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
12.5 mg/kg
oral gavage

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Tovorafenib (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.
In 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.
The 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.
The 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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Safety, pharmacokinetic and pharmacodynamic assessments[6]
Adverse 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).
Serial 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).
Based 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
Tovorafenib steady-state maximum concentration (Cmax) is 6.9 µg/mL (23%) and the area under the concentration-time curve (AUC) is 508 µg*h/mL (31%). The time to reach a steady state of tovorafenib is 12 days (33%). Tovorafenib exposure increases in a dose-proportional manner. Tovorafenib median (minimum, maximum) time to achieve peak plasma concentration (Tmax) is 3 hours (1.5, 4 hours), following a single dose with tablets or oral suspension. No clinically significant differences in tovorafenib Cmax and AUC were observed following administration of tablets with a high-fat meal (approximately 859 total calories, 54% fat) compared to fasted conditions, but the Tmax was delayed to 6.5 hours.

Route of Elimination
Following a single oral dose of radiolabeled tovorafenib, 65% of the total radiolabeled dose was recovered in the feces (8.6% unchanged) and 27% of the dose was recovered in the urine (0.2% unchanged).

Volume of Distribution
Tovorafenib apparent volume of distribution is 60 L/m² (23%). It crosses the blood-brain barrier.

Clearance
The apparent clearance is 0.7 L/h/m2 (31%).

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Metabolism / Metabolites
Tovorafenib is primarily metabolized by aldehyde oxidase and CYP2C8 in vitro. CYP3A, CYP2C9, and CYP2C19 metabolize tovorafenib to a minor extent.

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Biological Half-Life
Tovorafenib terminal half-life is approximately 56 hours (33%).

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Pharmacokinetics [6]
Mean (± standard deviation) plasma concentration–time profiles of Tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819) by QW dose group on days 1 and 22 of cycle 1 are shown in Fig. 2; cycle 1 day 22 plasma PK parameters are summarized by dose group in Table 4. Following multiple oral dosing of 600 mg QW, peak concentrations of tovorafenib were achieved at a median Tmax of 3 h post-dose (range 1–24 h) on cycle 1 day 22. Minimal to no apparent accumulation in terms of day 22 AUC168 over day 1 AUC168 was observed following repeated QW dosing. The mean plasma terminal half-life (t1/2) of tovorafenib was approximately 70 h (range 31–119 h) as defined in 20 evaluable patients receiving 600 mg QW. The relationship between dose and cycle 1 day 22 tovorafenib exposures (AUC168) is shown in Supplementary Figure S1. Steady-state exposures increased in an approximately dose-proportional manner over the 400 mg to 800 mg QW dose range with the 95% CI of the power model containing 1 (95% CI 0.55–2.04), with the coefficient of 1.30. For QW dosing regimens, minimum drug accumulation was observed and the geometric mean Rauc (accumulation ratio based on AUC0-last) was in the range of 1.03–1.09. With the Q2D dosing regimen at 200 mg, the geometric mean value of Rauc was ~ 2.55.
Similar PK analyses were carried out by Q2D dose group (Supplementary Figs. S1 and S2, and Supplementary Table S10). Steady-state Tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819)b AUC48 increased in an approximately dose-proportional manner over the dose ranges of 20 mg to 280 mg Q2D. While no apparent accumulation was observed with the QW dose regimens, Q2D administration resulted in approximately 2.5-fold accumulation in AUC48 at steady state.

The incidence of TEAEs and SAEs is summarized in Supplementary Table S5 and the most common TEAEs are listed in Table 2. Of note, only 1 of 149 treated patients (< 1%; Q2D dose expansion cohort) had squamous cell carcinoma of skin reported as a TEAE. The incidence of drug-related TEAEs according to dosing regimen is summarized in Supplementary Table S6. The two most common in the dose expansion phase were maculo-papular rash in the Q2D cohort (36%) and fatigue (42%) in the QW cohort. In the dose expansion phase, 68% of patients experienced a grade 3 or higher TEAE, including 73% of patients in the Q2D cohorts and 47% in the QW cohort. Grade 3 or higher TEAEs occurring in ≥ 5% of patients are listed in Supplementary Table S7. The two most commonly occurring overall were anemia (14%) and maculo-papular rash (8%). [6]

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In the Q2D expansion cohorts, drug-related TEAEs of grade 3 or higher occurred in 33 of 80 patients (41%); the most common were maculo-papular rash (9%) and anemia (8%). In the QW expansion cohort, drug-related TEAEs of grade 3 or higher occurred in 4 of 20 patients (20%); the most common was hyperbilirubinemia (10%).
In the dose escalation phase, drug-related treatment-emergent SAEs were reported in 2 of 30 patients (7%) in the Q2D cohort (280 mg dose level; grade 3 anemia in 1 patient, and grade 4 dyspnea and grade 5 respiratory failure in another patient) and 2 of 20 patients (10%) in the QW cohort (800 mg dose level; grade 3 rash, 1 patient, and grade 3 hyperbilirubinemia, 1 patient). In the dose expansion phase, drug-related treatment-emergent SAEs were reported in 12 of 80 patients (15%) in the Q2D cohorts and included acute kidney injury, macular rash, rash maculo-papular (grade 3 events in 2 patients each). In the QW dose expansion cohort, 4 of 19 patients (21%) had drug-related treatment-emergent SAEs, including grade 2 anemia and dyspnea in 1 patient, grade 2 nausea and grade 3 maculo-papular rash in another, and grade 3 erythema multiforme and macular rash in 1 patient each.
In the dose expansion phase, 15 of 80 patients (19%) in the Q2D cohort had TEAEs resulting in permanent discontinuation of Tovorafenib (MLN2480; BIIB-024; BSK1369; DAY-101; TAK-580; AMG-2112819). These included maculo-papular rash and sepsis (2 patients [3%] each). In the QW cohort of the dose expansion phase, 4 of 19 patients (21%) had TEAEs resulting in permanent discontinuation, including atrial flutter, dyspnea, erythema multiforme, and fatigue (1 patient each). In the dose expansion phase, 19 of 99 patients (19%) had TEAEs leading to dose reduction including 17 of 80 patients (21%) in the Q2D cohorts and 2 of 19 (11%) in the QW cohort, the most common of which were maculo-papular rash (5 of 99 patients, 5%) and generalized rash (3 patients, 3%).
There were 13 on-study deaths. The fatal SAEs associated with these deaths predominantly related to the underlying disease or complications thereof and are listed in Supplementary Table S8. Only one death, associated with respiratory failure in a patient in the 280 mg Q2D dose escalation cohort, was deemed by the study investigators to be treatment related.[6]

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Protein Binding
Tovorafenib is 97.5% bound to human plasma proteins in vitro.

[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-thiazolecarboxamide that is 2-[(1R)-1-aminoethyl]-1,3-thiazole-5-carboxylic acid in which the carboxy group undergoes formal condensation with the amino group of 5-chloro-4-(trifluoromethyl)pyridin-2-amine and in which the amino group undergoes formal condensation with the carboxy group of 6-amino-5-chloropyrimidine-4-carboxylic acid. It is a pan-RAF kinase inhibitor which is currently in clinical development for the treatment of radiographically recurrent or progressive low-grade glioma in children and young adults. It has a role as an antineoplastic agent, an apoptosis inducer and a B-Raf inhibitor. It is a chloropyridine, an organofluorine compound, a secondary carboxamide, an aminopyrimidine, a pyrimidinecarboxamide and a 1,3-thiazolecarboxamide.
Tovorafenib (TAK-580) is under investigation in clinical trial NCT02723006 (Study to Evaluate the Safety, Tolerability, and Pharmacodynamics of Investigational Treatments in Combination With Standard of Care Immune Checkpoint Inhibitors in Participants With Advanced Melanoma).
Tovorafenib is an orally available inhibitor of wild-type and certain mutant forms of A-Raf, B-Raf and C-Raf protein kinases, with potential antineoplastic activity. Upon administration, tovorafenib inhibits Raf-mediated signal transduction pathways, which may lead to an inhibition of tumor cell growth. Raf protein kinases play a key role in the RAF/MEK/ERK signaling pathway, which is often deregulated 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 areas of significant unmet medical need. MLN2480 is an investigational class II RAF kinase inhibitor and TAK-733 is an investigational allosteric MEK kinase inhibitor; each of which is the subject of a single agent phase I clinical trial. The present studies have characterized the combination activity of these agents in BRAF mutant and RAS mutant preclinical models of melanoma and colorectal cancer. Methods: Combination effects of MLN2480 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. Pharmacodynamic responses and growth inhibitory effects of the combination were studied in xenografts of the same cell lines, as well as in primary human tumor xenografts, of RAS mutant melanoma and CRC. Results: MLN2480 inhibits MAPK pathway signaling in BRAF mutant and some RAS mutant preclinical cancer models at concentrations that are tolerated in vivo. MLN2480 is most potent in BRAF mutant melanoma models but also has single agent activity in some RAS mutant models. 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. Conclusions: The activity of the RAF kinase inhibitor MLN2480 in preclinical models of BRAF and RAS mutant melanoma and CRC provides a rationale for clinical testing. The combination of MLN2480 with the MEK inhibitor TAK-733 represents an additional strategy for clinical research within these tumor types.[2]

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Upon activation by RAS, RAF family kinases initiate signaling through the MAP kinase cascade to control cell growth, proliferation, and differentiation. Among RAF isoforms (ARAF, BRAF, and CRAF), oncogenic mutations are by far most frequent in BRAF. The BRAFV600E mutation drives more than half of all malignant melanoma and is also found in many other cancers. Selective inhibitors of BRAFV600E (vemurafenib, dabrafenib, encorafenib) are used clinically for these indications, but they are not effective inhibitors in the context of oncogenic RAS, which drives dimerization and activation of RAF, nor for malignancies driven by aberrantly dimerized truncation/fusion variants of BRAF. By contrast, a number of “type II” RAF inhibitors have been developed as potent inhibitors of RAF dimers. Here, we compare potency of type II inhibitors tovorafenib (TAK-580) and naporafenib (LHX254) in biochemical assays against the three RAF isoforms and describe crystal structures of both compounds in complex with BRAF. We find that tovorafenib and naporafenib are most potent against CRAF but markedly less potent against ARAF. Crystal structures of both compounds with BRAFV600E or WT BRAF reveal the details of their molecular interactions, including the expected type II–binding mode, with full occupancy of both subunits of the BRAF dimer. Our findings have important clinical ramifications. Type II RAF inhibitors are generally regarded as pan-RAF inhibitors, but our studies of these two agents, together with recent work with type II inhibitors belvarafenib and naporafenib, indicate that relative sparing of ARAF may be a property of multiple drugs of this class. [6]

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Upon activation by RAS, RAF family kinases initiate signaling through the MAP kinase cascade to control cell growth, proliferation, and differentiation. Among RAF isoforms (ARAF, BRAF, and CRAF), oncogenic mutations are by far most frequent in BRAF. The BRAFV600E mutation drives more than half of all malignant melanoma and is also found in many other cancers. Selective inhibitors of BRAFV600E (vemurafenib, dabrafenib, encorafenib) are used clinically for these indications, but they are not effective inhibitors in the context of oncogenic RAS, which drives dimerization and activation of RAF, nor for malignancies driven by aberrantly dimerized truncation/fusion variants of BRAF. By contrast, a number of "type II" RAF inhibitors have been developed as potent inhibitors of RAF dimers. Here, we compare potency of type II inhibitors tovorafenib (TAK-580) and naporafenib (LHX254) in biochemical assays against the three RAF isoforms and describe crystal structures of both compounds in complex with BRAF. We find that tovorafenib and naporafenib are most potent against CRAF but markedly less potent against ARAF. Crystal structures of both compounds with BRAFV600E or WT BRAF reveal the details of their molecular interactions, including the expected type II-binding mode, with full occupancy of both subunits of the BRAF dimer. Our findings have important clinical ramifications. Type II RAF inhibitors are generally regarded as pan-RAF inhibitors, but our studies of these two agents, together with recent work with type II inhibitors belvarafenib and naporafenib, indicate that relative sparing of ARAF may be a property of multiple drugs of this class. [7] Mechanism of Action
Pediatric low-grade glioma, the most common childhood central nervous system (CNS) tumour, is often associated with BRAF genomic alterations, such as BRAF fusion or rearrangement. The BRAF kinase family is activated by RAS to phosphorylate MEK1/2, which phosphorylates ERK1/2 and promotes downstream signalling cascades that regulate multiple cellular processes, such as cell growth, proliferation, and differentiation. Oncogenic mutations in BRAF lead to an aberrant and hyperactivated RAS-RAF-MEK-ERK pathway, also known as the mitogen-activated protein kinase (MAPK) signalling pathway. Several RAF kinase inhibitors have been developed to treat cancers with BRAF mutations. These RAF inhibitors have been categorized into different "types" depending on their selectivity to a BRAF isoform and binding modes. Tovorafenib is a Type II RAF kinase inhibitor. RAF has a conserved three-residue segment (Asp-Phe-Gly) located at the N-terminus of the kinase activation loop called a DFG motif. In a state called a “DGF-out” conformation, the DFG motif is flipped in a way that reorients the phenylalanine residue, leaving a vacant site in which the drug can extend from the ATP site to insert a hydrophobic group. Tovorafenib is active against mutant BRAF V600E, wild-type BRAF, and wild-type CRAF kinases. Tovorafenib exhibited antitumor activity in cultured cells and xenograft tumour models harbouring BRAF V600E and V600D mutations, and in a xenograft model harbouring a BRAF fusion. Tovorafenib is not reported to induce paradoxical 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.)