B.R ified m and th D448 K723 vanced-stage melanom t ng a s c-Raf. 0307 648 340D an rotein kin by stabilizing the gphcaandohem by onco the active ed by sm were dete discovery ofPL g the ZYTE bih ical assay format (seled B-R inhibition lation of ERK by im This res s I umor regressi mpo ra or to treated m 205 p4720 mg C 05t ith the hope of wh PI Y d maintaining a X4720 ded in th ver )and all proce Experimental Procedures ote that det as SI Meth Me k210 100 hice co for 15 davs T mo/kg PLX47 synt size the inh he and and Crysta R-RAt To n,the E CDNAs (both V ME e E60 gmenter 0562RL588N.K6305.F667E.Y6735,A5eR.7065.0709R.57 98 and De -76SF00515,re k was s 1448to1.5.AR0518 (3)w the BRAFge 6 l( ay b 1325 W 17: ot al uted pyrazin 1045 -72 06)No RAF by 14 very of a etal (2000)The _JNa 13 sion in melanoma cells restores keratinocyte oR.A 551515-1525 30461www.pnas.org/egl/do/10.1073/pnas.0711741105 Tsaietal.affected by treatment with PLX4720. The results from these in vivo models validate our in vitro data and thereby support the use of PLX4720 as a next-generation therapeutic for patients with ad￾vanced-stage melanoma. Conclusions The mechanism of activating protein kinases by stabilizing the catalytically preferred conformation is exploited by oncogenic mutations such as B-RafV600E. Conversely, the selective targeting of the active DFG conformation can also be achieved by small￾molecule inhibitors, and this should enable the development of future therapeutic kinase inhibitors. The discovery of PLX4720 opens the door to future studies that seek to measure the effects of selective inhibition of B-RafV600E. Pharmacologic inhibition of oncogenic B-Raf blocks proliferation and causes tumor regressions. This finding is also consistent with recent studies showing the genetic depletion of B-Raf in B-RafV600E-bearing xenografted melanoma cells results in dramatic tumor regressions (28). Impor￾tantly, this efficacy is achieved without apparent toxicity to un￾transformed cells in vitro or to treated mice. The pharmaceutical properties of PLX4720 enable advancement to human clinical trials, with the hope of achieving clinical benefit and maintaining a significant therapeutic window. PLX4720 thus represents perhaps a first-in-class therapeutic molecule that selectively inhibits a target that exists only in tumors. Experimental Procedures Note that detailed methods are included as SI Methods. Synthesis of B-Raf Inhibitors. PLX4720 was synthesized by reacting commercially available 5-chloro-7-azaindole with propane-1-sulfonic acid (2,4-difluoro-3- formyl-phenyl)-amide followed by oxidation (SI Scheme 1). Similar procedures were used to synthesize the inhibitors listed in Fig. 1C by using appropriately substituted7-azaindoleandaldehydeasstartingmaterials(SIScheme2).Detailed synthetic methods are included as SI Methods. Cloning, Expression, Purification, and Crystallization of B-Raf. To enable crys￾tallization, theBRAF 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 pET 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 expressed, purified, crystallized, and the structures deter￾mined by using methods similar to those used for the reported for Pim-1 and Met (16). For enzymatic activity, B-Raf (residues D448–K723) with or without the V600E mutation and fused at the N terminus with six-residue HIS-tag, was coexpressed with CDC37 in insect cells by using a baculovirus vector. For compar￾ative enzymatic determinations, c-Raf-1 (residues Q307–F648, with Y340D and Y341D mutations, fused at N terminus with GST) from baculovirus-infected insect cells was used. For crystallographic data and refinement statistics, see SI Table 3. Biochemical and Cellular Assays. The in vitro Raf kinase activities were deter￾mined by measuring phosphorylation of biotinylated-MEK protein by using AlphaScreen Technology (16). In addition, 58 selected kinases were profiled for inhibition by PLX4720 by using the Z-LYTE biochemical assay format (Select￾Screen; Invitrogen) according to the manufacturer’s instructions. Cellular phosphorylation of ERK was determined by immunoassay, and cellu￾lar proliferation was determined by using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Melanoma cell proliferation, cell cycle analysis, apo￾ptosis assay, three-dimensional spheroid growth, and skin reconstruction exper￾iments were carried out as previously described (26, 27). COLO205 Tumor Xenograft. Female athymic mice (NCr nu/nu; Taconic) were implanted s.c. on day 0 with 30–60 mg COLO205 tumor fragments. Treatments began on day 11, when the mean estimated tumor mass was 104 mg (range, 95–113mg).Allanimalsweredosedwithvehicle(5%DMSO,1%methylcellulose) or PLX4720 suspended in vehicle by gavage daily for 14 consecutive days. Tumor burden (mg) was estimated from caliper measurements. This study was con￾ducted by Molecular Imaging Research (MIR), and all procedures were approved by the MIR Animal Care and Use Committee. Melanoma Tumor Xenografts. Metastatic melanoma cells (2  106) were s.c. injected into the flanks of SCID mice and allowed 2 weeks to reach 0.125 mm3 in volume. Subsequently, the animals received either 100 mg/kg PLX4720 (oral gavage) or vehicle control twice daily for 15 days. Tumor volume was recorded every 72 h. The average tumor size for each respective group was normalized to the tumor volume at the first day of treatment. 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