Discovery of a highly potent pan-RAF inhibitor IHMT-RAF-128 for cancer treatment

doi:10.21203/rs.3.rs-2376757/v1

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Discovery of a highly potent pan-RAF inhibitor IHMT-RAF-128 for cancer treatment

https://doi.org/10.21203/rs.3.rs-2376757/v1

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Although RAS mutations occur in about 30% of solid tumors, targeting RAS mutations other than KRAS-G12C is still challenging. As an alternative approach, developing inhibitors targeting RAF, the downstream effector of RAS signaling, is currently one of the main strategies for cancer therapy. Selective BRAF-V600E inhibitors Vemurafenib, Encorafenib, and Dabrafenib have been approved by FDA and received remarkable clinical responses, but these drugs are ineffective against RAS mutant tumors due to limited inhibition on dimerized RAF. In this study, we developed a highly potent pan-RAF inhibitor, IHMT-RAF-128, which exhibited similarly high affinities between RAF monomers and dimers, and showed potent anti-tumor efficacy against a variety of cancer cells harboring either RAF or RAS mutations, especially Adagrasib and Sotorasib (AMG510) resistant-KRAS-G12C secondary mutations, such as KRAS-G12C-Y96C and KRAS-G12C-H95Q. In addition, IHMT-RAF-128 showed excellent pharmacokinetic profile (PK), and the bioavailability in mice and rats were 63.9%, and 144.1%, respectively. Furthermore, IHMT-RAF-128 exhibited potent anti-tumor efficacy on xenograft mouse tumor models in a dose-dependent manner without any obvious toxicities. Together, these results support further investigation of IHMT-RAF-128 as a potential clinical drug candidate for the treatment of cancer patients with RAF or RAS mutations.

Cancer therapy

Pan-RAF

BRAF

Vemurafenib

RAS mutations

The constitutive activation of the mitogen-activated protein kinase (MAPK) pathway (RAS-RAF-MEK-ERK cascade) is closely associated to the occurrence and development of cancers. Although RAS mutations were reported to occur in 30% of cancers, direct targeting RAS faces great challenges due to high binding affinity to GTP and lack of appropriate binding pockets for conventional small molecules[1–3]. Up to now, no inhibitors targeting RAS mutations except for KRAS-G12C mutation has been approved by FDA[4]. RAFs (ARAF, BRAF, and CRAF) are serine/threonine protein kinases and the core members of the MAPK pathway[5, 6]. Once activated by RAS, RAF phosphorylates and activates the downstream MEK and ERK, which in turn regulate cell survival and proliferation[7]. Meanwhile, BRAF mutations are detected in many types of cancers, including 50% of melanomas[8–10], 35–70% of thyroid cancers[11], 5–15% of colorectal cancers[12], and 14–30% of ovarian cancers[13]. Therefore, to circumvent the difficulty in pharmaceutical inhibition against RAS, the development of inhibitors targeting RAF is currently one of the main strategies for the treatment of RAS and BRAF mutant tumors.

Until now, selective BRAF inhibitors Vemurafenib (PLX4032), Dabrafenib, and Encorafenib (LGX818) have been approved by FDA for the treatment of melanoma harboring BRAF-V600E/K mutations and obtained good clinical efficacy[14–16]. However, these drugs have week CRAF activities and show limited inhibition against RAS mutant tumors, in which the mutant RAS induces the dimerization of BRAF and CRAF, and selective inhibiting BRAF alone can activate CRAF-CRAF homodimers and BRAF-CRAF heterodimers, leading to the activation of RAF signaling[17]. Thus, developing a pan-RAF inhibitor, which could inhibit both BRAF and CRAF, is required for the treatment of RAS mutant tumors[18]. LY3009120 is a potent pan-RAF inhibitor that inhibits all RAF isoforms and exhibited promising preclinical efficacy against RAS-mutant cancers[19]. There are other pan-RAF inhibitors currently under pre-clinical and clinical trials, such as RAF709[20], TAK632[21], and BGB-283[22], etc. However, there is no pan-RAF inhibitor approved by FDA at present. Therefore, a potent pan-RAF inhibitor that has sufficient clinical efficacy is still needed.

Here, we described the discovery of a novel pan-RAF inhibitor IHMT-RAF-128, which achieves high potency against both BRAF and CRAF kinases. Cellular analyses showed that IHMT-RAF-128 could effectively inhibit the proliferation of BRAF and RAS mutant cancer cells. In addition, IHMT-RAF-128 displayed excellent PK and safety in a 14-day subacute toxicity test. In BRAF and RAS mutant xenograft mouse tumor models, IHMT-RAF-128 showed potent tumor growth inhibition without obvious toxicity. In conclusion, our data demonstrated that IHMT-RAF-128 is a potent pan-RAF inhibitor with therapeutic potential to treat patients with BRAF or RAS mutations.

Chemicals

IHMT-RAF-128 was in-house synthesized following the route shown in Scheme 1; Vemurafenib (PLX4032), LY3009120, Encorafenib, Adagrasib, Sotorasib, and RAF709 were purchased from MedChemExpress (Shanghai, P.R.China).

Antibodies and immunoblotting

Phospho-MEK1/2 (Ser217/221) antibody (#9154), MEK1/2 antibody (#9122), Phosphop-ERK1/2 (Thr202/Tyr204) (197G2) antibody (#4377), ERK1/2 (137F5) antibody (#4695), GAPDH (D16H11) XP antibody (#5174), PARP (46D11) antibody (#9532), and caspase-3 (8G10) antibody (#9665) were obtained from Cell Signaling Technology (Danvers, MA, USA).

BaF3 isogenic cell line generation

The functional BaF3 cell lines were constructed as described before[23]. In short, wild-type and mutant BRAF and RAS genes were cloned into the pMSCVpuro retroviral vector and co-transfected with two helper plasmids for virus production in HEK-293T cells. Culture supernatant containing virus particles was used to infect BaF3 cells, and stable BRAF or RAS-overexpression of BaF3 cells were then obtained by puromycin selection and IL-3 withdrawal.

Molecular modeling

The crystal structure of BRAF was obtained from Protein Data Bank (PDB ID: 4G9R). Water molecules and original ligand were manually removed using software[24]. CRAF structure of DFG-out type was modeled using MODELLER[25] based on the template of BRAF. The docking jobs were carried out by Autodock (version 4.2.6)[26]. The prepare_ligand4.py and prepare_recptor4.py scripts from AutoDockTools 1.5.6 were used to prepare the initial files including additions of charges and hydrogen atoms. A grid box of 60*60*60 with a spacing of 0.375Å was set to enclose the whole binding site. The Lamarckian genetic (LGA) was adopted to search the best binding poses. The specific docking settings were as follow: trials of 100 dockings, 300 individuals per population with a crossover rate of 0.8 and the local search rate was set to 0.06. Other parameters were set as default during the docking.

Cell lines and cell culture

H358, Capan-2, KYSE-30, PF382, ASH-3, H2087, BEAS-2B, MCF-10A, A375, HCT116, BxPC3, ASPC-1, HT29, and SK-Mel-28 cell lines were bought from ATCC (American Type Culture Collection). SK-GT-4, CAL62, MDA-MB-231, NALM-6, CAL-12T, 8305C, HEL, CHO cell lines were bought from Cobioer (Nanjing, China). CAL62, MDA-MB-231, CAL-12T, A375, HT29 and HCT116 cells were cultured in DMEM (Corning, USA) with 10% FBS; Capan-2 cells were cultured in McCoy's 5A (GIBCO, USA) with 10% FBS; KYSE-30 were cultured in RPMI1640: Ham’s F-12 (1:1), 2mM L-Glutamine, and 10% FBS; SK-GT-4, H358, PF382, NALM-6, H2087, HEL, BxPC3 and ASPC-1 cells were cultured in RPMI1640 with 10% FBS; ASH-3 cells were cultured in DMEM: RPMI1640 (1:1) with 10%FBS; 8305C, SK-Mel-28 cells were cultured in MEM (Corning, USA) with 10% FBS, NEAA, 1 mM sodium pyruvate, 2mM L-Glut. CHO cells were cultured in F12K (Corning, USA) with 10% FBS. BEAS-2B cells were cultured in BEGM (Lonza, USA) with 10% FBS; MCF-10A were cultured in MEGM (Lonza, USA) with 10% FBS. All cells were cultured in an incubator at 37℃and 5% CO₂.

Biochemical assay of kinase activity

The in vitro enzymatic assay was provided by the Beijing ICE Bioscience Company (Beijing, China). Briefly, BRAF (0.2ng/µL), CRAF (0.13ng/µL), and BRAF-V600E (0.1ng/µL) proteins were incubated with IHMT-RAF-128 (20-10000 nM), MEK1 substrates (BRAF: 2.5 mg/mL, CRAF: 7.1 mg/mL, BRAF-V600E: 2.5 mg/mL), and ATP (BRAF: 25 µM, CRAF: 10 µM, BRAF-V600E: 30 µM). The reactions were initiated by adding ATP to each test tube to and continued at room temperature for 1h. ADP-GLO reagent was then added to each well to terminate the reaction, and the remaining ATP was consumed for 40 minutes. Finally, the kinase detection reagent was added to the wells and incubated for 30 minutes to generate luminescence signals, which were then analyzed on microplate reader (Envision, PE, USA) and Prism 8.0 (GraphPad software) to calculate compound potencies.

Western blot analysis

A375, SK-Mel-28, ASPC-1, and HCT116 cells were treated with serially diluted IHMT-RAF-128, RAF709, and LY3009120 for 2h. Cells were then collected by centrifugation and lysed with RIPA buffer (Beyotime, China). The cell lysates were separated by 10% SDS-PAGE electrophoresis and blotted with antibodies after membrane transfer and blocking. At last, the ECL Western blot detection kit (Millipore) was used to examine the effects by drug treatment.

Cell cycle analysis

A375, SK-Mel-28, ASPC-1, and HCT116 cells were treated with serially diluted IHMT-RAF-128, RAF709, and LY3009120. 70% ethanol and PI/RNase staining buffer (BD, USA) were used to fix and stain cells, which were then analyzed on FACSCalibur™ flow cytometer and ModiFit software to test the effects of drug treatment on cell cycle.

Apoptosis detection

A375, SK-Mel-28, ASPC-1, and HCT116 cells were treated with continuously diluted IHMT-RAF-128, RAF709, and LY3009120 for 48h and 72h. The cells were then collected and measured by Western blot assays using PARP, cleaved-PARP, Caspase3, cleaved-Caspase3, and GAPDH antibodies to detect the effect of compounds on apoptosis.

Mouse xenograft study

Nude mice (5 weeks) were purchased from Vital River Laboratories (Beijing, China). The mouse xenograft studies complied with the animal ethics standards of the Hefei Institute of Physical Science, Chinese Academy of Sciences. Briefly, ten million SK-Mel-28, ASPC-1, and BxPC3 cells were first mixed 1:1 with Matrigel gel and injected into the right subcutaneous tissue of nude mice. When tumor sizes reached around 150 mm³, animals were randomly divided into groups of 4–5 mice each. IHMT-RAF-128 and LY3009120 were orally administered daily. The body weights and tumor sizes of the mice were measured daily. The formula (width² × length /2) was used to calculate the tumor volumes. Tumor growth inhibition (TGI) was calculated according to the formula: (W_vehicle-W_test) /W_vehicle × 100%, in which W is the tumor weight.

IHMT-RAF-128 is a highly potent pan-RAF inhibitor

To develop a highly potent pan-RAF inhibitor, we screened an in-house small-molecule compound library and identified IHMT-RAF-128 (synthetic route of IHMT-RAF-128 was shown in Scheme 1) through high-throughput screening using a house-made BaF3 isogenic cell library. We first examined the anti-proliferation effect of IHMT-RAF-128 against a panel of RAS mutant-transfected BaF3 cell lines (Fig. 1a, Supplementary Table 1). The results showed that IHMT-RAF-128 effectively inhibited the proliferation of BaF3 isogenic cells harboring RAS mutations including KRAS-G12C and NRAS-G12D, while no effects were seen with Vemurafenib (GI₅₀: 9 nM vs > 1000 nM; 14 nM vs > 1000 nM). Importantly, IHMT-RAF-128 displayed potent anti-proliferation efficacy against KRAS-G12C secondary mutations, such as KRAS-G12C-H95D, KRAS-G12C-H95Q, and KRAS-G12C-Y96C, which were resistant to KRAS-G12C inhibitors Adagrasib and partially to Sotorasib. Then, we evaluated IHMT-RAF-128 against a panel of BaF3 cell lines harboring multiple BRAF mutations. In Vemurafenib-sensitive BRAF-V600E cells, IHMT-RAF-128 showed more potent anti-proliferation effect than Vemurafenib (GI₅₀: 3.5 nM vs 14.7 nM). In cell lines that are resistant to Vemurafenib, such as K601E mutation located in the activation segment, G469A mutation located in the P-loop, and truncated form of V600E (V600E-P61), IHMT-RAF-128 also showed highly potent inhibitory activities on cell proliferation (GI₅₀:<0.3 nM vs 1800 nM; 4 nM vs 380 nM; 1300 nM vs > 10,000 nM) (Fig. 1b, Supplementary Table 2). To rule out general cell toxicity, we checked the effect of IHMT-RAF-128 on the proliferation of parental BaF3 cells, and the results showed that IHMT-RAF-128 did not affect parental Ba/F3 cells up to 10 µM (GI₅₀:>10 µM).

To verify the on-target effect of IHMT-RAF-128, we then used biochemical assays to test the inhibitory activity against BRAF, BRAF-V600E, and CRAF proteins. Our results showed that, compared with Vemurafenib, IHMT-RAF-128 exhibited more potent inhibitory effects against those three kinase proteins (IC₅₀:5.9 nM vs 17.5 nM; 5.9 nM vs 41.7 nM; 3.6 nM vs 23.5 nM; respectively) (Fig. 1c).

IHMT-RAF-128 is a selective pan-RAF inhibitor

To profile the selectivity of our compound in human kinome, we next used DiscoverX’s KINOMEScan assay to investigate the selectivity of IHMT-RAF-128 by comparing the affinities between IHMT-RAF-128 and 405 kinases. In this assay, IHMT-RAF-128 exhibited acceptable selectivity at the dose of 1 µΜ (Fig. 2a, Supplementary Table 3). Besides BRAF-V600E, IHMT-RAF-128 also exhibited affinities to a few other kinases, such as PDGFR, DDR1, DDR2, EPHB2, and CSF1R. To further confirm the kinome profiling result of IHMT-RAF-128 at the cellular level, we used a house-made BaF3 isogenic cell library and examined the anti-proliferation activity. The results demonstrated that IHMT-RAF-128 inhibited the proliferation of BRAF-V600E, PDGFRα, PDGFRβ, and EPHB2 cell lines at similar levels. While for other kinases, such as CSF1R, CKIT, VEGFR2, and FLT3, IHMT-RAF-128 exhibited 5 ~ 4667 folds of selectivity. Together, these data indicated that IHMT-RAF-128 is a relatively selective pan-RAF inhibitor (Fig. 2b).

IHMT-RAF-128 shows equipotent inhibitory efficacies against RAF monomers and dimers

To further investigate the binding mode of IHMT-RAF-128 to BRAF and CRAF proteins, we conducted computer-aided molecular modeling analysis. As we expected, IHMT-RAF-128 fits well in the ATP pockets of wild-type and V600E mutant BRAF as well as CRAF (Fig. 3a-b). The interactions are enhanced through three hydrogen bonds at the hinge region, DFG motif, and the αC helix. In addition, the trifluoromethyl phenyl group occupies the allosteric pocket formed by ‘DFG-out’ and ‘αC-in’ motifs. Moreover, the pyrazolyl moiety could exert π-stacking interaction with the side chain of Phe595 in BRAF (Phe487 in CRAF). It's worth noting that the V600E mutation of BRAF, as well as Val492 of CRAF at the homologous position, does not influence the interactions. In addition, we found that the inhibitor extends more closely to the solvent region residue Lys431 (CRAF) than Hid539 (BRAF), 4.5 Å toward Lys431 versus 7.3 Å toward Hid539, respectively. Since lysine residue could provide stronger electrostatic interaction, that may explain the slightly stronger inhibition of CRAF. In short, the results revealed that IHMT-RAF-128 is a type II inhibitor and binds to BRAF in DFG-out and αC-in conformation, which is different from Vemurafenib (DFG-in and αC-out) as reported[18].

As a type I inhibitor, Vemurafenib, the binding of which with the first protomer sterically hinders drug binding to the second protomer, was expected to be ineffective against RAF dimer[27]. To see whether our compound is able to inhibit dimerized RAF, we used a system developed by Yao et al, in which cells was pre-treated with Encorafenib for 1h to block the first site of the RAF dimer [18, 28]. For comparison, we selected A375 cells, in which RAF activates as monomers, with IHMT-RAF-128 and Vemurafenib to examine the inhibitory efficacy to RAF monomers. Meanwhile, we pre-treated HCT116 cells, in which RAF activates as dimers, with 1µM Encorafenib before the cells were used to test the binding ability of compound to the second site of RAF that is unoccupied. The results showed that IHMT-RAF-128 could inhibit the second site with a similar potency to RAF monomer, to which Vemurafenib was ineffective, which indicated that IHMT-RAF-128 would be effective in treating cancer cells harboring BRAF and RAS mutant, in which MEK-ERK pathway was activated by RAF dimers (Fig. 3c).

IHMT-RAF-128 inhibits RAF signaling and the feedback reactivation of the MAPK pathway

Previous studies have shown that the main reason why the efficacy of αC-out type of RAF inhibitors is limited, such as Vemurafenib, is the negative feedback of MAPK signal pathway[18]. To evaluate whether IHMT-RAF-128 could overcome the negative feedback of MAPK pathway, we first examined its effect on RAF signaling in BRAF-V600E and KRAS mutant cancer cells. The results showed that 2h treatments of both IHMT-RAF-128 and Vemurafenib could dose-dependently inhibited the phosphorylation of MEK and ERK, the downstream effectors of RAF in A375 and SK-MEL-28 cells harboring BRAF-V600E mutation. In HCT116 and H358 cells harboring KRAS-G13D or KRAS-G12C mutation, IHMT-RAF-128 also inhibited the phosphorylation of MEK and ERK in a dose-dependent manner. However, Vemurafenib enhanced the phosphorylation of MEK and ERK, which was due to the feedback reactivation of MAPK pathway as reported (Fig. 4a). In addition, while significant feedback effect was observed with prolonged treatment of Vemurafenib for 48h, IHMT-RAF-128 maintained potent inhibition on RAF signaling during this period of time in HT29 and HCT116 cells (Fig. 4b).

IHMT-RAF-128 effectively inhibits the proliferation of BRAF or RAS mutant cancer cells

To see what types of cancer cell lines are sensitive to IHMT-RAF-128, we next studied the anti-proliferative effects of IHMT-RAF-128 on a series of cancer cells with RAS and RAF mutations (Fig. 5a). The results showed that IHMT-RAF-128 exhibited similar efficacies to LY3009120, but it was more potent than Vemurafenib in both BRAF and RAS mutant cancer cells. At the same time, IHMT-RAF-128 showed no obvious effect on the proliferation of HEL cell harboring RAS-WT and normal cells, such as CHO, BEAS-2B and MCF-10A, which indicated that IHMT-RAF-128 had no obvious off-target cellular toxicity. To further explore the mechanism of IHMT-RAF-128 inhibition on cell proliferation, we examined its effects on cell cycle and apoptosis (Fig. 5b and 5c). As shown in Fig. 5B, IHMT-RAF-128 dose-dependently arrested cell cycle at the G0-G1 phase. In addition, IHMT-RAF-128 dramatically induced cell apoptosis in a dose-dependent manner with similar efficiencies to LY3009120 and RAF709. These results suggested that IHMT-RAF-128 inhibited the proliferation of BRAF or RAS mutant cells by blocking cell cycle and inducing apoptosis.

IHMT-RAF-128 displays ideal PK and safety profiles

To see whether the pharmacokinetics (PK) profile of IHMT-RAF-128 is suitable for in vivo anti-tumor evaluation, we first examined the PK in mice and rats (Table S5). At the dose of 10mg/kg by oral administration, the drug exposures of IHMT-RAF-128 were ideal with AUC_0−∞ at 4864 ng/mL·h and 11021 ng/mL·h, respectively. At the same time, the plasma concentration C_max reached peak values within 40 min in both mice and rats, which indicated that IHMT-RAF-128 could rapidly enter the systemic circulation. In addition, the bioavailability of IHMT-RAF-128 reached 63.9% and 144.1% in mice and rats, respectively, indicating that the drug was suitable for oral administration on mouse xenograft models.

To further explore the safe therapeutic window of IHMT-BRAF-128, we monitored its effects on mouse body weight, heart, liver, spleen, lung, and kidney at doses of 100 mg/kg and 500 mg/kg for 14 consecutive days (Fig. 6a-f). The results showed that, compared to the vehicle group, IHMT-BRAF-128 had no detectable effect on the weights of body and major organs up to 500 mg/kg dosage.

IHMT-RAF-128 displays anti-tumor activities in mutant BRAF and RAS mouse xenograft models

We next examined in vivo efficacy of IHMT-RAF-128 in mutant BRAF and RAS mouse xenograft models. In SK-Mel-28 melanoma mouse xenograft model, IHMT-RAF-128 showed no significant effect on animal weight at doses up to 100 mg/kg, and inhibited tumor growth in a dose-dependent manner with a tumor growth inhibition (TGI) rate of 78.7% at the dose of 100 mg/kg, which was more potent than LY3009120 at same dosages (TGI = 53.6%) (Fig. 7a). In BxPC3 pancreatic tumor mouse xenograft model, IHMT-RAF-128 dramatically inhibited tumor growth at the 100mg/kg dosage, and the inhibition rate was 91.8%, which was higher than LY3009120 (TGI = 64.0%) (Fig. 7b). In another pancreatic tumor mouse xenograft model derived from ASPC-1, IHMT-RAF-128 showed more efficient inhibition on tumor growth than LY3009120 at the same dose (TGI = 72.6% and 6.1%, respectively), and IHMT-RAF-128 exhibited dose-dependent anti-tumor potency without obvious toxicity (Fig. 7c). In addition, immunohistochemical (IHC) assay showed that IHMT-RAF-128 dose-dependently inhibited tumor cell growth and induced cell apoptosis (Supplementary Fig. S1).

Deregulation of RAS-RAF-MEK-ERK signaling pathway promotes cell proliferation and survival, and RAS and RAF mutations are common in various cancers, which promoted the development of ATP-competitive RAF inhibitors[29]. So far, three selective BRAF inhibitors, Vemurafenib, Encorafenib, and Dabrafenib, have been approved by FDA for the treatment of melanoma harboring BRAF-V600E/K and obtained good clinical efficacy[14–16]. However, these inhibitors adopted a binding mode of αC-out conformation, in which the binding of the first protomer would reduce the affinity of the second protomer by stabilizing the αC helix of the second protomer toward the IN position. Thus, these three inhibitors showed limited inhibition efficacy on RAF dimers and instead promoted phosphorylation of MEK and ERK in RAS-mutant cancer cells due to the feedback reactivation of MAPK pathway. In contrast, αC-in pan-RAF inhibitors, targeting both RAF dimers and monomers at the similar affinity, were predicted to be effective against RAS mutant cancer cells, in which dimeric RAF is known to be a major upstream effector of ERK signaling pathway.

Although Adagrasib and Sotorasib have shown excellent clinical efficacy in cancers harboring KRAS-G12C mutation, most patients would develop resistance after treatment with Adagrasib for ~ 11 weeks due to KRAS-G12C second-site mutations, such as H95D/Q, and Y96C, some of which also confer Sotorasib resistance as reported[30]. To overcome the acquired resistance to Adagrasib and Sotorasib, one of the strategies is to develop pan-RAF inhibitors. Currently, many pan-RAF inhibitors are undergoing clinical trials, such as RAF265, BGB-283, and PLX8394. However, there is no pan-RAF inhibitor approved by FDA so far. Therefore, developing novel pan-RAF inhibitors which are potently effective both in vitro and in vivo is still in great demand.

Here, we reported the discovery of a highly potent pan-RAF inhibitor IHMT-RAF-128, which stabilizes RAF in DFG-out and αC-in conformation and targets both BRAF and CRAF with similar potencies, thus inhibiting both RAF monomers and dimers, and avoiding resistance caused by paradoxical pathway activation. In vitro, IHMT-RAF-128 strongly inhibited the proliferation of a panel of BRAF and RAS mutant-transfected BaF3 cell lines, and induced cell cycle arrest and apoptosis in multiple mutant BRAF and RAS cancer cells, indicating that the compound is able to overcome resistance caused by paradoxical activation and KRAS-G12C second-site mutations. In vivo, IHMT-RAF-128 showed remarkable efficacy with little toxicity in mouse xenograft models harboring mutant BRAF and RAS, suggesting that IHMT-RAF-128 could become a candidate agent for treatment with patients harboring RAF or RAS mutations.

Funding

This work was supported by the National Natural Science Foundation of China (Grant Nos. 32171479, 82104239), the Plan for Major Anhui Provincial Science and Technology Project (Grant No. 202203a07020033), and the CASHIPS Director’s Found (Grant Nos. YZJJZX202011).

Authors’ contributions QL, JL, and WCW designed and supervised the project. ALW, JL, ZWW, and JYC. performed the biological study. XXL performed the chemical synthesis. FMZ, ZPQ, SQ, QWL, ZRJ, and JG performed the animal study. ALW, JL, BLW, and LW analyzed the data. ALW, JL, and BLW. drafted the manuscript. QSL, JL, and WCW revised the manuscript. All authors have read and approved the final manuscript.

Availability of data and materials

The data presented in this study are available on request from the corresponding author.

Ethics approval and consent to participate The research program was approved by the animal ethics standards of the Hefei Institute of Physical Science, Chinese Academy of Sciences.

Informed consent Not applicable.

Research involving human participants and/or animals The research program was approved by the animal ethics standards of the Hefei Institute of Physical Science, Chinese Academy of Sciences.

Conflicts of interest/Competing interests The authors declare no competing interests.

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Scheme 1 is available in Supplementary Files section.

No competing interests reported.

IHMTRAF128supplementary.docx
TableS3.csv
SupportingInformation.docx
Scheme1.png
Scheme 1: The synthetic route of IHMT-RAF-128.

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Discovery of a highly potent pan-RAF inhibitor IHMT-RAF-128 for cancer treatment

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Abstract

Figures

Introduction

Methods

Chemicals

Antibodies and immunoblotting

BaF3 isogenic cell line generation

Molecular modeling

Cell lines and cell culture

Biochemical assay of kinase activity

Western blot analysis

Cell cycle analysis

Apoptosis detection

Mouse xenograft study

Results

IHMT-RAF-128 is a highly potent pan-RAF inhibitor

IHMT-RAF-128 is a selective pan-RAF inhibitor

IHMT-RAF-128 shows equipotent inhibitory efficacies against RAF monomers and dimers

IHMT-RAF-128 inhibits RAF signaling and the feedback reactivation of the MAPK pathway

IHMT-RAF-128 effectively inhibits the proliferation of BRAF or RAS mutant cancer cells

IHMT-RAF-128 displays ideal PK and safety profiles

IHMT-RAF-128 displays anti-tumor activities in mutant BRAF and RAS mouse xenograft models

Discussion

Declarations

References

Scheme

Additional Declarations

Supplementary Files

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