Discovery of a novel pan-RAF inhibitor with potent anti-tumor activity in preclinical models of BRAFV600E mutant cancer

Life Sciences 183 (2017) 37–44

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Discovery of a novel pan-RAF inhibitor with potent anti-tumor activity in preclinical models of BRAFV600E mutant cancer Sung Pyo Hong, Soon Kil Ahn ⁎ Institute for New Drug Development, Division of Life Sciences, Incheon National University, Incheon 406-772, Republic of Korea

a r t i c l e

i n f o

Article history: Received 7 March 2017 Received in revised form 19 June 2017 Accepted 20 June 2017 Available online 21 June 2017 Keywords: Pan-RAF inhibitor BRAFV600E mutant RAS mutant Melanoma Colorectal cancer

a b s t r a c t Aims: BRAF mutations, especially BRAF V600E, are a frequent occurrence in malignant melanomas. The BRAF inhibitors are used as the care standard for BRAF-mutant metastatic melanomas. However, melanomas rapidly develop resistance to BRAF inhibitors after a median response duration of 6 months, and the subsequent rapid development of cutaneous toxicity is enhanced by the paradoxical activation of CRAF. In this study, we discovered a potent and selective pan-RAF inhibitor: INU-152. The goal of this study was to investigate whether the inhibition of pan-RAF with INU-152 completely disrupts the MAPK pathway in cancer cells bearing BRAF or RAS mutations. Main methods: Using a structure-based molecular modeling, we discovered INU-152, which is a potent and selective pan-RAF inhibitor. In kinase assays against RAF proteins, INU-152 exhibited a potent effect against RAF isoforms. INU-152 was tested for its inhibitory effect on the growth of human cancer cells bearing BRAFV600E. To study in vivo effects, INU-152 was administered using human melanoma and colorectal cancer xenograft models. To explore INU-152′s potential as a prospective drug candidate, pharmacokinetic studies and toxicity tests were performed using mice. Key findings: To inhibit and suppress paradoxical activation in mutant RAS cancer cells completely, it is important for RAF inhibitors to exhibit potent inhibitory activities against RAF isoforms. Significance: INU-152 inhibits all RAF isoforms and inhibits MAPK pathways in mutant BRAF cells. More importantly, INU-152 exhibits minimal paradoxical pathway activation in melanoma cells with mutant RAS. INU-152 exhibits anti-tumor activities in xenograft models carrying BRAF mutations. © 2017 Elsevier Inc. All rights reserved.

1. Introduction The RAF proteins are serine/threonine protein kinases that comprise the mitogen-activated protein kinase (MAPK) pathway, which controls cell proliferation, survival and metastasis. The RAF family members act downstream of RAS, activating MEK which, in turn, phosphorylates and activates ERK [1–4]. The RAF family is comprised of three members: ARAF, BRAF, and CRAF [5,6]. Heterodimerization or homodimerization of the RAF kinase domain is the principal RAS-regulated event in RAF activation [7–9]. The MAPK pathway is hyperactivated for a high percentage of tumors due to the frequent mutation activation of KRAS, NRAS, or BRAF genes [10]. BRAF is mutated in 50–80% of melanomas [11–13], 40% of thyroid cancers, 12% of colon cancers, and 7% of ovarian cancers. CRAF and ARAF are mutated in b 1% of human tumors [14]. The substitution of valine for glutamic acid at position 600 (V600E) accounts for N80% of the BRAF mutations identified in melanoma [15]. Many oncogenic

⁎ Corresponding author at: Division of Life Sciences, Incheon National University, Incheon 406-772, Republic of Korea. E-mail address: [email protected] (S.K. Ahn).

http://dx.doi.org/10.1016/j.lfs.2017.06.021 0024-3205/© 2017 Elsevier Inc. All rights reserved.

BRAF induce MEK-ERK signaling due to their enhanced ability to associate with endogenous CRAF in the presence of oncogenic RAS [16,17]. This suggests that BRAF could potentially cause a catalytic function of CRAF irrespective of BRAF's intrinsic kinase activity [7]. The RAF inhibitors, particularly vemurafenib and dabrafenib, are used as the standard of care for BRAF-mutant metastatic melanomas. However, these drugs frequently acquired resistance after a median response duration of 6–7 months, and rapid development of cutaneous toxicity is enhanced by the paradoxical activation of the MAPK pathway [18–20]. In addition, there are no treatment options for the 15–20% patients who bear a mutated RAS gene [21]. The development of cutaneous toxicity and the ineffectiveness of RAF inhibitors in patients with the RAS mutant both share a common problem: the paradoxical activation of CRAF. Thus, the development of more effective pan-RAF inhibitors is required to reduce or eliminate side effects and resistance. LY3009120 is a pan-RAF, EphA2, ZAK and p38 inhibitor under investigation in phase I clinical trials [22]. BGB-283 is another small molecule inhibitor of multiple kinases, including RAF isoforms and EGFR at both the biochemical and cellular level [23]. However, multi-kinase inhibitors have nonselective kinase inhibition, which resulted in side effects at doses well below those required to inhibit the MAPK signaling.

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In this study, we discovered INU-152, which is a potent and selective pan-RAF inhibitor. We have tested the effect of INU-152 on melanomas and colon cancer cell growth both in vitro and in vivo. INU-152 suppressed the growth of melanomas and colon cancer cells with the BRAF V600E mutation through inhibition of the RAF-MEK-ERK pathway. Interestingly, INU-152 reduced the capacity for paradoxical activation of the MAPK pathway in melanoma cells by activating RAS mutations. INU-152 also significantly reduced tumor volumes in the xenograft mouse model of human melanomas or colon cancers. Toxicology studies confirmed a wide safety margin consistent with a high degree of selectivity. In summary, our study identified INU-152 as a novel anticancer agent with a demonstrated inhibitory activity for BRAF V600E, BRAF and CRFA, which are ideal targets to enhance current melanoma treatments.

concentrations with a final DMSO concentration of 0.5% and incubated for 72 h. At the end of the incubation period, 15 μL of the dye solution was added into each well and cells were incubated at 37 °C for up to 4 h in a humidified, 5% CO2 atmosphere. After incubation, the Solubilization Solution/Stop Mix was added to each well. The absorbance was detected at 570 nm with a Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). The relative proliferation rate of the cells was expressed as the following ratio: (OD of experimental wells/OD of control wells) × 100. IC50 values were derived using a 12-point curve fitted with Prism (GraphPad Software). 2.5. Western blot analysis

INU-152 (N-(3-((3-(9H-purin-6-yl)pyridine-2-yl)amino)-2,4difluorophenyl)furan-3-sulfonamide) was synthesized at Incheon National University. Docking process was performed using the program AutoDock 4.2.

Proteins form total lysates or immunoprecipitated complexes were separated by 6–10% SDS-PAGE and blotted onto nitrocellulose using a Bio-Rad Mini Trans-Blot Electrophoretic Transfer cell. The antibodies used were obtained commercially from the following sources: antiACTIN (SC-4778); antibodies to MEK (9122, 9126), phospho-MEK1/2 (Ser217/221) (9154), ERK (4695), and phospho-ERK1/2 (Thr202/ Tyr204; 4370); and anti-rabbit IgG horseradish peroxidase (HRP)linked secondary antibody from Cell Signaling Technology. Antigen–antibody complexes were visualized using chemiluminescent substrate (Millipore) and detected with the ChemiDoc MP imaging system (BIO-RAD).

2.2. Biochemical kinase assays

2.6. GPCR, ion channel and phosphatase panel assay

The IC50 profiles of INU-152 were determined using three proteins (BRAF, BRAF V600E, and CRFA Y340D/Y341D). IC50 values were measured by testing 10 semi-log concentrations of test compounds using duplicate treatments, ranging from 1 × 10− 6 mol/L to 3 × 10− 11 mol/l. A radiometric protein kinase assay (33 PanQinase® Activity Assay) was used to measure the kinase activity of the three protein kinases. All kinase assays were performed using 50 μL reaction volumes in a 96-well FlashPlatesTM from Perkin Elmer (Boston, MA, USA). The reaction cocktail was pipetted in four steps in the following order: (1) 10 μL of non-radioactive ATP solution (in H2O), (2) 25 μL of assay buffer/[γ-33P]-ATP mixture, (3) 5 μL of test sample in 10% DMSO and (4) 10 μL of enzyme/substrate mixture. All enzyme assays contained 70 mM HEPES-NaOH, pH 7.5, 3 mmol/L MgCl2 , 3 mmol/L MnCl2, 3 μmol/L Na-orthovanadate, 1.2 m mol/L DTT, and ATP/[γ-33P]-ATP (in variable amounts, corresponding to the apparent ATP-km of the respective kinase and substrate). All protein kinases provided by ProQinase were expressed in Sf 9 insect cells or in E. coli as recombinant GST-fusion proteins or His-tagged proteins. All kinases were produced from human cDNAs. These kinase assays were performed at ProQinase GmbH. The potency of INU-152 against a select panel of 40 kinase enzymes was determined using a profiling service (EMD Millipore, MA, USA).

Potency of INU-152 against a selected panel of 76 GPCRs and 8 ion channels was determined using the profiling service (EMD Millipore, MA, USA). Briefly, FLIPR assays were conducted to profile INU-152 for agonist and antagonist activities on the GPCRs. Percentage activation and percentage inhibition values were determined for each GPCR. Electrophysiological assays were conducted to profile INU-152 for activities at 10 μM on the ion channel targets using the IonWorks Quattro and IonWorks HT electrophysiological platforms. A fluorogenic assay format utilizing the generic phosphatase substrate DiFMUP (6,8-difluoro-4methylumbelliferyl phosphate) was conducted to profile INU-152 for activities at 10 μM on the phosphatase targets.

2. Materials and methods 2.1. Chemical synthesis and molecular modeling of INU-152

2.3. Cell lines and cell culture Cell lines (A375P, SK-MEL-2, HEK-293, HT-29 and Colo-205) were obtained from the Korean Cell Line Bank (Korea). All cell lines were maintained in DMEM supplemented with 10% FBS, penicillin–streptomycin, and glutamine at 37 °C in a humidified, 5% CO2 incubator. For experimental purposes, cells were cultured in 60-mm tissue culture dishes until they reached 80% confluence.

2.7. Metabolic stability Human or mouse liver microsomes (0.5 mg protein/mL) in 100 mM potassium phosphate buffer (pH 7.4) were pre-incubated with 0.1, 1, or 10 μM INU-152 at 37 °C for 5 min, and then the reaction was initiated by adding NADPH regenerating solution (BD Biosciences). Samples were collected at 0 and 30 min, and each reaction was terminated by adding three volumes of ice-cold acetonitrile containing an internal standard (imipramine, 80 ng/mL) and mixing the resulting solution with a vortex. This solution was clarified by centrifugation at 10,000 × g for 3 min at 4 °C, and the clear supernatants were collected and transferred to liquid chromatography vials. The samples were analyzed by LC/MS/ MS for INU-152 quantification. Analysis was performed by the Korea Research Institute of Chemical Technology (Daejeon, Korea). 2.8. CYP450 liability profile Five major cytochrome P450 (CYP450) isoforms (1A2, 2C9, 2C19, 2D6, 3A4) were purchased for activity analysis (BD SCIENCE, USA). P450-Glo kit (Promega, USA) was used to assess CYP activities.

2.4. Cell growth assay 2.9. Pharmacokinetics The CellTiter 96® Non-Radioactive Cell Proliferation Assay kit (Promega) was used following the manufacturer's instructions. Briefly, 100 μL of the cell suspension (5 × 103 cells) was dispensed into each well of the 96-well plate (SPL Life Sciences, Korea) and incubated overnight. INU-152 or vemurafenib were then added at various

PK studies were conducted at Korea Research Institute of Chemical Technology. INU-152 was dissolved in DMSO/PEG400/Saline (5/40/55, v/v) to yield a nominal concentration (2 mg/mL, pH 7) for intravenous injection. For oral administration, INU-152 was dissolved in PEG400/

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NMP (9/1, v/v) to yield nominal concentrations (1, 3 and 6 mg/mL, pH 7). Male Sprague Dawley rats weighting 250–300 g were used for all the experiments. INU-152 was administered via either a single intravenous (IV) bolus injection or oral gavage. Blood samples (approximately 300 μL) were collected via cardiac puncture after the animals were euthanized by carbon dioxide inhalation at 0.033, 0.167, 0.5, 1, 2, 4, 6, 8, and 24 h after dose administration. Blood samples were placed into tubes containing sodium heparin and centrifuged at 8000 rpm for 6 min at 4 °C to separate the plasma. Following centrifugation, the resulting plasma was transferred to clean tubes and stored while frozen at − 80 °C until assay. A non-compartmental module of WinNonlin® Professional 5.2 was used to calculate parameters. Bioavailability was calculated with the following equation: F ð%Þ ¼ ðDoseiv  AUCoral ð0−∞ÞÞ=ðDoseoral  AUCiv ð0−∞ÞÞ  100%

2.10. Xenograft models Athymic nude mice (BALB/cAJc1-nu/nu) of approximately 5 weeks of age were obtained from Shanghai SINO-British SIPPR/BK Lab Animal Ltd. For subcutaneous-implanted tumor xenograft models, nude mice were injected with 5 × 106 cells (A375 or Colo205) per mouse. When tumor volumes reached a minimum threshold of 150 mm3, mice were randomly assigned to treatment groups. INU-152, PLX-4720, and sorafenib were dissolved in Cremophor EL/ethanol (50:50; Sigma Cremophor EL, 95% ethanol). CPT-11 was dissolved in 10% dimethylsulfoxide (DMSO). INU-152, PLX-4720, and sorafenib were sonicated for oral dosing and CPT-11 was diluted in saline for intraperitoneal dosing. The treatment lasted for 14 days. The animals were monitored an additional 14 days afterward. Percent tumor growth inhibition was calculated by the following formula: %TGI ¼ 100–ðVTreatment =VVehicle Þ  100 where VTreatment is the average tumor volume of the compound treated group and VVehicle is the average tumor volume of vehicle treated group. 2.11. Dose range-fining study and 14-day repeated dose study of INU-152 A toxicity study was conducted to determine both the maximum tolerated dose (MTD) of INU-152 in mice following a single oral administration and the repeated dose toxicity of INU-152 in mice following a 14-day repeated oral administration. Four treatment groups, each comprised of three male and three female ICR mice, were administered INU152 at respective dose levels of 100, 250, 500 and 1000 mg/kg for the MTD experiment. For the 14-day repeat-dose toxicity study, three treatment groups, each comprised of five male and five female mice, were administered INU-152 at respective dose levels of 30, 100 and 200 mg/kg. One additional group of ten animals (five animals per respective gender) received the vehicle as the control. INU-152 was administered to all groups via oral gavage at a dose volume of 10 mL/kg. Observations for morbidity, mortality, injury, and the availability of food and water were conducted twice daily for all animals across all dose administration trials. Clinical observations, body weights, and food consumption were checked and recorded once daily for 4 days during the MTD study and once daily during the 14-day repeated dose study. Blood samples for hematological evaluations were collected from all the main study animals at the conclusion of the 14-day repeated dose study. After the repeated dose study termination, the surviving animals in the MTD study were euthanized and the carcasses were discarded without further evaluation. However, the surviving animals from the 14-day repeated dose study were necropsied and their organ weights were recorded.

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3. Results 3.1. Novel pan-RAF inhibitor showed selective and potent biochemical activity Using a structure-based molecular modeling, we discovered INU152 as a potent and selective pan-RAF inhibitor containing a purine moiety as the key hinge-binding group [24–26] (Fig. 1A). To understand the binding mode of INU-152 with BRAF V600E, molecular docking was performed at the active site of the X-ray crystal structure of BRAF V600E (3OG7.pdb) [27] (Fig. 1B) and BRAF wild-type (3Q4C) (Fig. 1C) [28]. This model suggested a docking pose similar to the one observed in PLX-4032. Therefore, INU-152 is a type IIB inhibitor, which binds to a DFG-in and αC-helix out Raf conformation [29]. Specific interactions of INU-152 with the BRAF V600E include hydrogen bonding of the purine NH of ligand with the carbonyl oxygen of Cys532 in the hinge region and the sulfone oxygen of ligand with NH moieties of Asp 594 and Phe 595. In assays against recombinant RAF proteins, INU-152 showed a high potency against RAF family members (BRAF IC50 = 2.2 nmol/L, BRAFV600E IC50 = 2 nmol/L, CRAF IC50 = 1.2 nmol/L) (Table 1). To examine the selectivity of INU-152, the activity of INU-152 on other kinases was assessed by screening against a panel of 63 kinases, consisting of a wide range of tyrosine and serine/threonine targets, at a concentration of 10 μmol/L [30]. INU-152 exhibited selectivity in a kinase panel against other kinases. Furthermore, kinase inhibitory activity of N40% with 10 μM INU-152 was only observed against PKCμ and Abl (Table 2). 3.2. INU-152 preserved potency against oncogenic BRAF while minimizing the paradoxical activation of MAPK signaling INU-152 was tested for its activity to inhibit the growth of human cancer cells bearing BRAFV600E (Table 3). INU-152 had an approximately 10-fold increase in potent inhibitory activity than against BRAFV600E human melanoma and colon cancer cell lines. In addition, INU-152 had no cytotoxic effects against human embryonic kidney cells (HEK 293). To assess the inhibitory effect of INU-152 on cellular BRAFV600E activity, we studied the changes of phosphorylation in MEK and ERK, the direct downstream targets of BRAFV600E. As anticipated, INU-152 completely inhibited the phosphorylation of MEK-ERK in A375P cells bearing BRAFV600E when compared with vemurafenib (Fig. 2A). It has been reported that RAS-mutant cells develop resistance to growth suppression of specific BRAF inhibitors due to CRAF activation by RAS mutations [17]. Consistent with the previous report [31], vemurafenib induced the phosphorylation of MEK/ERK in SK-MEL-2 cells with NRAS mutation at both low and high doses. However, INU152 did not increase MEK/ERK phosphorylation at 3–10 μmol/L in these cells, although INU-152 slightly induced activation of MEK/ERK at low doses (0.5–1 μmol/L). Because BRAF inhibitors have been recently reported to cause a rapid recovery of phospho-ERK by reactivating the RAS-CRAF pathway or mitigating ERK-dependent negative feedback [32,33], we next investigated the time-course effect of INU-152 on pERK recovery. Rapid recovery of pERK was observed at 8 h in PLX4032-treated A375P cells, while INU-152 displayed a delayed recovery (24 h). 3.3. GPCR, phosphatase, ion channel panel assay, metabolic stability and CYP450 liability profile We then examined the possibility of off-target effects of INU-152 using a panel of GPCR, phosphatase, and ion channels (Supplementary Table S1). INU-152 exhibited no detectable agonistic activity N 15% on the GPCRs assayed at 12.5 μM. In the GPCR antagonist assay, INU-152 only inhibited A2B receptors with a mean percentage inhibition value of 53.7% at 10 μM. INU-152 exhibited no detectable antagonistic activity

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Fig. 1. Structure and binding mode of INU-152. (A) Chemical structure of INU-152. (B) The docking model of INU-152 was predicted on the basis of the X-ray crystal structure of the BRAF V600E (PDB accession code, 3OG7) and (C) BRAF wild-type (PDB accession code, 3Q4C). Dashed lines are hydrogen bonds.

N50% on the remaining GPCRs assayed at 10 μM. We conducted electrophysiological assays to evaluate the effect of INU-152 on activities for the eight ion channel targets. INU-152, at 10 μM, only inhibited the hERG current with a mean percentage inhibition value of 24%. In vitro assays using 5 CYP450 enzymes indicated that INU-152 is a modest to poor inhibitor of CYP450, having IC50 values N 10 mol/L for the 5 major human isoforms (CYP 1A2, 2C9, 2C19, 2D6 and 3A4) (Supplementary Table S2). To determine the in vitro metabolic stability of INU-152, we incubated INU-152 with mouse and human liver microsomes. We inferred that the t1/2 values were above 60 min because percentages of the remaining parent at 30 min were above 99% and 74%, respectively (Supplementary Table S3). 3.4. Pharmacokinetics parameters of INU-152 INU-152 was administered intravenously and orally at a dose of 10 mg/kg to SD male rats, respectively. Following IV administration of INU-152 at a dose of 10 mg/kg, the value of systemic clearance was 30.0 mL/h/kg. The value of AUC (0–∞) and half-life were 406.8 μg·hour/mL and 11.7 h, respectively. Following oral administration of Table 1 INU-152 is selective against RAF isoforms. Kinase

IC50 (nmol/L)

BRAF BRAFV600E CRAF

2.2 2 1.2

INU-152 at a dose of 10 mg/kg, the value of Cmax and Tmax were 8.6 μg/mL and 2.7 h, respectively. The value of AUC (0–∞) was 342 μg·hour/mL. As a result, the value for the bioavailability of INU-152 was 84% (Table 4). 3.5. In vivo antitumor activity of INU-152 To study the in vivo effect of INU-152 in a xenograft mouse model using melanomas and colon cancer cells with BRAFV600E, INU-152 was administered using A375 human melanoma xenografts. A

Table 2 Percentage inhibition data for INU-152 against a panel of kinases screened at 10 μM. Kinase

% Inhibition

PKCμ Abl PKG1β Lyn GSK3β Flt3 PKG1α

45 41 33 33 31 28 26

Kinase with percentage inhibition b13 at 10 μM: AMPKα1, CaMKIIβ, CaMKIIγ, CaMKIIδ, CaMKIV, CDK1/cyclinB, CDK2/cyclinA, CDK2/cyclinE, CDK3/ cyclinE, CDK5/p25, CDK5/p35, CDK7/cyclinH/MAT1, CDK9/cyclin T1, IR, LKB1, MAPK1, MAPK2, p70S6K, PhKγ2, PKA, PKBβ, PKCα, PKCβI, PKCβII, PKCγ, PKCδ, PKCε, PKCη, PKCι, PKCθ, PKCζ, PKG1α, ROCK-II, SAPK2a.

S.P. Hong, S.K. Ahn / Life Sciences 183 (2017) 37–44 Table 3 Antiproliferative activity of INU-152 versus solid tumor cell lines. EC50 (nmol/L)

A375P COLO 205 (BRAFV600E) (BRAFV600E)

INU-152 5 vemurafenib 80

1 16

HT-29 SK-MEL2 (BRAFV600E) (NRASQ61R)

HEK-293

5 80

N15,000 N15,000

4833 8607

PLX4032 analog, PLX4720, and CPT-11 were tested as standard drugs to compare in vivo effects for tumor suppression. For the A375 human melanoma xenograft model, administration of INU-152 at 30 mg/kg once daily resulted in the 78% inhibition of tumor growth relative to the

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controls, while sorafenib (50 mg/kg) and PLX-4720 (30 mg/kg) only exerted tumor suppressive effects of 32% and 43%, respectively (Fig. 3A). It was suggested that INU-152 has a more potent inhibitory effect on tumor growth than the two well-known RAF inhibitors. When we also investigated the dose-dependent in vivo effect of INU-152 using the A375 xenograft model, we observed a 69% and 79% reduction of tumor volume at 5 mg/kg and 20 mg/kg doses, respectively (Fig. 3B). We next studied the anti-tumor effects of INU-152 using the xenograft model with colorectal cancer, with Colo-205 bearing BRAFV600E. INU152 showed a 51% reduction of tumor volume compared with controls after once-daily oral administration of 20 mg of INU-152. In addition, a twice-daily administration of 10 mg/kg of INU-152 resulted in better

Fig. 2. Effect of INU-152 on MEK and ERK phosphorylation in melanoma cell lines. (A) A375P cells were treated without or with various concentrations of INU-152 or for 24 h. (B) SK-MEL-2 cells were treated without or with various concentrations of INU-152 or for 24 h. (C) A375P cells were treated with PLX4032 or INU-152 for increasing periods of time (0–48 h).

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Table 4 Pharmacokinetics parameters of INU-152 in SD rats (n = 3). Parameters

CL, mL/h/kg

Cmax, μg/mL

Tmax, hour

AUC (0–∞), μg·hour/mL

T1/2, hour

i.v. (10 mg/kg) p.o. (10 mg/kg)

30.0 ± 18.9

58.3 ± 2.5

0.03 ± 0.0

406.8 ± 183.0

11.7 ± 5.1

31.7 ± 11.2

8.6 ± 3.0

2.7 ± 2.9

342.0 ± 114.5

22.9 ± 13.6

antitumor efficacy with a 62% TGI (Fig. 3C), (Table 5). All dosage treatments were well-tolerated by the mice with no mortality and minimal body weight loss (less than5% relative to vehicle-matched controls). 3.6. Dose range-fining study and repeat-dose toxicity studies To evaluate the safety of INU-152, a dose range-finding study and a 14-day repeated dose study were conducted using mice. Mice body weights among each group decreased after treatments with INU-152 at single administration doses of 100 mg/kg, 250 mg/kg, 500 mg/kg, and 1000 mg/kg but subsequently increased gradually during the recovery period (Supplementary Fig. S1). After 14 days repeated oral administration of INU-152, the body weights of male animals dosed with 100 mg/kg and 200 mg/kg decreased significantly on day 4 when compared with the vehicle control. The body weights of female animals dosed with 100 mg/kg decreased significantly on days 5 and 7 when compared with the vehicle control. However, the body weights of the treatment animals increased gradually until the study termination (Supplementary Fig. S2). At the termination of the 14-day repeated dose study, the levels of RBC (erythrocytes), HGB (hemoglobin), HCT (hematocrit), and lymphocytes of males dosed with 200 mg/kg/day decreased significantly when compared to the vehicle control group. The levels of reticulocytes and neutrophils increased significantly when compared to the vehicle control group. The decrease of RBC, HGB, HCT and lymphocytes as well as the increase of reticulocytes and neutrophils in males dosed with 200 mg/kg/day were considered test article-related. No INU-152-related effects were identified in females from any of the dosage treatments (data not shown). Based on the data, the Maximum Tolerated Dose (MTD) for a single dose was 1000 mg/kg, and the Maximum Tolerated Dose (MTD) for a 14 days repeated dose was 100 mg/kg/day. 4. Discussion Progressive understanding of melanoma biology has led to the development of several targeted therapeutic drugs such as vemurafenib, ipilimumab, dabrafenib, and trametinib [34]. Selective BRAFV600E inhibitors, in particular vemurafenib and dabrafenib, were approved for metastatic melanoma patients with mutant BRAF genes [18–20,35,36]. They showed excellent clinical responses, such as improved progression free survival and overall survival in patients, when compared with dacarbazine chemotherapy [35,36]. It is suggested that disruption of BRAF kinase activity could benefit metastatic melanoma patients [37]. At higher vemurafenib exposures, N 80% inhibition of ERK phosphorylation in the tumors of patients correlated with clinical response [27]. In that respect, sorafenib did not ameliorate clinical outcomes for patients with melanoma [38,39]. This effect occurs because sorafenib is a less potent BRAFV600E inhibitor compared with vemurafenib and dabrafenib. These results suggest that a highly potent BRAFV600E inhibitor is needed to elicit significant clinical responses in melanoma. However, the weakness of vemurafenib and dabrafenib are their acquired resistances, which always develop after a median response duration of 6–7 months, leading to a rapid development of cutaneous squamous cell carcinomas [18–20]. These carcinomas are caused by the reactivation of ERK and the paradoxical activation of the MAPK pathway [17,33,40,41]. In addition, there are no treatment options for the 15–20% of patients who possess

F, %

84

a mutant RAS melanoma. The development of cutaneous toxicity and the ineffectiveness of RAF inhibitors in patients bearing RAS mutants both share the common problem of paradoxical CRAF activation. Binding of the BRAF inhibitor to BRAF during hetero-dimerization induces an allosteric change that transactivates the partner CRAF, which is an unbound member of the dimer by inhibitor. This activates the MEKERK-pathway in a continuous cycle [17,41]. Therefore, to overcome the paradoxical activation of the MAPK pathway, it is necessary to develop a pan-RAF inhibitor. Furthermore, as shown in the case of sorafenib, the new generation of pan-RAF inhibitors must have a higher potency for RAF family members than vemurafenib to demonstrate clinical efficacy [39]. INU-152 is a novel ATP-competitive, potent, and highly selective small-molecule inhibitor of the RAF family including BRAF, CRAF, and mutant BRAFV600E. RAF inhibitors are classified into two categories depending on their mode of action. Type 1 RAF inhibitors (such as vemurafenib) bind to the active conformation of RAF members, while type 2 RAF inhibitors (such as sorafenib) bind to the inactive conformation of RAF members [42,43]. Using a structure-based design, we discovered a potent and selective pan-RAF inhibitor bearing a purine moiety as the key hinge-binding group. INU-152 was designed to bind within the BRAF selective pocket, near to the ATP-binding site, suggesting that INU152 could be a type 1 RAF inhibitor. Vemurafenib, which acts as type 1 RAF inhibitor, has inhibitory potency for CRAF (IC50 48 nM), BRAFV600E (IC50 31 nM), and BRAF (IC50 100 nM), while INU-152 has a higher potency for the inhibition of CRAF (IC50 1.2 nM), BRAFV600E (IC50 2 nM), and BRAF (IC50 2.2 nM) [25,41], implying that INU-152 is a potent and selective pan-RAF inhibitor with an IC50 value in the sub-nanomolar range. Although INU-152 simultaneously inhibits RAF family members, it showed selectivity against non-Raf kinases. In the cellular assay, INU-152 exhibited an approximately 10-fold higher inhibitory activity than vemurafenib against melanoma and colon cancer cell lines bearing BRAFV600E, suggesting that INU-152 has a more potent in vitro effect on growth inhibition of cells with BRAFV600E than vemurafenib. However, Colo205, as a cell line lacking PI3K/AKT pathway mutation, appears to be more sensitive to INU-152 than HT-29 which have mutant PIK3CA. There is a possibility that combined treatment of INU-152 with PI3K inhibitors may significantly reduce cell viability compared with single agent treatment in colorectal cancer cells [44]. INU-152 consistently completely inhibited MEK-ERK phosphorylation in A375P cells at lower doses than vemurafenib. Despite moderate sensitivity on growth inhibition of NRAS-mutant melanoma cells, INU-152 demonstrated a reduced capacity for paradoxical activation MAPK pathway in these cells. Because it is pan-RAF inhibitor blocking both BRAF and CRAF activities [17]. Therefore, these data suggest that INU-152 might be an ideal BRAF V600E inhibitor without the side effects of vemurafenib. INU-152 caused a significant and dose-dependent reduction of tumor volume in xenograft mice using BRAFV600E mutant cells. Especially, administration of INU-152 in the A375 xenograft models caused a more enhanced anti-tumor effect than that of PLX-4720 or sorafenib in parallel with the in vitro effects. Notably, INU-152 showed no toxicity in any tumor xenograft model tested. Furthermore, to evaluate the potential toxicity of INU-152, we conducted single and repeated-dose toxicity studies in mice. The maximum tolerated dose (MTD) for single and

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Table 5 Antitumor efficacy of INU-152 in xenograft models. Tumor

Route

Dose(mg/kg)

TGI (%)

A375

PO PO PO PO PO

5 Q.D. 20 Q.D. 30 Q.D. 10 B.I.D. 20 Q.D.

69.47 78.85 78.44 61.87 50.79

COLO205

Abbreviations: Q.D., once a day dosing; B.I.D., twice a day dosing; TGI, tumor growth inhibition.

5. Conclusions In the present study, we describe the biochemical and pharmacological properties of INU-152, which is a well-tolerated, orally active panRAF inhibitor with a decreased capacity for paradoxical activation of the RAF-MEK pathway. Therefore, our findings provide a rationale for the use of INU-152 as a pan-RAF inhibitor for the treatment of melanoma, and these results suggest an innovative strategy to develop nextgeneration therapeutic agents against advanced melanoma. Acknowledgements This work was supported by the Incheon National University Research Grant in 2014. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.lfs.2017.06.021. References

Fig. 3. In vivo antitumor activity of INU-152. (A) and (B) Mice bearing established subcutaneous tumors derived from A375 cell lines were treated daily from day 0 to 14 with sorafenib, PLX-4720 or INU-152. (C) Mice bearing established subcutaneous tumors derived from Colo205 cell lines were treated daily or twice from day 0 to 14 with CPT-11 (QD, 30 mg/kg) or INU-152 (QD, 20 mg/kg; BID, 10 mg/kg).

14-day repeated dose was approximately 1000 mg/kg and 100 mg/kg/day, respectively. These cumulative results suggest that INU-152 has an acceptable margin of safety as well as a selective potential for treating melanoma.

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