RAF709

RAF709 is a potent inhibitor of B/C RAF kinase with almost equivalent IC50 values of 0.4 nM for B-RAF and C-RAF, showing a high level of selectivity, demonstrating greater than 99% on-target binding to BRAF, BRAFV600E, and CRAF at 1 μM and very few off-targets with DDR1 (>99%), DDR2 (86%), FRK (92%), and PDGFRb (96%), the only kinases with binding >80% at 1 μM.

CAS No. 1628838-42-5

サイズ	価格(税別)	在庫状況
5mg	JPY 23100	国内在庫あり
25mg	JPY 69600	国内在庫あり
100mg	JPY 141600	国内在庫あり

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文献中Selleckの製品使用例（3）

製品安全説明書

現在のバッチを見る： S869001 DMSO] 100 mg/mL] false] Ethanol] 100 mg/mL] false] Water] Insoluble] false 純度： 99.86%

99.86

RAF709関連製品

関連ターゲット	C-Raf/Raf-1 B-Raf A-raf	もっと見る
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関連化合物ライブラリー	Kinase Inhibitor Library MAPK Inhibitor Library Cell Cycle compound library TGF-beta/Smad compound library Anti-alzheimer Disease Compound Library	もっと見る

シグナル伝達経路

Rafシグナル伝達経路

Raf阻害剤の選択性比較

生物活性

製品説明

RAF709 is a potent inhibitor of B/C RAF kinase with almost equivalent IC50 values of 0.4 nM for B-RAF and C-RAF, showing a high level of selectivity, demonstrating greater than 99% on-target binding to BRAF, BRAFV600E, and CRAF at 1 μM and very few off-targets with DDR1 (>99%), DDR2 (86%), FRK (92%), and PDGFRb (96%), the only kinases with binding >80% at 1 μM.

Targets

C-Raf ^[1] (Cell-free assay)	BRAF(V600E) ^[2] (Cell-free assay)	B-Raf ^[2] (Cell-free assay)
0.4 nM	1 nM	1.5 nM

In Vitro
In vitro	RAF709 appears to have very slow dissociation kinetics (T_1/2 > 6.5 h) using the rapid dilution method to measure its dissociation rate constant. In cellular assays, the dose−response of pMEK and pERK are measured in Calu-6 cells with EC50 of 0.02 and 0.1 μM with minimal paradoxical activation and inhibition of proliferation with EC50 of 0.95 μM. RAF709 stabilizes BRAF−CRAF dimers with an EC50 of 0.8 μM. Of the 456 kinases tested, RAF709 shows a high level of selectivity, demonstrating greater than 99% on-target binding to BRAF, BRAFV600E, and CRAF at 1 μM and very few off-targets with DDR1 (>99%), DDR2 (86%), FRK (92%), and PDGFRb (96%), the only kinases with binding >80% at 1 μM^[1]. RAF709 shows equal activity against both RAF monomers and dimers. In in vitro biochemical assays, RAF709 exhibits potent inhibitory activity targeting BRAF, BRAFV600E, and CRAF with IC50 values ranging between 0.3 to 1.5 nmol/L. RAF709 treatment leads to a dose-dependent induction of B/CRAF heterodimerization in HCT116, but inhibits MEK and ERK phosphorylation, in line with the ability of RAF709 to effectively inhibit the RAF dimers. RAF709 selectively inhibits oncogenic signaling and proliferation in tumor cells with BRAF, NRAS, or KRAS mutations with minimal paradoxical activation^[2].
Kinase Assay	CRAF kinase assay
Kinase Assay	The CRAF kinase assay was carried out using 10 nM kinase-dead MEK1 protein substrate (carrying a K97R mutation), 3 μM ATP, and 10 pM CRAF Y340E/Y341E. The reaction buffer contained 50 mM Tris pH 7.5, 10 mM MgCl2, 0.05% BSA, 50 mM NaCl, 0.01% Tween-20, and 1 mM DTT. The reactions were carried out at room temperature in a volume of 10 μL in white 384-shallow-well plates for 40 min and stopped by adding 5 μL/well quench solution (50 mM Tris pH 7.5, 50 mM EDTA). Terminated reactions received 5 μL/well detection reagents consisting of 50 mM Tris pH 7.5, 0.01% Tween-20, 1:1000 diluted antiphospho MEK1/2 S217/S221 antibody, 0.01 mg/mL each of AlphaScreen Protein A-coated acceptor beads, and streptavidin-coated donor bead. Plates were read in an EnVision plate reader after overnight incubation at room temperature. In compound inhibition studies, compounds were tested over a concentration range of 25 μM to 1.74 × 10⁻⁶ μM in 16-point, 3-fold format. DMSO was at a final concentration of 0.5%. Compounds were preincubated with CRAF for 30 min before adding substrates to start the reaction. Inhibition data were fit to a four-parameter logistic equation to calculate the IC50 of the compounds.
細胞実験	細胞株	HCT116 cells
	濃度	0.1, 1, 10 μM
	反応時間	1 hour
	実験の流れ	HCT116 cells are treated with DMSO or RAF709 at indicated concentrations for 1 hour; 1 mmol/L dabrafenib treatment is included for comparison. BRAF/CRAF dimerization is assessed by immunoprecipitating BRAF or CRAF, followed by Western blot analysis of BRAF and CRAF. Levels of pMEK and pERK in whole-cell lysates (WCL) are determined by Western blot analysis. GAPDH level is included as a loading control.

In Vivo
In Vivo	RAF709 is well tolerated and efficacious in KRAS mutant xenograft models. It is reasonably stable in plasma after a 3 h incubation at 37℃ across species [plasma stability (%remaining): rat 85%, mouse 82%, dog 95%, human 101%], and plasma protein binding is measured to be 98% across species. In pharmacokinetic experiments, RAF709 has moderate clearance in mouse (35 mL/min/kg) and dog (14 mL/min/kg) and high clearance in rat (50 mL/min/kg). Cmax in mouse (1 μM), dog (0.5 μM), and rat (0.5 μM) reach pharmacologically active concentrations, and acceptable oral availability is observed in mouse (68%), rat (24%), and dog (48%). In the Calu-6 xenograft nude mouse model, treatment with RAF709 results in dose-dependent antitumor activity with 10 mg/kg being subefficacious (%T/C = 92%), 30 mg/kg resulted in measurable antitumor activity (% T/C = 46%), and 200 mg/kg resulted in mean tumor regression of 92%, while the same high dose is not efficacious in the PC3, KRAS WT mode^[1].
動物実験	動物モデル	Calu-6 model (tumor bearing mice)
	投与量	10, 30, or 200 mg/kg
	投与経路	oral administration

参考
[1] Nishiguchi GA, et al. J Med Chem. 2017, 60(12):4869-4881. [2] Shao W, et al. Cancer Res. 2018, 78(6):1537-1548.

参考

化学情報

分子量	542.55	化学式	C₂₈H₂₉F₃N₄O₄
CAS No.	1628838-42-5	SDF	--
Smiles	CC1=C(C=C(C=N1)NC(=O)C2=CC(=CC=C2)C(F)(F)F)C3=CC(=C(N=C3)OC4CCOCC4)N5CCOCC5
保管	3年-20°C粉	溶液の保管冷凍と解凍の繰り返しを避けるため小分けにしてください	1年-80°Cin solvent
保管	3年-20°C粉	溶液の保管冷凍と解凍の繰り返しを避けるため小分けにしてください	1个月-20°Cin solvent

In vitro
Batch:

DMSO : 100 mg/mL ( (184.31 mM)；吸湿したDMSOは溶解度を減少させます。新しいDMSOをご使用ください。)

Ethanol : 100 mg/mL

Water : Insoluble

モル濃度計算器

in vivo
Batch:

Add solvents to the product individually and in order.

投与溶液組成計算機

実験計算

モル濃度計算器

質量	濃度	体積	分子量
=	×	×

希釈計算器分子量計算器

投与溶液組成計算機(クリア溶液)

ステップ１：実験データを入力してください。（実験操作によるロスを考慮し、動物数を1匹分多くして計算・調製することを推奨します）

投与量 mg/kg 動物平均体重 g 投与体積（動物毎）μL 動物数匹

ステップ２：投与溶媒の組成を入力してください。（ロット毎に適した溶解組成が異なる場合があります。詳細については弊社までお問い合わせください）

% DMSO + % + % Tween 80 + % ddH₂O

%DMSO+ %

計算結果：

投与溶媒濃度： mg/ml;

DMSOストック溶液調製方法： mg 試薬を μL DMSOに溶解する（濃度 mg/mL, 注：濃度が当該ロットのDMSO溶解度を超える場合はご連絡ください。 )

投与溶媒調製方法：Take μL DMSOストック溶液に μL PEG300，を加え、完全溶解後μL Tween 80，を加えて完全溶解させた後 μL ddH₂O，を加え完全に溶解させます。

投与溶媒調製方法：μL DMSOストック溶液に μL Corn oil，を加え、完全溶解。

注意：１.ストック溶液に沈殿、混濁などがないことをご確認ください；
２.順番通りに溶剤を加えてください。次のステップに進む前に溶液に沈殿、混濁などがないことを確認してから加えてください。ボルテックス、ソニケーション、水浴加熱など物理的な方法で溶解を早めることは可能です。

C1=C0/X	C1:	LOG(C1):
C2=C1/X	C2:	LOG(C2):
C3=C2/X	C3:	LOG(C3):
C4=C3/X	C4:	LOG(C4):
C5=C4/X	C5:	LOG(C5):
C6=C5/X	C6:	LOG(C6):
C7=C6/X	C7:	LOG(C7):
C8=C7/X	C8:	LOG(C8):