Raf kinase

MLN2480 inhibits MAPK pathway signaling at concentrations that are tolerated in vivo in BRAF mutant and some RAS mutant preclinical cancer models[1].
At very low concentrations, it is found to activate phosphorylated MEK, whereas at higher concentrations, it inhibits this same activity. It has been discovered that MLN-2480's inhibitory effects differ between models and genetic contexts[2].
In vitro testing of the drug combination of MLN2480 and TAK-733 (an investigational allosteric MEK kinase inhibitor) in cell proliferation assays shows synergistic activity. Additionally, western blot analysis shows how MLN2480 reverses the feedback activation of MEK in response to TAK-733, resulting in more concerted MAPK pathway inhibition. PRAK is only weakly inhibited by MLN-2480 [1][2].

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Potency of Tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) and naporafenib across RAF isoforms [7]
To better understand the RAF selectivity of tovorafenib and naporafenib, we measured their inhibition of the RAF complexes described above using our adapted TR-FRET assay. BRAFWT, BRAFV600E, and CRAFSSDD were assayed at a concentration of 1 nM, while CRAFWT and ARAFSSDD were assayed at 4 nM and 10 nM, respectively, due to their lower enzymatic activities. An ATP concentration of 200 μM was used for all assays, and the WT MEK1 substrate concentration was 250 nM. Measured IC50 values and calculated Ki values are provided in Table 1, and representative concentration-response curves from which they were derived are shown in Figure 2. [7]
Both Tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) and naporafenib were most potent as inhibitors of CRAF, with IC50 values of 94.2 nM and 3.7 nM, respectively, against the WT CRAF kinase. Potency against the CRAFSSDD mutant was essentially the same as for CRAFWT (Table 1). Both agents exhibited intermediate potency against BRAFWT and BRAFV600E (633 nM for tovorafenib and 13.4 nM for naporafenib on BRAFWT) and were much weaker inhibitors of ARAFSSDD. Tovorafenib did not completely inhibit ARAFSSDD even at 10 μM, the highest inhibitor concentration we could achieve in this assay. While tovorafenib and naporafenib share potency trends across the RAF isoforms, naporafenib is consistently more potent than tovorafenib against each enzyme by at least an order of magnitude. A prior study of naporafenib activity against purified ARAF, BRAF, and CRAF reported relative potencies similar to those we observe but with markedly lower IC50 values (0.07 nM for CRAF) (42). Reaction conditions were not provided for this study, precluding meaningful comparison with our results. [7]
The very steep concentration-response curves for Tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) and naporafenib against CRAF and WT BRAF suggest positive cooperativity of inhibition of these RAF dimers (Fig. 2). Fitting of these curves with a four-parameter model to allow for a variable Hill slope resulted in Hill slopes ranging from −2.6 to −3.2 (Table 1). These values indicate that tovorafenib and naporafenib inhibit BRAF and CRAF dimers with marked positive cooperativity; that is, that binding of inhibitor to the active site of one protomer increases the affinity for inhibitor binding to the second protomer in the RAF dimer. We did not observe this effect with either ARAFSSDD or with BRAFV600E, which is monomeric in this assay (Table 1).

MLN2480 exhibits antitumor activity in vivo in xenograft models for pancreatic, lung, colon, and melanoma cancer[3].
MLN-2480 (37.5 mg/kg) in a tumor xenograft model is tolerable. An SK-MEL-30 xenograft model benefits from the combination of MLN-2480 (12.5 mg) and TAK-733 (1 mg/kg), but neither drug by itself has much of an impact[2].

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The dose expansion phase provided a preliminary indication of Tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) efficacy. Partial responses were seen in 8 (50%) of 16 patients in the BRAF mutation-positive, RAF and MEK inhibitor-naïve cohort who received the Q2D RP2D. This level of monotherapy activity is in line with that seen in phase 1 studies of first-generation agents in a similar setting. The PK analyses showed that Tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) has a moderately fast absorption rate, with an overall median Tmax of 2–4 h post-dose. Overall mean accumulation following 21 days of Q2D dosing was 2.5-fold. By contrast, QW dose administration was associated with minimal to no apparent accumulation of tovorafenib in systemic circulation in the dose range of 400 mg to 800 mg. Steady-state AUC increased in an approximately dose-proportional manner for both Q2D and QW dose ranges tested. The plasma terminal half-life (t1/2) of tovorafenib was approximately 70 h.[6]

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The combination of MLN2480 with TAK-733 inhibits the growth of a broader range of RAS mutant tumor models than single agent MLN2480, including primary human tumor xenograft models of melanoma and CRC. In vitro analysis of this drug combination in cell proliferation assays demonstrates synergistic activity. Western blot analysis demonstrated the effect of MLN2480 in reversing feedback activation of MEK in response to TAK-733, leading to more concerted MAPK pathway inhibition. [2]

Kinase inhibition assays [7]
Inhibition assays were performed using a modified HTRF KinEASE tyrosine kinase assay kit. Rather than the provided kit substrate, we purified MEK135-393 and biotinylated it (MEK-B) in-house using birA enzyme. Inhibitors were dispensed into black 384-well plates using an HP300e dispenser and normalized to 1% final DMSO concentration per well. Kit assay buffer was supplemented with purified RAF at a final concentration of 1 nM for MEK1SASA:BRAFKD:14-3-3 and MEK1SASA:CRAFSSDD:14-3-3, 4 nM for MEK1:CRAFKD:14-3-3, and 10 nM for MEK1SASA:ARAFSSDD-14-3-3 𝜀, as well as purified biotinylated MEK-B at a final concentration of 250 nM. Supplemented kinase buffer was dispensed into 384-well plates using a Multidrop combi dispenser and incubated with inhibitors at room temperature for 40 min before reactions were initiated by 200 uM ATP dispensed using the Multidrop combi dispenser. Plates were quenched after 30 min at room temperature using the kit detection buffer supplemented with XL665 and PAb Anti-phospho MEK1/2-Eu. The FRET signal ratio was measured at 665 and 620 nm using a PHERAstar microplate reader and processed using GraphPad Prism fit to a three-parameter dose-response model with Hill Slope constrained to −1 and a four-parameter dose-response model that fits the Hill Slope to the data. Assays were performed in triplicate three independent times.

In vitro, MLN-2480 is effective against both wild-type and B-raf Val600Glu. At very low concentrations, MLN-2480 is found to activate phosphorylated MEK, but at higher concentrations, it inhibits this same activity. High concentrations of MLN-2480 block the signaling pathway in the human malignant melanoma A-375 mutant B-raf Val600Glu cell line. MLN-2480's inhibitory effects are found to vary depending on the model and genetic context; it only mildly inhibits PRAK. High levels of apoptotic biomarkers were seen when MLN-2480 and TAK-733 were combined in NRAS mutant human malignant melanoma cell lines (SK-MEL-2).

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Combination effects of Tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) and TAK-733 on cell viability were studied using an ATP-based cell viability assay across a panel of BRAF and RAS mutant melanoma and CRC cell lines. Western blot analysis was used to compare effects on MAPK pathway signaling and response markers in cell lines showing a range of sensitivity to this combination. [2]

C57BL/6J mice
\n12.5 mg/kg
\noral gavage

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\n\nTovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) was administered orally (tablet formulation), with patients fasting (except for water) for at least 2 h before and at least 2 h after taking their dose. Treatment was to be continued until disease progression, unacceptable toxicity, or the patient discontinued for any other reason, for a maximum duration of 12 months. Treatment could be continued beyond 12 months if it was determined that a patient would derive benefit from such continued therapy.\n
\nIn the dose escalation phase, a 3 + 3 design was used to evaluate Tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) administered with continuous dosing on Q2D and QW dosing regimens. Prior to the initiation of QW dose escalation, the initial cycle length of 22 days was changed to 28 days by protocol amendment to improve clinical feasibility and better facilitate future combination studies. Patients enrolled prior to this protocol amendment in an ongoing Q2D dose escalation cohort continued on the 22-day cycle schedule until the cohort was full and all patients had been evaluated for dose-limiting toxicity (DLT). For both Q2D and QW regimens, dose escalation progressed according to the incidence of DLT in the first treatment cycle (either 22 days or 28 days). DLTs were defined as: grade 4 neutropenia lasting ≥ 7 consecutive days; febrile neutropenia (defined as an ANC ≤ 1000 cells/μL and fever ≥ 38.5 °C) or documented infection ≥ grade 3 with ANC ≤ 1000 cells/μL; grade 4 thrombocytopenia (platelet count < 25,000/μL), tovorafenib-related thrombocytopenia requiring platelet transfusion, or tovorafenib-related bleeding requiring medical attention; treatment delays of ≥ 14 days due to any toxicity; ALT and AST toxicities (ALT or AST > 7.5 × ULN for greater than 14 days or ALT or AST > 7.5 × ULN accompanied by an elevation in total bilirubin of > 3 × ULN [not explained by obstruction] regardless of duration); nonhematological toxicity ≥ grade 3 (with the exception of: nausea, vomiting, and diarrhea except if they persisted at ≥ grade 3 for > 3 days despite adequate supportive care measures [at the investigator’s discretion, patients who experienced nausea, vomiting, or diarrhea after taking tovorafenib could receive antiemetic or antidiarrheal medication prior to subsequent doses]; isolated laboratory abnormalities ≥ grade 3 that resolved to ≤ grade 1 in ≤ 7 days without clinical sequelae or the need for therapeutic intervention; fatigue ≥ grade 3 for ≤ 7 days; development of keratoacanthomas or skin carcinoma unless unusually aggressive or metastatic), provided the site investigator considered such events were at least possibly related to study treatment. The MTD was defined as the highest dose level that generated DLT in 0/3 or 1/6 patients. On a case-by-case basis, the sponsor in collaboration with the principal investigators determined if intrapatient dose escalation was appropriate. Patients who had any dose reductions were not permitted to dose escalate.
\n\nThe starting dose for the Q2D dose escalation phase was 20 mg, which was equivalent to one-tenth of the highest non-severely toxic dose (HNSTD) established in monkey toxicology studies. Dose escalation included planned dose levels of 40 mg, 80 mg, 135 mg, 200 mg, and 280 mg. Once the MTD and/or RP2D of Q2D tovorafenib was established, patients with melanoma were enrolled into 1 of 6 Q2D melanoma expansion cohorts (approximately 16 patients per cohort), based on tumor genotype and treatment history (Supplementary Table S1). In addition, a seventh Q2D cohort was to enroll sufficient patients (approximately 16) with any advanced solid tumor (excluding lymphoma) to ensure that 12 patients completed protocol-specified dosing and PK assessments scheduled during cycle 1.\n
\nThe study was initially designed to investigate a Q2D schedule. Subsequently, a protocol amendment introduced planned QW dose escalation cohorts. The alteration in the dosing regimen from Q2D to QW was expected to reduce drug accumulation and increase Cmax while maintaining similar steady-state AUC. In addition, it was hypothesized that the increased Cmax might lead to a higher degree of pathway inhibition for a window of time within the dosing interval, without compromising overall dose density. Planned QW doses to be administered on days 1, 8, 15, and 22 of a 28-day cycle were a starting dose of 400 mg, followed by dose level increases of 200 mg (i.e., doses of 600 mg, 800 mg, and 1000 mg) in each subsequent cohort until the MTD/RP2D was reached. Once the MTD and/or RP2D of Q2D Tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) was established, and following a further protocol amendment, a single expansion cohort of up to 16 patients with NRAS-mutated cutaneous melanoma, naïve to prior therapy with RAF and MEK inhibitors was enrolled. [6]

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\n\n\nSafety, pharmacokinetic and pharmacodynamic assessments[6]
\n\nAdverse events were coded using the Medical Dictionary for Regulatory Activities (MedDRA) Version 19.0 and were graded according to the National Cancer Institute (NCI) Common Terminology Criteria (CTC) for adverse events (CTCAE) (Version 4.03). The assessment period for treatment emergent adverse events (TEAEs) was from the first dose of study treatment to 30 days after the last dose of study medication, or until the start of subsequent antineoplastic therapy, whichever occurred first. Following baseline evaluation, response was assessed by investigators every two cycles by computed tomography or magnetic resonance imaging according to Response Evaluation Criteria in Solid Tumors (version 1.1).
\nSerial blood samples were collected before and after Tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) dosing on days 1 and 21 (Q2D dosing) or days 1 and 22 (QW dosing) of cycle 1 for plasma PK analysis. In addition, for patients on Q2D schedules, predose or trough samples were collected on days 9 and 15 (Q2D dosing) or days 8 and 15 (QW dosing) to evaluate time to steady state. A validated liquid chromatography coupled to tandem mass spectroscopy (LC–MS/MS) method was used to quantify plasma concentrations of tovorafenib [24]. The concentrations of tovorafenib were determined using a fully validated bioanalytical method (QPS 96-1116) with a lower limit of quantification at 0.5 ng/mL in plasma. This bioanalytical method used protein precipitation extraction of tovorafenib and its stable labeled internal standard from human plasma with positive ionization mode in mass spectrometry. Plasma concentration–time analysis was performed using noncompartmental analysis. The plasma PK parameters were estimated using a validated version of Phoenix WinNonlin software (Version 6.3 or above, Pharsight Corporation, Raleigh, NC). Terminal half-life was calculated based on the equation: t1/2 = ln2/kel (kel = elimination rate constant determined by linear regression analysis of selected time points in the apparent terminal phase of the log plasma concentration versus time curve).\n
\nBased on tissue availability, pharmacodynamic assays included assessment of pERK expression levels in paired biopsy samples (baseline and day 21) from patients in the melanoma dose expansion cohorts. The level of staining was assessed both by a pathologist (semi-quantitative measurements according to H-score assessment) and by quantitated image analysis.

Absorption
The steady-state maximum concentration (Cmax) of tovorafenib was 6.9 µg/mL (23%), and the area under the concentration-time curve (AUC) was 508 µgh/mL (31%). The time required to reach steady-state plasma concentrations of tovorafenib was 12 days (33%). Exposure to tovorafenib increased in a dose-dependent manner. The median time to peak plasma concentration (Tmax) of tovorafenib after a single dose of tablet or oral suspension was 3 hours (min, maxima) (1.5, 4 hours). Compared with the fasting state, there was no clinically significant difference in Cmax and AUC of tovorafenib when co-administered with a high-fat meal (approximately 859 calories, 54% fat), but Tmax was delayed to 6.5 hours.
Excretion Route
After a single oral dose of radiolabeled tovorafenib, 65% of the total radiolabeled dose is excreted in feces (8.6% unchanged) and 27% in urine (0.2% unchanged).
Volume of Distribution
The apparent volume of distribution of tovorafenib is 60 L/m² (23%). It crosses the blood-brain barrier.
Clearance
The apparent clearance is 0.7 L/h/m² (31%).
Metabolism/Metabolites
Tovorafenib is primarily metabolized in vitro by aldehyde oxidase and CYP2C8. CYP3A, CYP2C9, and CYP2C19 have weaker metabolic effects on tovorafenib.
Biological Half-Life
The terminal half-life of tovorafenib is approximately 56 hours (33%).

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Pharmacokinetics [6]
Figure 2 shows the mean (± standard deviation) plasma concentration-time curves of tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) divided by QW dose groups on days 1 and 22 of cycle 1; Table 4 summarizes the plasma pharmacokinetic parameters on day 22 of cycle 1 by dose group. After multiple oral doses of 600 mg once weekly (QW), the peak concentration of tovorafenib was reached on day 22 of cycle 1, with a median time to peak (Tmax) of 3 hours after administration (range 1–24 hours). After repeated QW administration, no significant drug accumulation was observed on day 22 AUC168 compared to day 1 AUC168. In 20 evaluable patients treated with 600 mg QW, the mean plasma terminal half-life (t1/2) of tovorafenib was approximately 70 hours (range 31–119 hours). The relationship between dose and tovorafenib exposure (AUC168) on day 22 of cycle 1 is shown in Supplementary Figure S1. Steady-state exposure increased approximately dose-proportional across the once-weekly (QW) dose range of 400 mg to 800 mg, with a 95% confidence interval of 1 (95% CI 0.55–2.04) and a coefficient of 1.30 for the power function model. Minimal drug accumulation was observed with the QW dosing regimen, with a geometric mean Rauc (accumulation ratio based on AUC0-last) ranging from 1.03 to 1.09. In the 200 mg every two days (Q2D) dosing regimen, the geometric mean Rauc was approximately 2.55. Similar pharmacokinetic analyses were performed on the Q2D dose groups (Supplementary Figures S1 and S2, and Supplementary Table S10). Steady-state AUC48 of tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) increased approximately dose-proportionately across the 20 mg to 280 mg Q2D dose range. While no significant drug accumulation was observed with the QW dosing regimen, the Q2D dosing regimen resulted in approximately a 2.5-fold accumulation of AUC48 at steady state.

The incidence of treatment-associated adverse events (TEAEs) and serious adverse events (SAEs) occurring during treatment is summarized in Supplementary Table S5, with the most common TEAEs listed in Table 2. Notably, only 1 out of 149 treated patients (< 1%; Q2D dose expansion cohort) reported cutaneous squamous cell carcinoma, which was classified as a TEAE. The incidence of drug-related TEAEs according to dosing regimen is summarized in Supplementary Table S6. The two most common adverse events during the dose expansion phase were maculopapular rash (36%) in the Q2D cohort and fatigue (42%) in the QW cohort. During the dose expansion phase, 68% of patients experienced grade 3 or higher TEAEs, with 73% in the Q2D cohort and 47% in the QW cohort. Grade 3 or higher TEAEs with an incidence ≥ 5% are listed in Supplementary Table S7. Overall, the two most common adverse events were anemia (14%) and maculopapular rash (8%). [6] In the every two days (Q2D) extended cohort, 33 out of 80 patients (41%) experienced grade 3 or higher treatment-related adverse events (TEAEs); the most common were maculopapular rash (9%) and anemia (8%). In the weekly (QW) extended cohort, 4 out of 20 patients (20%) experienced grade 3 or higher treatment-related adverse events (TEAEs); the most common was hyperbilirubinemia (10%). During the dose escalation phase, 2 out of 30 patients (7%) in the Q2D group (280 mg dose group) reported drug-related serious adverse events (SAEs) during treatment (one patient experienced grade 3 anemia, and the other experienced grade 4 dyspnea and grade 5 respiratory failure); 2 out of 20 patients (10%) in the QW group (800 mg dose group) reported drug-related serious adverse events (SAEs) during treatment (one patient experienced grade 3 rash, and the other experienced grade 3 hyperbilirubinemia). During the dose extension phase, 12 out of 80 patients (15%) in the Q2D group reported drug-related serious adverse events (SAEs) during treatment, including acute kidney injury, maculopapular rash, and maculata rash (2 patients each experienced grade 3 adverse events). In the once-weekly (QW) dose expansion cohort, 4 out of 19 patients (21%) experienced drug-related treatment-associated serious adverse events (SAEs), including one patient with grade 2 anemia and dyspnea, one patient with grade 2 nausea and grade 3 maculopapular rash, and one patient with grade 3 erythema multiforme and maculopapular rash, respectively. In the dose expansion phase, 15 out of 80 patients (19%) in the twice-daily (Q2D) cohort experienced treatment-associated adverse events (TEAEs) leading to permanent discontinuation of tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819). These adverse events included maculopapular rash and sepsis (2 cases each [3%]). In the weekly dosing group (QW group) during the dose expansion phase, 4 out of 19 patients (21%) permanently discontinued treatment due to treatment-specific adverse events (TEAEs), including atrial flutter, dyspnea, erythema multiforme, and fatigue (1 case each). During the dose expansion phase, 19 out of 99 patients (19%) reduced their dose due to TEAEs, including 17 out of 80 patients (21%) in the every-two-day dosing group (Q2D group) and 2 out of 19 patients (11%) in the weekly dosing group (QW group). The most common adverse events were maculopapular rash (5 out of 99 patients, 5%) and generalized rash (3 out of 3%). A total of 13 deaths occurred during the study period. Fatal serious adverse events (SAEs) associated with these deaths were primarily related to underlying diseases or their complications, as detailed in Supplementary Table S8. Only one death was associated with respiratory failure in a patient in the 280 mg Q2D dose escalation cohort, which the researchers considered treatment-related. [6]

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Protein binding
Tovorafenib binds to human plasma proteins in vitro at a rate of 97.5%.

[1].Drugs. 2024 Aug;84(8):985-993.

[2]. Mol Cancer Ther (2013) 12 (11_Supplement): C146.

[3]. J Clin Oncol 31, 2013 (suppl; abstr 2547)

[4]. Sci Signal. 2015 Dec 15;8(407):ra129..

[5]. Oncotarget. 2017 Feb 14;8(7):11460-11479.

[6]. Clinical Trial Cancer Chemother Pharmacol. 2023 Jul;92(1):15-28.

[7]. J Biol Chem. 2023 May;299(5):104634.

TAK-580 is a 1,3-thiazolyl carboxamide, chemically named 2-[(1R)-1-aminoethyl]-1,3-thiazolyl-5-carboxylic acid, in which the carboxyl group undergoes a condensation reaction with the amino group of 5-chloro-4-(trifluoromethyl)pyridine-2-amine, and the amino group further undergoes a condensation reaction with the carboxyl group of 6-amino-5-chloropyrimidine-4-carboxylic acid. It is a pan-RAF kinase inhibitor currently under clinical development for the treatment of radiographically recurrent or progressive low-grade gliomas in children and adolescents. It possesses antitumor, apoptosis-inducing, and B-Raf activity-inhibiting effects. It is a chloropyridine, organofluorine compound, secondary amide, aminopyrimidine, pyrimidine carboxamide, and 1,3-thiazolyl carboxamide.
Tovorafenib (TAK-580) is being investigated in the clinical trial NCT02723006 (a study evaluating the safety, tolerability, and pharmacodynamics of investigational treatment in combination with standard immune checkpoint inhibitors in patients with advanced melanoma).
Toporafenib is an orally administered inhibitor of wild-type and certain mutant forms of A-Raf, B-Raf, and C-Raf protein kinases with potential antitumor activity. After administration, toporafenib inhibits Raf-mediated signaling pathways, which may lead to suppression of tumor cell growth. Raf protein kinases play a crucial role in the RAF/MEK/ERK signaling pathway, which is frequently aberrantly regulated in human cancers and plays a key role in tumor cell proliferation and survival.

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Background: RAS-mutant melanoma and colorectal cancer represent a significant unmet medical need. MLN2480 is an investigational class II RAF kinase inhibitor, and TAK-733 is an investigational allosteric MEK kinase inhibitor; both are currently in Phase I monotherapy clinical trials. This study aims to characterize the combined activity of these drugs in preclinical models of BRAF-mutant and RAS-mutant melanoma and colorectal cancer. Methods: An ATP-based cell viability assay was used to investigate the effect of the combined action of MLN2480 and TAK-733 on cell viability in BRAF and RAS mutant melanoma and colorectal cancer cell lines. Western blotting was used to analyze and compare changes in MAPK pathway signaling and response markers in different cell lines, which showed varying sensitivities to the combination drug. The pharmacodynamic response and growth inhibition of the combination drug were studied in xenografts of the same cell lines and in primary human tumor xenografts of RAS mutant melanoma and colorectal cancer. Results: MLN2480 inhibited MAPK pathway signaling at the tolerated concentration in BRAF mutant and some RAS mutant preclinical cancer models. MLN2480 exhibited the strongest activity in the BRAF mutant melanoma model, but also showed single-drug activity in some RAS mutant models. Compared with MLN2480 alone, the combination of MLN2480 and TAK-733 inhibited the growth of a wider range of RAS-mutant tumor models, including primary human tumor xenograft models of melanoma and colorectal cancer. In vitro cell proliferation assays showed that the combination of drugs had synergistic activity. Western blot analysis showed that MLN2480 could reverse TAK-733-induced MEK feedback activation, thereby more effectively inhibiting the MAPK pathway. Conclusion: The activity of the RAF kinase inhibitor MLN2480 in preclinical models of BRAF and RAS-mutant melanoma and colorectal cancer provides a theoretical basis for clinical trials. The combination of MLN2480 and the MEK inhibitor TAK-733 provides a new strategy for clinical research on these tumor types. [2]

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After RAS activation, RAF family kinases initiate the MAP kinase cascade signaling pathway, thereby controlling cell growth, proliferation and differentiation. Among the RAF subtypes (ARAF, BRAF, and CRAF), BRAF oncogenic mutations are the most common. BRAFV600E mutations drive more than half of malignant melanomas and are also present in many other cancers. Selective inhibitors of BRAFV600E (vemurafenib, dabrafenib, and encorafenib) have been used clinically to treat these indications, but they are ineffective against oncogenic RAS (driving RAF dimerization and activation) and malignancies driven by truncated/fusion variants of aberrant BRAF dimerization. In contrast, many "type II" RAF inhibitors have been developed as potent inhibitors of RAF dimers. In this article, we compared the inhibitory potency of the type II inhibitors tovolafenib (TAK-580) and naproxenfenib (LHX254) against the three RAF subtypes using biochemical experiments and described the crystal structures of these two compounds in their BRAF complexes. We found that tovolafenib and naproxenfenib exhibited the strongest inhibitory activity against CRAF, but significantly weaker inhibitory activity against ARAF. The crystal structures of these two compounds with BRAFV600E or wild-type BRAF revealed details of their molecular interactions, including the expected type II binding mode, where both subunits of the BRAF dimer are fully occupied. Our findings have important clinical implications. Type II RAF inhibitors are generally considered pan-RAF inhibitors, but our study of these two drugs, along with recent studies of type II inhibitors bevacirafenib and naproxenfenib, suggests that relative non-inhibition of ARAF may be a common characteristic of this class of drugs. [6]

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RAF family kinases initiate the MAP kinase cascade signaling pathway upon RAS activation, thereby controlling cell growth, proliferation, and differentiation. Among the RAF subtypes (ARAF, BRAF, and CRAF), oncogenic mutations are most common in BRAF. BRAFV600E mutations drive more than half of malignant melanomas and are also present in many other cancers. Selective inhibitors of BRAF V600E (vemurafenib, dabrafenib, and encorafenib) have been used clinically to treat these indications, but they are ineffective against oncogenic RAS (driving RAF dimerization and activation) and malignancies driven by truncated/fusion variants of aberrant BRAF dimerization. In contrast, many “type II” RAF inhibitors have been developed as potent inhibitors of RAF dimers. In this article, we compared the inhibitory potency of the type II inhibitors tovolafenib (TAK-580) and naproxenfenib (LHX254) against three RAF subtypes using biochemical experiments and described the crystal structures of these two compounds in their BRAF complexes. We found that tovolafenib and naproxenfenib exhibited the strongest inhibitory activity against CRAF, but significantly weaker inhibitory activity against ARAF. The crystal structures of these two compounds with BRAF V600E or wild-type BRAF revealed details of their molecular interactions, including the expected type II binding mode, where both subunits of the BRAF dimer are fully occupied. Our findings have important clinical implications. Type II RAF inhibitors are generally considered pan-RAF inhibitors, but our studies of these two drugs, as well as recent studies of type II inhibitors bevacirafenib and naproxenfenib, suggest that relative non-inhibition of ARAF may be a common characteristic of this class of drugs. [7] Mechanism of Action
Low-grade gliomas in children are the most common central nervous system (CNS) tumors in children and are often associated with BRAF genomic alterations, such as BRAF fusions or rearrangements. Upon activation by RAS, the BRAF kinase family phosphorylates MEK1/2, which in turn phosphorylates ERK1/2, thereby activating downstream signaling cascades that regulate a variety of cellular processes, such as cell growth, proliferation, and differentiation. Oncogenic mutations in BRAF lead to aberrant activation of the RAS-RAF-MEK-ERK pathway, also known as the mitogen-activated protein kinase (MAPK) signaling pathway. A variety of RAF kinase inhibitors have been developed for the treatment of BRAF-mutant cancers. These RAF inhibitors can be classified into different “types” based on their selectivity for BRAF subtypes and their binding patterns. Tovorafenib is a type II RAF kinase inhibitor. The N-terminus of the RAF kinase activation loop contains a conserved three-amino acid residue segment (Asp-Phe-Gly) called the DFG motif. In a conformation called "DGF-out," the DFG motif flips, causing the phenylalanine residue to be reoriented, leaving a vacancy that allows the drug to extend from the ATP-binding site and insert a hydrophobic group. Tovorafenib is active against BRAF V600E mutant, wild-type BRAF, and wild-type CRAF kinases. Tovorafenib has shown antitumor activity in cultured cells and xenograft models carrying BRAF V600E and V600D mutations, as well as in xenograft models carrying BRAF fusion genes. It has been reported that tovorafenib does not induce anomalous activation of the MAPK pathway.

C17H12CL2F3N7O2S

506.29

505.01

C, 40.33; H, 2.39; Cl, 14.01; F, 11.26; N, 19.37; O, 6.32; S, 6.33

1096708-71-2

25161177

White to off-white solid powder

1.64

5.024

3

11

5

32

695

1

ClC1C(N([H])[H])=NC([H])=NC=1C(N([H])[C@]([H])(C([H])([H])[H])C1=NC([H])=C(C(N([H])C2C([H])=C(C(F)(F)F)C(=C([H])N=2)Cl)=O)S1)=O

VWMJHAFYPMOMGF-ZCFIWIBFSA-N

InChI=1S/C17H12Cl2F3N7O2S/c1-6(28-15(31)12-11(19)13(23)27-5-26-12)16-25-4-9(32-16)14(30)29-10-2-7(17(20,21)22)8(18)3-24-10/h2-6H,1H3,(H,28,31)(H2,23,26,27)(H,24,29,30)/t6-/m1/s1

2-[(1R)-1-[(6-amino-5-chloropyrimidine-4-carbonyl)amino]ethyl]-N-[5-chloro-4-(trifluoromethyl)pyridin-2-yl]-1,3-thiazole-5-carboxamide

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: ≥ 0.67 mg/mL (1.32 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 6.7 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: ≥ 0.67 mg/mL (1.32 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 6.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 0.67 mg/mL (1.32 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 6.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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