B-Raf (V600E) (IC50 = 13 nM); B-Raf (IC50 = 160 nM); BRK (IC50 = 130 nM); ; FRK (IC50 = 1300 nM); Csk (IC50 = 1500 nM); Src (IC50 = 1700 nM); FAK (IC50 = 1700 nM); FGFR (IC50 = 1900 nM); KDR (IC50 = 2300 nM); HGK (IC50 = 2800 nM); CSF1R (IC50 = 3300 nM); Aurora A (IC50 = 3400 nM)

PLX-4720 exhibits >10 times selectivity against wild-type B-Raf and >100 times selectivity over other kinases, including Frk, Src, Fak, FGFR, and Aurora A, with an IC50 of 1.3-3.4 μM. PLX-4720 significantly reduces the ERK phosphorylation in cell lines expressing B-RafV600E, but not in cells expressing wild-type B-Raf (IC50 = 14–46 nM). PLX-4720 significantly slows the expansion of tumor cell lines that carry the B-RafV600E oncogene, including COLO205, A375, WM2664, and COLO829, with GI50 values of 0.31 μM, 0.50 μM, 1.5 μM, and 1.7 μM, respectively. Additionally, treatment with PLX-4720 at 1 μM only causes cell cycle arrest and apoptosis in B-RafV600E-positive 1205Lu cells while having no effect on B-Raf wild-type C8161 cells[1]. In comparison to PTEN-cell lines (4-fold), PLX-4720 treatment (10 μM) significantly increases the expression of BIM in PTEN+ cells by > 14 times, which provides an explanation for PTEN-cells' resistance to PLX-4720's ability to induce apoptosis[2].

In B-RafV600E-dependent COLO205 tumor xenografts, oral administration of PLX-4720 at 20 mg/kg/day results in significant tumor growth delays and regressions, with no overtly harmful side effects in mice, even at doses as high as 1 g/kg. While having no effect on the C8161 xenografts containing wild-type B-Raf, PLX-4720 at 100 mg/kg twice daily almost completely eradicates the 1205Lu xenografts bearing B-RafV600E. When PLX-4720 is applied to cells carrying the V600E mutation, the anti-tumor effects are correlated with the blockade of the MAPK pathway[1]. Treatment with PLX-4720 at 30 mg/kg/day significantly inhibits the growth of 8505c xenograft tumors by >90% and significantly lowers distant lung metastases[3].

In 20 mM Hepes (pH 7.0), 10 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 100 nM biotin-MEK protein, varying ATP concentrations, and increasing concentrations of PLX-4720, 20-μL reactions are carried out for each enzyme (0.1 ng). At 2, 5, 8, 10, 20, and 30 minutes, reactions are stopped using 5 μL of a solution containing 20 mM hepes (pH 7.0), 200 mM sodium chloride, 80 mM EDTA, and 0.3% BSA. The AlphaScreen Protein A Detection Kit's phospho-MEK Antibody, Streptavidin-coated Donor beads, and Protein A Acceptor beads are also included in the stop solution. For 30 minutes, the antibody and beads are preincubated in stop solution at room temperature in the dark. The final antibody dilution is 1/2000, and the final bead concentration is 10 g/mL. The assay plates are read on a PerkinElmer AlphaQuest reader after an hour of room temperature incubation.

PLX-4720 is applied to cells at different concentrations for 24, 48, and 72 hours. The MTT assay or CellTiter-Glo Luminescent Cell Viability Assay are used to measure cell proliferation. Supernatant and cells are collected, pelleted, and fixed with 70% ethanol for cell cycle analysis. Cells are incubated for 1 hour at 37°C in 0.5 mg/mL RNase I to remove any remaining RNA contamination before staining with propidium iodide (10 μg/mL). Following that, samples are examined using the EPICS XL device. Media and cells are collected, pelleted, and stained with annexin-FITC and propidium iodide to determine the level of apoptosis. The EPICS XL instrument is then used to analyze the samples.

SCID mice have their flanks injected subcutaneously with 2×106 metastatic melanoma cells, which are given approximately two weeks to reach a volume of 0.125 mm3. The animals then either receive 100 mg/kg PLX4720 (oral gavage) or vehicle control twice daily for 15 days. The tumor volume is measured every 72 hours. Normalized to the tumor volume on the first day of treatment, the average tumor size for each respective group. Animals are put to death 15 days into the treatment process, and tumors are removed, formalin-fixed, paraffin-embedded, and immunohistochemically examined.

[1]. Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc Natl Acad Sci U S A, 2008, 105(8), 3041-3046.

[2]. PTEN loss confers BRAF inhibitor resistance to melanoma cells through the suppression of BIM expression. Cancer Res, 2011, 71(7), 2750-2760.

[3]. B-Raf(V600E) and thrombospondin-1 promote thyroid cancer progression. Proc Natl Acad Sci U S A, 2010, 107(23), 10649-10654.

[4]. Neuropilin-1 upregulation elicits adaptive resistance to oncogene-targeted therapies. J Clin Invest. 2018 Aug 31;128(9):3976-3990.

PLX-4720 is a pyrrolopyridine that is vemurafenib in which the p-chlorophenyl group has been replaced by chlorine. It is a potent and selective inhibitor of the Raf kinase B-Raf(V600E). It has a role as a B-Raf inhibitor and an antineoplastic agent. It is a pyrrolopyridine, a sulfonamide, a difluorobenzene, an organochlorine compound and an aromatic ketone.

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BRAF(V600E) is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting "active" protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-Raf(V600E) with an IC(50) of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-Raf(V600E) kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-Raf(V600E)-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-Raf(V600E)-positive cells. In B-Raf(V600E)-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-Raf(V600E)-driven tumors.[1]

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This study addresses the role of PTEN loss in intrinsic resistance to the BRAF inhibitor PLX4720. Immunohistochemical staining of a tissue array covering all stages of melanocytic neoplasia (n = 192) revealed PTEN expression to be lost in >10% of all melanoma cases. Although PTEN expression status did not predict for sensitivity to the growth inhibitory effects of PLX4720, it was predictive for apoptosis, with only limited cell death observed in melanomas lacking PTEN expression (PTEN-). Mechanistically, PLX4720 was found to stimulate AKT signaling in the PTEN- but not the PTEN+ cell lines. Liquid chromatography multiple reaction monitoring mass spectrometry (LC-MRM) was performed to identify differences in apoptosis signaling between the two cell line groups. PLX4720 treatment significantly increased BIM expression in the PTEN+ (>14-fold) compared with the PTEN- cell lines (four-fold). A role for PTEN in the regulation of PLX4720-mediated BIM expression was confirmed by siRNA knockdown of PTEN and through reintroduction of PTEN into cells that were PTEN-. Further studies showed that siRNA knockdown of BIM significantly blunted the apoptotic response in PTEN+ melanoma cells. Dual treatment of PTEN- cells with PLX4720 and a PI3K inhibitor enhanced BIM expression at both the mRNA and protein level and increased the level of apoptosis through a mechanism involving AKT3 and the activation of FOXO3a. In conclusion, we have shown for the first time that loss of PTEN contributes to intrinsic BRAF inhibitor resistance via the suppression of BIM-mediated apoptosis.[2]

C17H14CLF2N3O3S

413.83

413.041

C, 49.34; H, 3.41; Cl, 8.57; F, 9.18; N, 10.15; O, 11.60; S, 7.75

918505-84-7

PLX-4720-d7;1304096-50-1

24180719

white solid powder

1.5±0.1 g/cm3

621.4±65.0 °C at 760 mmHg

329.6±34.3 °C

0.0±1.8 mmHg at 25°C

1.646

3.14

2

7

6

27

648

0

O=C(C1C(F)=C(NS(CCC)(=O)=O)C=CC=1F)C1C2C(=NC=C(C=2)Cl)NC=1

YZDJQTHVDDOVHR-UHFFFAOYSA-N

InChI=1S/C17H14ClF2N3O3S/c1-2-5-27(25,26)23-13-4-3-12(19)14(15(13)20)16(24)11-8-22-17-10(11)6-9(18)7-21-17/h3-4,6-8,23H,2,5H2,1H3,(H,21,22)

N-[3-(5-chloro-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluorophenyl]propane-1-sulfonamide

PLX 4720; PLX4720; 918505-84-7; PLX-4720; PLX4720; N-(3-(5-chloro-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide; PLX 4720; N-[3-[(5-Chloro-1H-pyrrolo[2,3-b]pyridin-3-yl)carbonyl]-2,4-difluorophenyl]-1-propanesulfonamide; N-[3-(5-chloro-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluorophenyl]propane-1-sulfonamide; MFCD14635203; PLX-4720

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.03 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.03 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 2% DMSO +50% PEG 300 +5% Tween 80 +ddH2O: 5mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)