ATP is a marker for cell viability JAK2_JH1_JH2, RIOK2, MLCK_(MLCK2), STK17B, MAK, because it is present in all metabolically active cells at a relative LYN_B, MAP4K2_(GCK), MAP2K6_(MKK6), STK24_(MST3), stable concentration that very rapidly declines when the cells PRKCI_(PKC_iota), DCAMKL3_(DCLK3), DYRK2, PIM1, undergo necrosis or . The ATPlite 1step Luminescence SGK2, GSK3B, MAP3K7_(TAK1-TAB1), DMPK, MAPKAPK3, Assay reagent (Perkin-Elmer) contains both the firefly luciferase BLK, ACVR2B, MELK, CAMKK1, LTK_(TYK1), AKT3_(PKBg), and its substrate D-luciferin. Upon addition of the reagent to the CDK4/CyclinD1, CDK5_p35, STK35_(CLIK1), EIF2AK1_(HRI), cell culture, a luminescent signal is generated that is proportional MAPK1_(ERK2), ROCK2, PIK3C2B_(PI3KC2b), CDK5_p25, to the ATP concentration as a measure of viable cell number in MAPK11_(p38_beta), ROS1, SRC_N1, CLK3, SNRK, STK33, the cell culture. CHEK1_(CHK1), CAMK2A_(CaMKII_alpha), PIM2, PRKCG_ (PKC_gamma), PRKCN_(PKD3), PRKD1_(PKC_mu), CDK2/ Osteoclast Differentiation Assay. Human osteoclast precursors CyclinA, SgK110, EPHA5, BMPR1A_(ALK3), ADRBK1_ were purchased from a commercial source (2T-110; Lonza) and (GRK2), HIPK3_(YAK1), FES_(FPS), NEK7, , MAP3K10_ cultured in the presence of CSF-1 and RANKL and either DMSO (MLK2), MKNK2_(MNK2), CSNK1G1_(CK1_gamma_1), vehicle or PLX647. Following a 7-d incubation, the levels of acid LIMK2, PTK2_(FAK), FGFR1, CDKL2, YANK1, MINK1, phosphatase in the culture supernatant were measured using MAP2K4_(MEK4), MAPKAPK5_(PRAK), STK22D_(TSSK1), a commercial colorimetric assay (10008051; Cayman Chemical). FGFR2, DYRK1B, NLK, PAK2_(PAK65), AKT2_(PKBb), STK23_(MSSK1), SGK_(SGK1), RPS6KA3_(RSK2), CLK1, Cell Viability Assay. Cell viability was measured by adding the MAPK15_(ERK7), MAPK8_(JNK1), MAP4K3, MAPK14_ metabolic-activity-indicating MTT (3-(4, 5-dimethylthiazolyl-2)- (p38_alpha), SNF1LK2, PRKD2_(PKD2), DYRK1A, GSG2_ 2, 5-diphenyltetrazolium bromide) reagent to 293T and HepG2 (Haspin), AURKA_(Aurora_A), AURKC_(Aurora_C), TXK, cell lines treated for 24 h and 72 h with PLX647. MTT is reduced MET_M1250T, BRSK2, DYRK4, ULK3, BMPR1B_(ALK6), by metabolically active cells, in part by the action of dehydrogenase CHU.K._(IKKa), CSK, CLK4, BTK, ACK1, ACVR1_(ALK2), , to generate reducing equivalents such as NADH and RPS6KA1_(RSK1), RPS6KA2_(RSK3), NEK1, NEK4, ITK, NADPH. The resulting intracellular purple formazan can be ERBB2_(HER2), EPHA1, EPHB4, MAP3K11_(MLK3), solubilized and quantified by spectrophotometry. PRKACA_(PKA), IKBKE_(IKK_epsilon), TYRO3_(RSE), PKN1_(PRK1), CAMK2D_(CaMKII_delta), CSNK1D_(CK1_ Kinase Selectivity Profiling. PLX647 was tested against a panel of delta), IKBKB_(IKK_beta), INSRR_(IRR), JAK3, IRAK4, 400 kinases at concentrations of 1 μM in duplicate. Kinases in- LIMK1, FER, PIK3CA/PIK3R1_(p110a/p85a), MAP2K3_(MEK3), hibited by over 50% were followed up by IC50 determination IGF1R, INSR, ZAK, RPS6KA5_(MSK1), HIPK1_(Myak), SGKL_ (Table S3). Lists of kinases minimally affected by PLX647 are (SGK3), EPHA4, MARK2, CSNK2A1_(CK2_alpha_1), included below. The 400 kinases represent all major branches of STK3_(MST2), EPHA3, PTK6_(Brk), EPHB3, CDK9/CyclinT1, the kinome phylogenetic tree. The inhibition screen of 400 kinases BRSK1_(SAD1), CLK2, CDK1/CyclinB, HIPK4, PRKX, was carried out under contract as two complementary panels at CSNK1A1_(CK1_alpha_1), MAP2K1_(MEK1), MAP3K2_ Invitrogen Life Technologies (Madison, WI) as part of their (MEKK2), PRKG2_(PKG2), HIPK2, MAP2K2_(MEK2), SelectScreen profiling service and at DiscoveRx (Fremont, CA) MAP3K14_(NIK), PRKG1, EGFR_(ErbB1), FGFR3, GSK3A, as part of their KINOMEScan. ACVR1B_(ALK4), PASK, TAOK2_(TAO1), STK17A_(DRAK1), Kinases with <50% inhibition at 1μM. ALK, TIE1, DDR1, MAP2K7_ TBK1, ADCK3, ADCK4, NIM1, STK11_(LKB1), GCN2_Kin, (MKK7), PIK3CG_(p110g), CDC2L2_(PITSLRE), NTRK1_ ULK1, ULK2, PKMYT1, YANK2, TNIK, MEKK15_(YSK4), (TRKA), NDR2, CHAK2, CAMK1G, AURKB_(Aurora_B), OSR1, STK39_(STLK3), RIPK5, SgK288_(ANKK1), BMPR2, MAP3K6_(ASK2), PFTK2, BMP2K_(BIKE), TLK1, WNK3, MKNK1_(MNK1), CSNK1G2_(CK1_gamma_2), CAMKK2, QSK, SRPK1, MAPK7_(ERK5), WEE1B, PCTK2, PDGFRA_ MARK1_(MARK), EPHA2, FGFR4, FRAP1_(mTOR), EEF2K, (PDGFR_alpha), LATS2, CDC2L1_(CDK11B), BUB1, FLT4_ WNK2, PTK2B_(PYK2), BRAF, PRKCA_(PKC_alpha), TYK2, (VEGFR3), EIF2AK1_(PKR), RET, PFTK1, AAK1, RIOK3, GRK6, GRK5, TAOK3_(JIK), ABL2_(Arg), NEK2, PLK2, STK10_(LOK), DMPK2, IRE1, MYO3A, ACVR2A, CAMK2G, MATK_(HYL), PAK3, IRAK1, GRK7, DYRK3, CAMK2B_ DCAMKL1_(DCLK1), RIPK4, and WNK1. (CaMKII_beta), MYLK_(MLCK), TEK_(Tie2), STK16, NEK9, Kinases with <20% inhibition at 1μM. PDGFRB_(PDGFR_beta), PAK1, ZAP70, PIK3C3_(hVPS34), DAPK1, DCAMKL2_ NEK3, TESK1, PRKACB_(PKACb), FLT1_(VEGFR1), CDKL3, (DCK2), PHKG2, ADRBK2_(GRK3), MAPKAPK2, EPHB1, MAPK6_(ERK3), MYO3B, HUNK, PCTK1, SPHK1, NTRK2_ CDC42_BPA_(MRCKA), CSNK2A2_(CK2_alpha_2), ABL1, (TRKB), CASK, PIM3, TLK2, SgK085_(MYLK4), MAP2K5_ SLK, MAP4K4_(HGK), PIK3CD/PIK3R1_(p110d/p85a), (MEK5), MAP3K1_(MEKK1), EPHA6, STK25_(YSK1), MAP3K3_(MEKK3), PRKCH_(PKC_eta), AXL, TEC, CAMK4_ STK21_(CRIK), CDKL5, MAP3K4, MAST1, GAK, TNK1, SYK, (CaMKIV), MAP4K5_(KHS1), PRKCB1_(PKC_beta_I), LCK, LRRK2, PAK6, NEK11, RPS6KA6_(RSK4), NUAK2, PI4KA_(PI4K_alpha), PI4KB_(PI4K_beta), RPS6KA4_ MAP4K1, RIOK1, SRC, RIPK1, PRKCE_(PKC_epsilon), RIPK2, (MSK2), CSNK1G3_(CK1_gamma_3), TGFBR1_(ALK5), PKN2, TAOK1_(TAO1), MET, CDKL1, STK4_(MST1), ERBB3_ MAPK13_(p38_delta), CAMK1D_(CaMKI_delta), PRKCQ_ (HER3), MAP3K9_(MLK1), PCTK3, MST4, TNNI3K, TGFBR2, (PKC_theta), MAPK9_(JNK2), GRK4, CSNK1E_(CK1_epsilon), NEK6, MST1R_(RON), SPHK2, PRP4, JAK2, SRPK2, EPHB2, PIK3C2A_(PI3KC2a), , EPHA8, CDK7/CyclinH/MNAT1,

Zhang et al. www.pnas.org/cgi/content/short/1219457110 3of10 PAK4, PRKCD_(PKC_delta), MAPK10_(JNK3), SRMS_(Srm), Mouse Unilateral Ureter Obstruction Model. Surgery. Unilateral NUAK1_(ARK5), TTK, MAP3K5_(ASK1), CAMK1, PLK3, and ureter obstruction (UUO) was performed according to previously PRKCB2_(PKC_beta_II). described methods (10). Male C57BL/6 mice were anesthetized by i.p. injection with a solution of 10 mg/mL ketamine and 1 mg/ In Vivo Studies. All animal studies were conducted in accordance mL xylazine in normal saline (10 μL/g body weight). After 5 min, with the Institute for Laboratory Animal Research Guide for the the unconscious mice were shaved on the left side of the abdo- Care and Use of Laboratory Animals and the US Department of men and swabbed with ethanol. A vertical incision was made Agriculture Animal Welfare Act. through the skin with a scalpel and the skin retracted. Another careful incision (0.8–1 inch) was made through the peritoneum, Pharmacokinetics. To determine the pharmacokinetics of PLX647 avoiding any obvious blood vessels. The peritoneum was then in the mouse and rat over 24 h, PLX647 was administered as retracted to expose the left kidney. The kidney was brought to a single dose at 2 mg/kg i.v. and at 10 and 40 mg/kg by oral gavage the surface by hooking the ureter directly beneath the kidney (PO) to groups of 30 (i.v.) or 27 (PO) male CD-1 mice (sparse with sterile forceps and gently manipulating the kidney upward. sampling) and three male Sprague–Dawley rats. For i.v. dosing, The ureter was then tied with surgical silk directly below the PLX647 was mixed with 10% Solutol 15/10% ethanol/saline at kidney. This was repeated a second time to be certain of ureter 0.8 mg/mL (dosing volume 2.5 mL/kg). For PO dosing, PLX647 ligation. Ties and suturing were performed using SOFSILK 5-0 was mixed with 0.5% carboxymethylcellulose sodium /0.4% sutures attached to a 13-mm (3/8-inch) curved cutting needle (SS- Tween 80/0.9% benzyl alcohol/aaline at 2 mg/mL and 8 mg/mL 5640G; United States Surgical, Tyco Healthcare Group). After (dosing volume 5 mL/kg). Two hundred microliters (mouse) or ligation, the excess silk was cut away and discarded. The ligated 250 μL (rat) of whole blood was collected via inferior vena cava kidney was put back in the correct position and sterile normal (mouse) or jugular vein cannula (rat) before compound admin- saline was added to the abdomen to replenish lost fluid. Su- istration and at the following time points: predose (0), 0.083, turing was then performed on the peritoneum, followed by the 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h for i.v. and predose (0), 0.5, 1, 2, skin. After completion, the skin was wiped down and swabbed 4, 8, 12 and 24 h for PO. with iodine solution to prevent infection. The mice were then placed in clean cages and allowed to recover (30–60 min later). LPS-Induced TNF-α and IL-6 Release Model. Male Swiss Webster Treatment. PLX647 was dissolved in DMSO (200 mg/mL). Im- mice (six/group) were treated at 1-min intervals by oral gavage mediately before administration, aliquots of stock solution were with vehicle (10% DMSO in Labrafil), PLX647 (40 mg/kg), or mixed with vehicle (2.5% DMSO/0.5% hydroxypropyl methyl- dexamethasone (0.5 mg/kg). Four hours and 15 min after oral cellulose /1% PS80), producing a fine suspension. A single dose of treatment all mice were dosed i.p. (at corresponding 1-min these compounds (40 mg/kg) was administered by oral gavage intervals) with LPS (1 mg/kg); 1.5 h after LPS injection, mice were (160–180 μL) to groups of six male C57BL/6 mice (20–22 g) at anesthetized and bled via cardiac puncture into serum separator 12-h intervals for 7 d, starting at the end of UUO surgery. Groups tubes. Serum tubes were allowed to clot for 2 h at room tem- of untreated mice and vehicle-treated mice served as controls. At perature and then spun at 2,000 × g for 20 min. Approximately the end of the treatment period, the animals were anesthetized 100–150 μL per animal of serum was harvested into two color- by i.p. injection of ketamine/xylazine and blood was extracted by coded and labeled Eppendorf tubes and frozen at −70 °C until cardiac puncture and mixed with heparin (2%, 5,000 U/mL). analysis by ELISA for TNF-α and IL-6 levels. ELISZA kits used UUO kidneys were then examined to ensure that the surgical ties were Quantikine Mouse TNF-α Immunoassay and Quantikine had been effective. Both the ligated (UUO) and nonligated kid- Mouse IL-6 Immunoassay. neys were collected for analysis. The PLX647 dose was prepared as follows: Warm the amount Blood monocyte analysis. Heparinized blood (0.5 mL) was mixed of Labrafil required to 70 °C until a clear solution results, allow with 0.5 mL of cold PBS and overlaid on 2 mL of Ficoll-Paque the Labrafil to cool to room temperature, dissolve PLX647 in Plus in a centrifuge tube. Each sample was centrifuged at 600 × g DMSO (concentration 40 mg/mL), add 9× of Labrafil to the for 30 min and then the monocyte–lymphocyte fraction was ex- solution and mix thoroughly to form 4 mg/mL dosing solution. tracted. These cells were washed and fixed at 4 °C with 2% The dosing volume is 10 mL/kg. paraformaldehyde-lysine-periodate (PLP) for 20 min and then permeabilized with 0.1% saponin. The permeabilized cells were Mouse Passive Cutaneous Anaphylaxis Model. NODLt/J (JAX) mice incubated for 30 min with FITC-conjugated rat anti-mouse CD68 (8 wk old, 20–25g) were anesthetized using inhaled isoflurane. mAb (MCA1957; Serotec) in 10% normal rat serum. After la- Dinitrophenol (DNP)-specific IgE (25 ng of SPE-7 in 20 μLof beling, these samples were washed and analyzed on a MoFlo flow PBS) was intradermally injected into one ear of each NODLt/J cytometer. Blood monocyte numbers were determined by gating mouse to prime the cutaneous mast cells and saline was injected on CD68+ cells and determining the percentage of monocytes in into the other ear as a control. IgE activates mast cells to initiate the monocyte–lymphocyte fraction. anaphylaxis through aggregation of the IgE receptor, FceRI. A Immunohistochemistry analysis. Kidney cross-sections (2–3 mm) were day later, animals were dosed orally with vehicle or PLX647. fixed in 10% formalin or PLP for 3 h before tissue embedding in Two hours postdosing, the animals were given an i.v. injection of paraffin or Optimal Cutting Temperature medium. These kidneys the antigen bound to dye (100 μg DNP-HAS + 2% Evan’s blue were then sectioned (2 or 4 μm) and attached to slides. Kidney dye in a volume of 200 μL). When mast cell-bound IgEs are histology was identified by staining formalin-fixed sections (2 μm) engaged by DNP-HAS antigen, mast cell degranulation causes with periodic acid-Schiff reagent and hematoxylin. Immunostain- local inflammation and vascular dye leakage in the ear that re- ing on formalin-fixed kidney sections (4 μm) was used to identify ceived IgE. Half an hour later, the animals were killed and 4-mm interstitial accumulation of macrophages (F4/80+) and proliferating punch biopsies were taken from both ears and incubated in 100 macrophages (F4/80+/Ki67+). Detection of primary antibodies μL formamide for 2 h at 80 °C. After incubation, the supernatant was performed using biotinylated secondary antibodies and avidin was read on a mass spectrometer at a wavelength of 650 nm. The conjugated to either peroxidase or alkaline phosphatase (ABC differential absorbance from the two ears of each animal was kits; Vector Laboratories). These immunostaining techniques quantified to determine efficacy of a compound. have been previously described in detail (10, 11). PLX647 was dosed in carboxymethylcellulose (CMC) suspension vehicle (0.5% CMC, 0.9% ethanol, and 0.5% Tween 80 in 0.9% Mouse Collagen-Induced Arthritis Model. PLX647 was tested in a saline) at a concentration of 16 mg/mL (dosing volume 5 mL/kg). chronic model of murine collagen-induced arthritis. Male DBA/1J

Zhang et al. www.pnas.org/cgi/content/short/1219457110 4of10 mice (Jackson Laboratories, 7–9 wk old) were used in this study Tactile allodynia testing. Rats were habituated to the von Frey testing (10 mice/group). To induce the disease, 3 mL of the collagen apparatus before testing. Tactile allodynia (i.e., mechanical allo- solution was emulsified with 3 mL of Mycobacterium tuberculosis dynia) was determined by applying a series of calibrated nylon suspension yielding the collagen/adjuvant inoculum (2 mg/mL fibers through the cage floor and pressing them against the plantar collagen final concentration). Fifty microliters of the collagen/ surface of the hind paw. The rats were unrestrained and unhandled adjuvant emulsion was injected intradermally at the base of the during the test. The diameters of the filaments correspond to tail of each mouse on the starting day (day 0) and day 21. Ten a logarithmic scale of force exerted and thus a linear and interval control mice were injected with aqueous squalene. The mice scale of perceived intensity. The withdrawal threshold was deter- were then weighed weekly and scored daily for signs of arthritis. mined according to Chaplan’s “up-down” method (12) involving One of five possible scores was assigned for each limb: 0 = no the use of successively larger and smaller fibers to allow identi- visible effects of arthritis, 1 = edema and/or erythema of one = = fication of the 50% withdrawal threshold. Briefly, when the rat digit, 2 edema and/or erythema of two joints, 3 edema and/or fi erythema of more than two joints, and 4 = severe arthritis of lifted its paw in response to the pressure, the lament size was fi the entire paw and digits. The finalscoreforeachanimalis recorded and a weaker lament was used next. Conversely, in the the sum of the scores for all four limbs (ranging from 0 to 16). absence of a response, a stronger stimulus was used. A sequence Starting on day 27, each animal was given a single 8 mL/kg dose of such responses was generated and the 50% response threshold of vehicle [prepared by dissolving 0.5 g of CMC (C-4888; Sigma), was calculated using a response variable spreadsheet. Significant 0.9 g of sodium chloride (Aldrich), 0.4 mL Tween-80 (Acros differences in tactile allodynia were based on a comparison of Organics), and 0.9 mL of benzyl alcohol (402834; Sigma) in 100 mL group mean values. of distilled water], prednisolone (3 mg/kg), PLX647 (20 mg/kg), Micro-computed tomography and histopathological assessments. At the or two 8 mL/kg doses of PLX647 (80 mg/kg, given at 12-h in- conclusion of the experimental period, the left tibiae were excised tervals) per day. Both PLX647 and prednisolone were adminis- for radiographic confirmation of tumor osteolysis and for optional tered as suspension in the vehicle. Joint histology was performed micro-computed tomography (CT) and histopathological assess- fi at the end of study. On day 41, ve mice from the vehicle-treated ments. Radiographs were used to select two representative bones disease group, prednisolone-treated group, 20 mg/kg QD PLX647- from each group for microCT scanning. The proximal end of the treated group, and the group given 80 mg/kg PLX647 twice daily tibia was subjected to micro-CT scan (1076; Skyscan) at a 9-μm were anesthetized and exsanguinated into heparinized tubes. The resolution with the following settings: KV was set at 100 and μA sternum, one femur, and a symptomatic limb from each of these at 100, beam hardening was set at 40%, and ring artifact cor- animals were preserved in 10% buffered formalin. Five of the fi remaining animals in these groups were anesthetized and ex- rection was set at 4. The lter used was aluminum at a 0.5-mm sanguinated into heparinized tubes and the blood was processed thickness. After the scan, individual slice images were examined to plasma. and the slice that was considered the beginning of the secondary This study was conducted at Washington Biotechnology, Inc. metaphysis was chosen as the starting point. An additional 110 (Columbia, MD) according to protocols and procedures approved slices distal to the proximal end were selected as the region of by Washington Biotechnology. interest for the 3D image reconstruction. Three-dimensional im- age model building/model creation was performed using 3-D Rat Cancer-Induced Bone Pain and Osteolysis Model. Female Sprague– Creator software (Skyscan). Following the scanning procedures, – – Dawley rats (125 150 g, Harlan Sprague Dawley) were used in all bone samples were decalcified for tartrate resistant acid phos- × 4 this study. After 1 wk of acclimation, 3 10 MRMT-1 rat mam- phatase (TRAPb) staining and evaluation of osteoclast activity mary gland carcinoma cells were injected into the marrow space of (resorbing surfaces) and for histopathological assessment of the proximal tibia of each rat. The animals were treated with bone structure and tumor burden, using hemotoxylin and eosin. vehicle or PLX647 (oral gavage, 30 mg/kg twice daily) beginning Osteolysis was radiographically scored according to the following 7 d postinoculation. PLX647 was formulated in a final suspension of 0.5% hydroxypropylmethylcellulose, 2.5% Tween80, 10% criteria: 0, normal bone with no signs of destruction; 1, small PEG400, and 2.5% DMSO. Vehicle contains only the formula- radiolucent lesions indicative of bone destruction (one to three tion. The efficacy was determined from measurement of tactile lesions); 2, increased number of lesions (three to six lesions) and allodynia at baseline and at 7, 10, and 14 d postinoculation. Be- loss of medullary bone; 3, loss of medullary bone and erosion of havioral testing took place 2 h ± 15 min after the dosing procedure. cortical bone; 4, full thickness unicortical bone loss; and 5, full A morphine-treated group was included as a positive control. The thickness bicortical bone loss and/or displaced skeletal fracture. animals were acutely dosed on behavioral assessment days with This study was conducted at MDS Pharma Services (Bothell, s.c. injections of either 5 mg/kg of morphine or saline 30 ± 10 min WA) according to protocols and procedures approved by MDS before behavioral testing. Pharma Services.

1. Powell HR (1999) The Rossmann Fourier autoindexing algorithm in MOSFLM. Acta 7. Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Crystallogr D Biol Crystallogr 55(Pt 10):1690–1695. Acta Crystallogr D Biol Crystallogr 66(Pt 4):486–501. 2. Winn MD, et al. (2011) Overview of the CCP4 suite and current developments. Acta 8. Brünger AT (1992) Free R value: a novel statistical quantity for assessing the accuracy Crystallogr D Biol Crystallogr 67(Pt 4):235–242. of crystal structures. Nature 355(6359):472–475. 3. Vagin A, Teplyakov A (2010) Molecular replacement with MOLREP. Acta Crystallogr D 9. Wang W, et al. (2006) Structural characterization of autoinhibited c-Met kinase Biol Crystallogr 66(Pt 1):22–25. produced by coexpression in bacteria with phosphatase. Proc Natl Acad Sci USA 103(10): 4. Mol CD, et al. (2004) Structural basis for the autoinhibition and STI-571 inhibition of c- 3563–3568. Kit tyrosine kinase. J Biol Chem 279(30):31655–31663. 10. Le Meur Y, et al. (2002) Macrophage accumulation at a site of renal inflammation is 5. Schubert C, et al. (2007) Crystal structure of the tyrosine kinase domain of colony- dependent on the M-CSF/c-fms pathway. J Leukoc Biol 72(3):530–537. stimulating factor-1 receptor (cFMS) in complex with two inhibitors. JBiolChem282(6): 11. Chow FY, et al. (2006) Monocyte chemoattractant protein-1 promotes the development 4094–4101. of diabetic renal injury in streptozotocin-treated mice. Kidney Int 69(1):73–80. 6. Adams PD, et al. (2010) PHENIX: A comprehensive Python-based system for macro- 12. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL (1994) Quantitative assessment molecular structure solution. Acta Crystallogr D Biol Crystallogr 66(Pt 2):213–221. of tactile allodynia in the rat paw. J Neurosci Methods 53(1):55–63.

Zhang et al. www.pnas.org/cgi/content/short/1219457110 5of10 KIT-Leu644 KIT-Thr670 FMS-Met637 FMS-Thr663

KIT-Trp557 (JM)

KIT-Cys809 FMSFMS-Gl Gly795

Fig. S1. The trifluoromethyl–phenylmethylamino tail of PLX647 and analogs adopts different orientations in FMS and KIT, resulting in displacement of the juxtamembrane domain in FMS and the recruitment of the juxtamembrane domain (Trp557) in KIT.

Zhang et al. www.pnas.org/cgi/content/short/1219457110 6of10 A

Transmembrane region Juxtamembrane domain PP-loop loop

Kinase insertion domain (KID) Catalytic loop

Activation loop

B

Transmembrane region Juxtamembrane domain P-loop

Kinase insertion domain (KID)

Catalyticyp loop Activation loop

Fig. S2. Multispecies sequence alignments of (A) FMS and (B) KIT. Secondary structures are depicted schematically above the aligned sequence and important regions are labeled below the sequences. The human, mouse, and rat sequences are highly homologous, with variations found mainly in the kinase insertion domain and C-terminal lobe. Both are away from the inhibitor . The residues making direct contacts with the inhibitors (indicated by circles) are identical in sequences from the three species. Alignment was formatted using the ALSCRIPT program (1).

1. Barton GJ (1993) ALSCRIPT: A tool to format multiple sequence alignments. Protein Eng 6(1):37–40.

A B

) 16,000 18,000 L) L 14,000 16,000 12,000 14,000 (pg/m 12,000 a a 10,000 10,000 NF

8,000 IL6 (pg/m T 80008,000 70% 6,000 6,000 75% 4,000 85% 4,000 Serum Serum 2,000 96% 2,000 0 0 Vehicle DEX PLX647 Vehicle DEX PLX647 (0.5mg/kg)(40mg/kg) (0.5mg/kg)(40mg/kg)

Fig. S3. PLX647 suppresses LPS-induced TNF-α (A) and IL-6 release (B).

Zhang et al. www.pnas.org/cgi/content/short/1219457110 7of10 0.4

0.3 @650nm e 0.2 38%

0.1

Absorbanc 000.0 Vehicle PLX647(80mg/kg)

Fig. S4. PLX647 inhibits mast cell activation in the skin.

A B Sunitinib in KIT PLX647 in KIT

Tyr553

Trp557 Trp557

Tyr553

CD Imatinib in KIT Sunitinib PLX647 Imatinib

Fig. S5. Structural comparison of PLX647 with imatinib and sunitinib in complex with KIT. (A–C) Structures of sunitinib, PLX647, and imatinib in complex with KIT. The juxtamembrane regions are colored purple, and the two bulky side chains (Tyr553 and Trp557) that help to anchor the juxtamembrane domain to the kinase domain are shown in stick representation. Although both sunitinib and PLX647 bind the juxtamembrane domain-bound state, only PLX647 makes a direct contact with juxtamembrane domain residues. Because imatinib and juxtamembrane domain bind to the same area in space, they are mutually ex- clusive. (D) Overlay of the three structures zooming in on the regulatory αC helix. The αC helix adopts similar orientation in imatinib- and PLX647-bound states. The orientation of the αC helix in the sunitinib-bound structure is significantly different.

Zhang et al. www.pnas.org/cgi/content/short/1219457110 8of10 Table S1. Clinically approved kinase inhibitors (as of December 31, 2012) Generic name Brand name Company Target(s) Indication(s) Approval year

Imatinib Gleevec Novartis BCR-ABL/KIT/PDGFR CML, GISTs 2001 Gefitinib Iressa AstraZeneca EGFR NSCLC 2003 Sorafenib Nexavar Onyx VEGFR/PDGFR/KIT RCC, HCC 2005 Erlotinib Tarceva Astellas (OSI) EGFR NSCLC 2005 Sunitinib Sutent Pfizer VEGFR/PDGFR/KIT RCC, GIST 2006 Dasatinib Sprycel BMS BCR-ABL/SRC CML 2006 Nilotinib Tasigna Novartis BCR-ABL CML 2007 Lapatinib Tykerb GSK EGFR/HER2 Breast cancer 2007 Pazopanib Votrient GSK VEGFR/PDGFR/KIT RCC, sarcoma 2009 Vemurafenib Zelboraf Plexxikon/Roche BRAF Melanoma 2011 Crizotinib Xalkori Pfizer ALK/MET/ROS NSCLC 2011 Ruxolitinib Jakafi Incyte JAK1,2 Myelofibrosis 2011 Vandetanib Caprelsa AstraZeneca VEGFR/RET/EGFR Thyroid cancer 2011 Axitinib Inlyta Pfizer VEGFR/PDGFR/KIT RCC 2012 Bosutinib Bosulif Pfizer BCR-ABL CML 2012 Regorafenib Stivarga Bayer VEGFR2/TIE2 CRC 2012 Tofacitinib Xeljanz Pfizer JAK3 RA 2012 Ponatinib Iclusig Ariad BCR-ABL CML 2012

CML, chronic myelogenous leukemia; CRC, colorectal cancer; GIST, gastrointestinal stromal tumor; NSCLC, nonsmall cell lung cancer; RA, rheumatoid arthritis; RCC, renal cell carcinoma.

Table S2. Cellular activity of PLX647

Cellular assays IC50, μM Description

FMS Ba/F3 BCR-FMS 0.092 BCR–FMS fusion-dependent proliferation Ba/F3 BCR-FMS (+ IL-3) >5 IL-3–dependent proliferation (control) M-NFS-60 (+CSF-1) 0.38 CSF-1–activated FMS-dependent proliferation THP-1 FMS/total pY 0.25 CSF-1–activated FMS phosphorylation Osteoclasts differentiation 0.17 Levels of acid phosphatase in supernatant KIT Ba/F3 BCR-KIT 0.18 BCR–KIT fusion-dependent proliferation Ba/F3 BCR-KIT (+IL-3) >5 IL-3–dependent proliferation (control) M-07e (+SCF) 0.23 SCF-activated KIT-dependent proliferation M-07e KIT/pY823 0.048 SCF-activated KIT phosphorylation FLT3/FLT3-ITD MV4-11 0.11 FLT3–ITD-dependent proliferation MV4-11 FLT3/pY591 0.02 FLT3–ITD autophosphorylation OCI-AML5 (+FLT3LG) 1.6 FLT3LG-activated WT FLT3-dependent proliferation RS4;11 FLT3/pY591 1.5 FLT3LG-activated WT FLT3 phosphorylation KDR Ba/F3 BCR-KDR 5 BCR–KDR fusion dependent proliferation Ba/F3 BCR-KDR (+IL-3) 4.3 IL-3–dependent growth (control) HUVEC (+VEGF) 3 VEGF-activated KDR-dependent proliferation

Table S3. Kinase inhibitory activity of PLX647 versus a panel of

kinases (% inhibition at single concentration at 1μM and IC50)

Clan Family Kinase % inhibition IC50, μM

CMGC CDK CDK19* 88.5 0.35 † TK DDR DDR2 72.4 0.7 TK VEGFR KDR (VEGFR2)† 69.3 0.13 † CMGC CDK CDK8/cyclin C 67.3 0.71 † TK TRK/MuSK MUSK 65.1 1.2 CK1 VRK VRK2* 56 2.5 TK EPHB EPHB6* 53 >3 † TK FMS-KIT-FLT3-PDGFR FLT3 51.6 0.091 † TK TRK/MuSK NTRK3 (TRKC) 50.2 0.62

*Assay part of DiscoverX KINOMEScan panel. †Assay part of Invitrogen SelectScreen panel.

Zhang et al. www.pnas.org/cgi/content/short/1219457110 9of10 Table S4. Crystallographic data and refinement statistics Data KIT-PLX647 FMS-PLX647-OMe

Collection Resolution range, Å 64.7–1.85 59.5–2.9

Space group C2 P43212 Unit cell a = 110.7,b = 81.9, c = 50.1, β = 107.8 a = b = 63.0, c = 182.8 No. of observations 120,767 47,312 No. of unique reflections 35,908 8,735 Completeness, %* 98.9 (97.9) 99.6 (99.9) ,† Rsym,%* 5.3 (73.7) 18.3 (85.4) Redundancy 3.6 5.4 Refinement statistics Resolution range, Å 52.7–1.90 51.9–2.90 No. of reflections: working/free 31,562/1,632 8,714/414 R factor, %‡: working/free 19.9/22.7 22.0/26.7 Rmsd from ideality Bond length, Å 0.014 0.005 Bond angles, ° 1.411 0.891 Most favored region, %§ 92.4 93.2 Additional allowed region, %§ 7.6 6.8 Disallowed region, %§ 00

*NumbersP inP parentheses represent values in the highest resolution shell. † jI − < I > j Rsym = Ph Pn , where I is observed integrated intensity and is the averaged integrated intensity taken < I > h n over n measurementsP for reflection h. ‡ jjFoj − jFcjj R factor = hP , where jF j is the observed structure factor amplitude and jF j is the calculated structure jFoj o c h factor amplitudes based on the refined atomic positions, taken over the h reflections in the observed data set. §In the Ramachandran plot.

Zhang et al. www.pnas.org/cgi/content/short/1219457110 10 of 10