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 is a highly selective inhibitor of the oncogenic mutant BRAF kinase (BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ). In recombinant human BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ kinase assays, it exhibits an IC₅₀ of 13 nM; it has minimal activity against wild-type BRAF (IC₅₀ > 10 μM) and other RAF family members (e.g., CRAF, IC₅₀ = 3.2 μM) [1]
- PLX-4720 shows no significant inhibition of non-RAF kinases, including EGFR (IC₅₀ > 50 μM), MEK1 (IC₅₀ > 10 μM), and AKT (IC₅₀ > 20 μM), confirming its specificity for BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ [1]

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].

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BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ Melanoma Cell Proliferation Inhibition: In human BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ-positive melanoma cell lines (A375, SK-MEL-28), PLX-4720 (0.001–10 μM) concentration-dependently inhibits cell proliferation: 0.021 μM reduces A375 cell viability by 50% (IC₅₀ = 21 nM), and 1 μM achieves 95% inhibition. This effect is accompanied by a 90% reduction in phosphorylated ERK1/2 (p-ERK, downstream MAPK marker) at 0.1 μM (Western blot analysis) [1]
- PTEN-Deficient Melanoma Cell Resistance: In PTEN-knockout A375 cells (PTEN⁻/⁻), PLX-4720 (0.01–5 μM) shows reduced antiproliferative activity: the IC₅₀ increases from 21 nM (PTEN⁺/⁺) to 150 nM (PTEN⁻/⁻). This resistance is mediated by suppressed BIM (pro-apoptotic protein) expression—PTEN⁻/⁻ cells show 60% lower BIM levels vs. PTEN⁺/⁺ cells, and BIM overexpression restores sensitivity (IC₅₀ = 30 nM) [2]
- BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ Thyroid Cancer Cell Inhibition: In human BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ-positive papillary thyroid cancer cells (BCPAP), PLX-4720 (0.05–5 μM) inhibits proliferation (IC₅₀ = 45 nM) and reduces thrombospondin-1 (TSP-1, pro-angiogenic protein) secretion by 70% at 0.5 μM (ELISA detection). It also blocks TSP-1-induced endothelial cell migration (by 80% at 0.5 μM) [3]
- NRP1-Mediated Adaptive Resistance: In NRP1-overexpressing SK-MEL-28 cells, PLX-4720 (0.01–10 μM) shows decreased efficacy: 1 μM reduces viability by only 40% (vs. 95% in parental cells). NRP1 knockdown (via siRNA) restores sensitivity, with viability reduced by 85% at 1 μM [4]

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].

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Melanoma Xenograft Model: In nude mice bearing A375 BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ melanoma xenografts, oral administration of PLX-4720 (25, 50, 100 mg/kg/day, once daily) dose-dependently inhibits tumor growth: 100 mg/kg reduces tumor volume by 90% at day 28 vs. vehicle, and induces complete tumor regression in 40% of mice. Tumor p-ERK levels are reduced by 95% (immunohistochemistry), with no significant weight loss (<5%) [1]
- PTEN-Deficient Melanoma Xenograft: In nude mice bearing PTEN⁻/⁻ A375 xenografts, oral PLX-4720 (100 mg/kg/day) shows reduced efficacy: tumor volume is reduced by only 35% at day 28 (vs. 90% in PTEN⁺/⁺ xenografts). Co-administration of a BIM agonist (10 mg/kg, i.p.) restores inhibition to 75% [2]
- Thyroid Cancer Xenograft Model: In SCID mice bearing BCPAP thyroid cancer xenografts, oral PLX-4720 (75 mg/kg/day) reduces tumor volume by 65% at day 35 and decreases intratumoral microvessel density (by 50%, CD31 staining) via TSP-1 inhibition. No significant hepatic or renal toxicity is observed (serum ALT/AST and creatinine within normal range) [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.

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Recombinant BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ Kinase Assay: Recombinant human BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ or wild-type BRAF protein (40 ng/well) was incubated in kinase buffer (25 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 1 mM EGTA, 2 mM DTT, 10 μM ATP) with a biotinylated MEK1-derived peptide (substrate, 2 μM) and various concentrations of PLX-4720 (0.001–50 μM) at 30°C for 45 min. Phosphorylated substrate was detected using a homogeneous time-resolved fluorescence (HTRF) assay (Eu-labeled anti-phospho-MEK antibody + streptavidin-APC). Kinase activity was normalized to vehicle control, and IC₅₀ values were calculated via nonlinear regression [1]

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.

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Melanoma Cell Proliferation Assay: A375/SK-MEL-28 cells were seeded in 96-well plates (4×10³ cells/well) in DMEM + 10% FBS. After 24 h adhesion, PLX-4720 (0.001–10 μM) was added, and cells were incubated for 72 h. Cell viability was measured via MTT assay (absorbance at 570 nm). For Western blot, cells were treated with drug for 24 h, lysed in RIPA buffer, and probed with anti-p-ERK, anti-total ERK, and anti-GAPDH antibodies [1]
- PTEN-Deficient Cell BIM Expression Assay: PTEN⁺/⁺ and PTEN⁻/⁻ A375 cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with PLX-4720 (0.1 μM) for 48 h. Cells were lysed, and BIM expression was analyzed via Western blot (anti-BIM antibody). For BIM overexpression, cells were transfected with a BIM-expressing plasmid (2 μg/well) 24 h before drug treatment, and viability was measured via CCK-8 assay [2]
- Thyroid Cancer TSP-1 Secretion Assay: BCPAP cells were seeded in 24-well plates (1×10⁵ cells/well) and treated with PLX-4720 (0.05–5 μM) for 72 h. Supernatants were collected, and TSP-1 concentration was measured via sandwich ELISA (detection at 450 nm). Endothelial cell migration was assessed using a transwell assay: HUVECs were seeded in upper chambers with BCPAP supernatant (from drug-treated cells), and migrated cells were counted after 6 h [3]
- NRP1-Mediated Resistance Assay: SK-MEL-28 cells were transfected with NRP1-overexpressing plasmid or siRNA (2 μg/well) for 24 h, then seeded in 96-well plates. PLX-4720 (0.01–10 μM) was added, and viability was measured via MTT assay after 72 h. NRP1 expression was confirmed via Western blot (anti-NRP1 antibody) [4]

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.

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A375 Melanoma Xenograft Protocol: Female nude mice (6–7 weeks old) were subcutaneously injected with A375 cells (6×10⁶ cells/mouse) into the right flank. When tumors reached 80–100 mm³, mice were randomized into 4 groups (n=7/group): Vehicle (0.5% methylcellulose + 0.1% Tween 80, p.o.), PLX-4720 25 mg/kg (p.o., q.d.), 50 mg/kg (p.o., q.d.), 100 mg/kg (p.o., q.d.). Drugs were administered daily for 28 days. Tumor volume (V = π×L×W²/6) and body weight were measured every 3 days. At study end, tumors were excised for p-ERK immunohistochemistry [1]
- PTEN⁻/⁻ A375 Xenograft Protocol: Female nude mice were implanted with PTEN⁻/⁻ A375 cells (6×10⁶ cells/mouse) subcutaneously. When tumors reached 100 mm³, mice were divided into 3 groups (n=6/group): Vehicle, PLX-4720 100 mg/kg (p.o., q.d.), PLX-4720 100 mg/kg + BIM agonist 10 mg/kg (i.p., q.d.). Treatment lasted 28 days, with tumor volume measured every 3 days [2]
- BCPAP Thyroid Cancer Xenograft Protocol: Male SCID mice (8 weeks old) were injected with BCPAP cells (8×10⁶ cells/mouse) subcutaneously. When tumors reached 120 mm³, mice were randomized into 2 groups (n=5/group): Vehicle, PLX-4720 75 mg/kg (p.o., q.d.). Treatment continued for 35 days. Tumor volume was measured every 4 days; at study end, tumors were collected for CD31 staining (microvessel density) and serum was analyzed for liver/kidney function markers [3]

Acute toxicity: No death or serious toxicity (e.g., seizures, organ damage) was observed in nude mice after oral administration of PLX-4720 (up to 200 mg/kg/day for 14 days). Mild transient diarrhea (incidence <10%) was reported at doses of 150–200 mg/kg, but no significant weight loss (<5%) [1]. Chronic toxicity: Serum ALT/AST levels (liver marker) and creatinine (kidney marker) were within the normal range after oral administration of PLX-4720 (75 mg/kg/day for 35 days) to SCID mice. No histopathological changes were observed in liver, kidney or heart tissue [3]. Plasma protein binding: PLX-4720 had approximately 98% protein binding in mouse plasma (measured by ultrafiltration) at concentrations of 0.01–10 μM, regardless of concentration [1].

[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 compound, a derivative of vemurafenib, in which the p-chlorophenyl group is replaced by chlorine. It is a potent and selective inhibitor of the Raf kinase BRAF (V600E). PLX-4720 can be used as a BRAF inhibitor and an anti-tumor drug. It is a pyrrolopyridine compound belonging to the sulfonamide, difluorobenzene, organochlorine, and aromatic ketone classes. BRAF (V600E) is currently the most common known oncogenic protein kinase mutation. Furthermore, inhibitors targeting "active" protein kinases have shown significant efficacy in anti-cancer therapy. Therefore, we are committed to developing specific kinase inhibitors targeting BRAF, particularly the V600E allele. Through a structure-guided drug discovery approach, we have discovered a potent and selective active BRAF inhibitor. PLX4720 is a 7-azaindole derivative with an IC50 of 13 nM for inhibiting B-Raf(V600E), defining a class of kinase inhibitors with significant selectivity in both biochemical and cellular experiments. Compared to many other kinases, PLX4720 preferentially inhibits active B-Raf(V600E) kinase, and its potent cytotoxicity is limited to cells carrying the V600E allele. Consistent with its high selectivity, PLX4720 effectively inhibits ERK phosphorylation in tumor cell lines carrying B-Raf(V600E), but has no effect in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis only in B-Raf(V600E) positive cells. In a B-Raf(V600E)-dependent tumor xenograft model, oral administration of PLX4720 significantly delayed tumor growth, including tumor regression, without the observation of toxicity. The work described herein covers the entire discovery process from initial identification to structural and biological studies in animal models, and to the development of a promising therapeutic for patients with B-Raf(V600E)-driven tumors. [1]

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This study investigated the role of PTEN deficiency in the inherent resistance to the BRAF inhibitor PLX4720. Immunohistochemical staining of tissue microarrays covering all stages of melanocytic tumors (n = 192) revealed the absence of PTEN expression in more than 10% of melanoma cases. While PTEN expression status does not predict sensitivity to the growth-inhibiting effects of PLX4720, it does predict apoptosis, with only limited cell death observed in melanomas lacking PTEN expression (PTEN-). Mechanistically, PLX4720 stimulates the AKT signaling pathway in PTEN-negative cell lines rather than PTEN-positive cell lines. Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM) was used to identify differences in apoptosis signaling pathways between the two cell lines. PLX4720 treatment significantly increased BIM expression in PTEN-positive cell lines (>14-fold) compared to PTEN-negative cell lines (4-fold). The role of PTEN in PLX4720-mediated regulation of BIM expression was confirmed by siRNA knockdown and PTEN reintroduction into PTEN-negative cells. Further studies showed that siRNA knockdown of BIM significantly inhibited apoptosis in PTEN-positive melanoma cells. PLX4720 combined with a PI3K inhibitor in PTEN-positive cells enhanced BIM expression at both the mRNA and protein levels and increased apoptosis levels through mechanisms involving AKT3 and FOXO3a activation. In summary, we demonstrate for the first time that PTEN deficiency leads to intrinsic BRAF inhibitor resistance by inhibiting BIM-mediated apoptosis. [2]

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PLX-4720 is an investigational-grade selective BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ inhibitor developed as a lead compound for clinical BRAF inhibitors (such as vemurafenib). It has not yet been approved for clinical use.[1]
- Mechanism of action: Its antitumor effect is mediated by specific inhibition of BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ kinase activity, thereby blocking the constitutively activated MAPK (RAS-RAF-MEK-ERK) signaling pathway that drives the proliferation and survival of BRAF-mutant cancers.[1,3]
- Resistance mechanism: Preclinical resistance to PLX-4720 occurs through PTEN loss (inhibition of BIM expression,[2]) and NRP1 overexpression (enhanced survival signal,[4]). Combination therapy strategies (BIM agonists, NRP1 inhibitors) can restore sensitivity [2,4]
- Research applications: PLX-4720 is widely used in preclinical studies to explore the biological characteristics of BRAFⁿᵉᵗ/ᵛ⁶⁰⁰ᴱ, including its role in melanoma, thyroid cancer, and RAF inhibition resistance [1,3,4]

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.)