BRAF(V600E) (IC50 = 3.8 nM); BRAF (IC50 = 14 nM); CRAF (IC50 = 23 nM)
BRAF kinase (inhibitor). [3]

PLX8394 is a next-generation, orally available small-molecule BRAFi that inhibits both monomeric and dimeric BRAFV600 and BRAFnon-V600 protein signaling and does not cause the RAF/MEK/ERK paradoxical activation[1].
Unlike first-generation BRAFi, PLX8394 [a] does not induce paradoxical activation of the RAF/MEK/ERK pathway in cells with stimulated RAS signaling, and [b] blocks signaling from both monomeric BRAFV600 and dimeric BRAFnon-V600 mutations.[1]
PLX8394, a new generation BRAF inhibitor, selectively inhibits BRAF in colonic adenocarcinoma cells and prevents paradoxical MAPK pathway activation. [3]
PLX8394 overcame RAF inhibitor resistance in BRAF fusions characterizing paediatric astrocytomas28 and maintained activity against cells that are vemurafenib-resistant through secondary mutation in NRAS. [2]

---

In the BRAF V600E-mutant melanoma cell line LM-MEL-64, treatment with PLX8394 at 1 µM resulted in strong MAPK pathway inhibition, demonstrated by an 80.3% ± 2.4% reduction in phosphorylated ERK (pERK) levels compared to control. In the BRAF wild-type (wt)/RAS wt melanoma cell line LM-MEL-39, PLX8394 caused little or no change in pERK levels. [3]
In BRAF wt/KRAS G12D-mutant colorectal cancer (CRC) cell lines (LM-COL-1, ALA, LS513, HCT 116), treatment with 1 µM PLX8394 had minimal effect on the phosphorylation levels of MEK1/2 (pMEK) and ERK1/2 (pERK), in contrast to the first-generation BRAF inhibitor vemurafenib which caused a paradoxical increase (e.g., pERK increased by 160.2% ± 18.0% in LM-COL-1). [3]
In BRAF V600E-mutant/KRAS wt CRC cell lines (LIM2405, COLO 201), treatment with PLX8394 decreased pMEK and pERK levels, similar to the effect of vemurafenib. [3]
In functional proliferation assays, treatment of BRAF wt/KRAS G12D CRC cell lines (ALA, LS513, LM-COL-1, HCT 116) with PLX8394 across a range of concentrations (0.1, 0.5, 1 µM) for 72 hours did not enhance cell proliferation. This was in contrast to vemurafenib, which stimulated proliferation in these cell lines. [3]
In BRAF V600E-mutant CRC cell lines (LIM2405, COLO 201), treatment with PLX8394 inhibited cell proliferation in a dose-dependent manner over 72 hours. [3]

PLX8394 is a small molecule BRAF inhibitor of the newest generation that can be taken orally and has antineoplastic potential.
Of 26 patients who received at least 1 dose of PLX8394, 15 patients (10 with BRAF mutation) were enrolled to cohorts with PLX8394 monotherapy: 450mg BID (n=8), 900mg BID (n=3), 450mg TID (n=4). PK studies demonstrated lower than expected drug exposures; the protocol was amended and 11 additional patients (8 with BRAF mutation) were enrolled in cohorts with PLX8394 coadministered with cobicistat: 450mg BID (n=5) and 900mg BID (n=6). As of 31 May 2017 cutoff, 15 subjects terminated the study: 14 for disease progression, 1 subject withdrawal. Duration of study drug exposure was a median of 56 (22 - 727) days. Reversible Grade (G) 3 transaminitis in a patient treated with PLX8394 900mg BID + cobicistat was the only dose-limiting toxicity (DLT); rechallenge with PLX8394 alone was tolerated without recurrence of transaminitis. PLX8394 900mg BID + cobicistat was declared as RP2D. Treatment-related G>3 AEs other than DLT included G3 diarrhea (n=1), G3 anorexia (n=1), both in PLX8394 monotherapy cohorts and both manageable with supportive care. There were no cutaneous or ocular AEs. Cobicistat coadministration resulted in a 2-3 fold increase in PLX8394 systemic exposure, with RP2D achieving the projected efficacious exposure range based on preclinical models. There were no objective responses in PLX8394 monotherapy cohorts; however, to date 2 (28%) of 7 evaluable BRAF mutant patients treated with PLX8394 + cobicistat achieved partial responses (BRAFV600E-mutated colorectal cancer, 42% and BRAFV600E-mutated glioma, 65%; both BRAFi naive), which were confirmed by 8-wk serial scans. Tumors without BRAF mutation showed no response. Conclusion: PLX8394 with cobicistat was very well tolerated and showed promising activity in refractory solid tumors with BRAF mutation. A phase II study in BRAF-mutated cancers is ongoing. [1]

---

To construct therapeutic strategies for treating cancers harboring LLRins506 or VLRins506 mutation, we next determined the efficacies of RAF inhibitors (vemurafenib, dabrafenib, and PLX8394) and MEK inhibitor (trametinib) against fibroblastomas induced by LLRins506 or VLRins506 mutant. Although both RAF inhibitors and MEK inhibitor impaired the growth of BRAF(V600E)–induced fibroblastomas by different extends (trametinib > dabrafenib > vemurafenib > PLX8394), only MEK inhibitor exhibited a strong inhibitory effect on that of LLRins506/VLRins506 mutant–induced fibroblastomas (Fig. 6, D to F). Consistent with this finding, our immunohistochemical staining revealed that administrating with MEK inhibitor, trametinib, but not with RAF inhibitors significantly decreased the level of phospho-ERK1/2 and Ki67 in LLRins506/VLRins506 mutant–induced fibroblastomas (Fig. 6G). Together, these data demonstrated that LLRins506 and VLRins506 are real driver mutations in cancer genomes and cancers harboring this type of mutations could be treated effectively with MEK inhibitor, trametinib.[4]

---

In a xenograft tumor mouse model using NOD-SCID mice injected with fibroblasts expressing BRAF(V600E), oral administration of Plixorafenib (PLX8394) (150 mg/kg daily) impaired tumor growth, although its efficacy was less than that of dabrafenib, vemurafenib, and trametinib. [4]
In contrast, xenograft tumors induced by fibroblasts expressing LLRins306 or VLRins306 BRAF mutants were resistant to treatment with Plixorafenib (PLX8394) (150 mg/kg daily), showing no significant reduction in tumor growth. [4]

For in vitro kinase assays of BRAF mutants, the immunoprecipitants were washed once with kinase reaction buffer (25 mM Hepes, 10 mM MgCl2, 0.5 mM Na3VO4, 0.5 mM dithiothreitol, pH 7.4) and then incubated with 20-μl kinase reaction mixture [2-μg His-tagged MEK1(K97A) and 100 μM adenosine 5′-triphosphate in 20-μl kinase reaction buffer] per sample at room temperature for 10 min. Kinase reaction was stopped by adding 5 μl per sample of 5× Laemmli sample buffer and determined by immunoblotting. Otherwise, the immunoprecipitants were directly mixed with 2× Laemmli sample buffer before detection. The immunoblotting was carried out as described before [4].

---

MST analysis [4]
The affinity of BRAF(V600E) and LLRins506 mutants with different RAF inhibitors (vemurafenib, dabrafenib, and PLX8394) was measured using the Monolith NT.115 from NanoTemper Technologies. The MST analysis was carried out as described before. Briefly, BRAF mutants purified from 293T transfectants were labeled with a fluorescent dye NT-647 (Cysteine Reactive) according to the manufacturer’s protocol. Then, a series of protein solutions with different concentrations were prepared by consecutive twofold dilutions in buffer containing 25 mM Hepes (pH 7.5), 150 mM NaCl, 0.2% IGEPAL, and 0.1 mM Tris(2-carboxyethyl)Phosphine (TCEP). The labeled proteins were mixed with unlabeled drugs at a volume ratio of 1:1 and loaded into silica capillaries after a short incubation at room temperature. The measurements were performed at 25°C by using 20% light-emitting diode power and 40% MST power. The laser-on and laser-off intervals were 30 and 5 s, respectively. NanoTemper Analysis (x86) software was used to fit the data and to determine the apparent dissociation constant values.

Cellular proliferation assay [3]
Cellular proliferation assay of colon cancer cell lines was performed on Falcon® 96 well clear plates. Cells were seeded at 3000 cells per well. Cells were incubated with the inhibitors vemurafenib or PLX8394 at concentrations of 0 µM (DMSO), 0.1 µM, 0.5 µM, and 1 µM. Proliferation assays were performed using the MTS-based CellTiter 96® AQueous One Solution Cell Proliferation Assay. Briefly, cells were incubated for 1 hour in diluted CellTiter mixture (1:5 dilution in growth media) prior to measurement of absorbance at 490nm wavelength and subtraction of background as measured from no-cell control wells. Readings were performed at day 1 before treatment and after 72 hours (3 days) of inhibitor treatment. Absorbance were normalised within cell lines to untreated controls. [3]

---

Western Blot Analysis of MAPK Pathway: Cells were treated with DMSO (control), 1 µM vemurafenib, or 1 µM PLX8394 for 6 hours. Following treatment, cells were lysed, and proteins were extracted. Western blotting was performed to detect total and phosphorylated forms of MEK1/2 and ERK1/2. Signal intensity was quantified by densitometry, and protein levels were expressed relative to the DMSO-treated control. [3]
Cell Proliferation Assay: Cells were treated with BRAF inhibitors at concentrations of 0 (DMSO control), 0.1, 0.5, and 1 µM for 72 hours. After the treatment period, relative cell numbers were measured and normalized to the DMSO-treated control. The effect on proliferation was expressed as a percentage change relative to control. [3]

For xenograft experiments, female NOD-SCID mice (6 to 8 weeks) were subcutaneously injected with 5 × 106 cells per mice in 1:1 Matrigel. Tumor volumes were monitored by digital calipers twice a week and calculated using the formula: volume = (width)2 × length/2. Vemurafenib (120 mg/kg), dabrafenib (200 mg/kg), PLX8394 (150 mg/kg), and trametinib (3 mg/kg) were administered orally and daily when tumors reached an average volume of ~100 mm3 according to the previous studies. At the experiment endpoint, mice were euthanized, and tumors were harvested for ex vivo analysis and subsequent histology. All operations were approved by the Animal Ethics Committee of National Cancer Centre Singapore (NCCS) with Institutional Animal Care and Use Committee (IACUC)–approved animal protocol. [4]

---

Trial protocol PLX120-03 (NCT02428712) incorporates an open-label, multicenter, phase I study using a 3+3 design to determine the recommended phase II dose (RP2D) in patients with refractory or relapsed solid tumors with or without BRAF mutation. Other endpoints are safety, including dermatologic and ophthalmic adverse events (AEs), pharmacokinetics (PK), and response per RECIST1.1. Dose escalation includes cohorts with either PLX8394 monotherapy or PLX8394 coadministered with an oral CYP3A4 inhibitor, cobicistat 150mg, to enhance systemic PK. Enrollment at the RP2D continues in the subsequent phase II study.[1]

---

For in vivo xenograft studies, female NOD-SCID mice (6-8 weeks old) were subcutaneously injected with 5×10^6 immortalized fibroblasts (reconstituted BRAF-/- fibroblasts expressing specific BRAF mutants) suspended in a 1:1 mixture with Matrigel. Tumor volume was monitored twice weekly using digital calipers and calculated using the formula: volume = (width)^2 × length / 2. When the average tumor volume reached approximately 100 mm³, mice were randomly assigned to treatment groups. Plixorafenib (PLX8394) was administered orally at a dose of 150 mg/kg, daily. The control group received vehicle only. Treatment continued until the experimental endpoint, after which mice were euthanized, and tumors were harvested for weight measurement and histopathological analysis (e.g., immunohistochemistry for phospho-ERK1/2 and Ki67). [4]

[1]. Mol Cancer Ther (2018) 17 (1_Supplement): B176.

[2]. Nature . 2015 Oct 22;526(7574):583-6.

[3]. Mol Cancer . 2017 Jun 28;16(1):112.

[4]. Sci Adv . 2021 Jun 9;7(24):eabg0390.

PLX8394 is currently undergoing clinical trial NCT02428712 (a study of PLX8394 monotherapy in patients with advanced unresectable solid tumors). Prisorafenib is an orally bioavailable serine/threonine protein kinase B-raf (BRAF) protein inhibitor and specific dimer disruptor with potential antitumor activity. After oral administration, prisorafenib selectively binds to and inhibits the activity of dimeric BRAF mutants (including BRAF fusions and splice variants) and BRAFV600 monomers without affecting RAF function in normal cells. This inhibits the proliferation of tumor cells expressing these BRAF mutants. BRAF is a member of the raf family of serine/threonine protein kinases and plays a role in the regulation of mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) signaling pathways; mutations in the BRAF gene can lead to persistent activation of these pathways. Mutated and fusion forms of BRAF are associated with a variety of neoplastic diseases. BRAF-induced oncogenic activation promotes tumor growth by persistently enhancing RAS-independent mitogen-activated protein kinase (MAPK) pathway signaling. Therefore, RAF inhibitors have significantly improved personalized treatment for metastatic melanoma. However, these targeted therapies have also revealed an unintended consequence: stimulating the growth of certain cancers. Structurally diverse ATP-competitive RAF inhibitors can either inhibit or anomalously activate the MAPK pathway, depending on whether the activation is caused by BRAF mutations or upstream events such as RAS mutations or receptor tyrosine kinase activation. Here we identify a new generation of RAF inhibitors (referred to as “paradox breakers”) that inhibit BRAF-mutant cells without activating the MAPK pathway in cells carrying upstream activation. In cells expressing the same HRAS mutations common in squamous cell carcinoma patients treated with RAF inhibitors, the first-generation RAF inhibitor vemurafenib stimulated cell growth and induced the expression of MAPK pathway-responsive genes both in vitro and in vivo; in contrast, the “paradox breakers” PLX7904 and PLX8394 did not exhibit this effect. Paradox breakers can also overcome several known mechanisms of resistance to first-generation RAF inhibitors. Separating MAPK pathway inhibition from paradoxical activation may offer greater safety and more durable efficacy than first-generation RAF inhibitors, a concept currently being evaluated in human clinical trials using PLX8394. [2]

---

BRAF inhibitors (BRAFi) are the standard of care for BRAF V600 mutation-driven metastatic melanoma, but may lead to paradoxical activation of the mitogen-activated protein kinase (MAPK) signaling pathway. This may promote the development of precancerous lesions and secondary tumors, primarily (but not limited to) associated with pre-existing mutations in the RAS gene. We previously reported a case of a patient with both BRAF-mutant metastatic melanoma and BRAF wild-type/KRAS G12D metastatic colorectal cancer (CRC) who experienced CRC recurrence and progression after treatment with the BRAF inhibitor dabrafenib (GSK2118436). We constructed the cell line LM-COL-1 using resected colorectal cancer metastases, which directly and reliably mimics clinical conditions, including paradoxical activation of the MAPK signaling pathway following dabrafenib treatment, leading to increased cell proliferation. Novel BRAF inhibitors (PLX8394 and PLX7904) are called "paradox breakers" and are designed to inhibit V600-mutant oncogenic BRAFs while avoiding paradoxical activation of the MAPK pathway. In this study, we used the LM-COL-1 model and several other colorectal cancer cell lines with different mutation backgrounds to demonstrate that the paradox breaker PLX8394 can target and inhibit mutant BRAF V600 without paradoxically promoting the MAPK signaling pathway. [3] Despite the encouraging results achieved in cancer treatment using RAF inhibitors targeting BRAF mutants, resistance remains a huge challenge, and the underlying molecular mechanisms have not been fully elucidated. This paper characterizes a group of previously unknown oncogenic BRAF mutants with in-frame insertions (LLRins506 or VLRins506) in the αC-β4 loop. Through structural modeling and molecular dynamics simulations, we found that these insertions form a large hydrophobic network that can stabilize the R-spine domain, thereby activating the catalytic activity of BRAF. In addition, these insertions disrupt the BRAF dimer interface and inhibit dimerization. Unlike BRAF (V600E), these BRAF mutants with low dimer affinity exhibit strong resistance to all RAF inhibitors in clinical or clinical trials, which is due to their stable R-spine domain. As predicted by molecular docking, the stable R-spine domain in other BRAF mutants also confers resistance. Our data collectively suggest that the stability of R-spine, rather than dimer affinity, determines the resistance of oncogenic BRAF mutants to RAF inhibitors. [4]

---

PLX8394 is a new generation BRAF inhibitor, known as the "paradox breaker". Its development aims to inhibit oncogenic BRAF V600 mutations while avoiding paradoxical activation of the MAPK signaling pathway—a common side effect of first-generation BRAF inhibitors such as vemurafenib and dabrafenib. This paradoxical activation promotes the growth of cells with pre-existing RAS mutations. [3]
At the time of this study, PLX8394 was undergoing a clinical trial (NCT02428712) for unresectable solid tumors. [3]

C25H21F3N6O3S

542.5329

542.134

C, 55.35; H, 3.90; F, 10.51; N, 15.49; O, 8.85; S, 5.91

1393466-87-9

1393466-87-9 (PLX8394); 1652573-86-8 (PLX7683, a paradox breaker); 918505-61-0 (Vemurafenib/ PLX4032 analog)

90116675

White to off-white solid powder

1.6±0.1 g/cm3

786.8±70.0 °C at 760 mmHg

429.6±35.7 °C

0.0±2.7 mmHg at 25°C

1.703

0.97

2

11

7

38

976

1

S(N([H])C1C([H])=C([H])C(=C(C(C2=C([H])N([H])C3=C2C([H])=C(C([H])=N3)C2C([H])=NC(C3([H])C([H])([H])C3([H])[H])=NC=2[H])=O)C=1F)F)(N1C([H])([H])C([H])([H])[C@]([H])(C1([H])[H])F)(=O)=O

YYACLQUDUDXAPA-MRXNPFEDSA-N

InChI=1S/C25H21F3N6O3S/c26-16-5-6-34(12-16)38(36,37)33-20-4-3-19(27)21(22(20)28)23(35)18-11-32-25-17(18)7-14(8-31-25)15-9-29-24(30-10-15)13-1-2-13/h3-4,7-11,13,16,33H,1-2,5-6,12H2,(H,31,32)/t16-/m1/s1

(3R)-N-[3-[5-(2-cyclopropylpyrimidin-5-yl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl]-2,4-difluorophenyl]-3-fluoropyrrolidine-1-sulfonamide

PLX-8394; PLX 8394; PLX8394; 1393466-87-9; Plixorafenib; (R)-N-(3-(5-(2-cyclopropylpyrimidin-5-yl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluorophenyl)-3-fluoropyrrolidine-1-sulfonamide; UNII-J2L7Z273SG; FORE8394; PLX8394

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)

DMSO: ~100 mg/mL (~184.3 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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