H-Ras·GTP; Ras-Raf interaction ( Ki = 149 μM )
Target: Kobe-2602 targets the Ras-Raf interaction by competitively inhibiting the binding of H-Ras·GTP to the c-Raf-1 RBD (Ras-binding domain) with a Ki value of 149±55 μM. By disrupting this critical protein-protein interaction in the MAPK signaling pathway, it inhibits downstream Raf-MEK-ERK signaling. Kobe-2602 inhibits Ras·GTP but not Ras·GDP, indicating specificity for the active GTP-bound form of Ras. The compound inhibits cellular Ras-Raf binding with an IC50 value of approximately 10 μM.

Kobe2602 (2–20 μM; 1 hour) shows Ras–Raf–binding inhibition in NIH 3T3 cells[1].
Kobe2602 inhibits cellular Ras-Raf binding with an IC50 value of roughly 10 μM[1].
Kobe2602 (20 μM) effectively suppresses the phosphorylation of Raf downstream kinases MEK and ERK in NIH 3T3 cells that are transiently expressing H-Ras^G12V[1].
Kobe2602 inhibits Ras⋅GTP but not Ras⋅GDP[1].
Kobe2602 (20 μM) prevents H-Ras^G12V-transformed cells from proliferating anchorage-dependently[1].

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In Vitro Activity: Kobe-2602 (2-20 μM; 1 hour) demonstrates Ras-Raf binding inhibition in NIH 3T3 cells. At 20 μM, it effectively suppresses the phosphorylation of Raf downstream kinases MEK and ERK in NIH 3T3 cells transiently expressing H-RasG12V. It inhibits cellular Ras-Raf binding with an IC50 of roughly 10 μM. Kobe-2602 (20 μM) prevents H-RasG12V-transformed cells from proliferating in an anchorage-dependent manner. It efficiently suppresses H-Ras(G12V)-transformed NIH 3T3 cells' anchorage-dependent and independent growth and induces apoptosis. This is accompanied by down-regulation of downstream molecules including MEK/ERK, Akt, and RalA, as well as the upstream molecule Son of Sevenless.

Kobe2602 (80 mg/kg; p.o.; five consecutive days per week; for 17 days) demonstrates antitumor activity against a xenograft of K-Ras^G12V-carrying human colon carcinoma SW480 cells[1].

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In Vivo Activity: Oral administration of Kobe-2602 (80 mg/kg; p.o.; five consecutive days per week; for 17 days) demonstrates antitumor activity against a xenograft of human colon carcinoma SW480 cells carrying the K-ras(G12V) gene. The compound displays antitumor activity by interfering with Ras-GTP-effector interaction. Kobe-2602 and its analog Kobe0065 exhibit inhibitory activity toward H-Ras·GTP-c-Raf-1 binding both in vivo and in vitro. Kobe-2602 might act as a building block for the creation of more potent and selective Ras inhibitors.

Biochemical Assays[1]
H-Ras (residues 1–166) and GST-c-Raf-1-Rasbinding doman (RBD; residues 50–131) were produced in Escherichia coli and purified as described previously. For the in vitro binding inhibition assays, H-Ras(1–166), preloaded with [γ35S]GTPγS, was incubated with GST-c-Raf-1-RBD(50–131) at 25 °C for 30 min in the presence of the compound and the amount of bound H-Ras was quantified as the radioactivity pulled down by glutathione-sepharose resin. The Ki value for the compound was calculated as described in Fig. S1. For in vivo assays, NIH 3T3 cells were transfected with pEF-BOS-HA-H-RasG12V or pEF-BOS-HAK-RasG12V, cultured for 18 h at 10% (vol/vol) FBS, and then incubated in the presence of the compound at 2% (vol/vol) FBS for 1 h. Cells were lysed in 50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 10% (vol/vol) glycerol, 1 mM EDTA, 1 mM DTT, phosphatase inhibitor mixture, and protease inhibitor mixture and subjected to detection of c-Raf-1 coimmunoprecipitated with an anti-H-Ras antibody (C-20) by Western blotting with an anti-cRaf-1 antibody (C-12), of phosphorylated MEK and ERK with anti-pMEK1/2 (p217/p221) and antiERK1/2 (p202/204) antibodies, and of phosphorylated Akt with an anti-pAkt antibody (Ser473) and of RalA·GTP pulled down with GST-Sec5(1–99) immobilized on glutathione-sepharose resin by an anti-RalA antibody. HA-tagged H-RasG12V·GTP was detected by an antiHA antibody. In vitro assays for the kinase activity of recombinant c-Raf-1 were performed by using a Raf-1 Kinase Assay kit. In vitro GDP–GTP exchange assays were done by incubating 600 nM GST-H-Ras(1–166)·GDP immobilized on glutathione-sepharose resin with 11 μM [γ35S]GTPγS (1,500 cpm/pmol) at 25 °C in the presence of purified 6×His-tagged mouse Son of sevenless (mSos)1(563–1,049) (180 nM each), wildtype, or a W729E mutant (5) in buffer B [50 mM Tris·HCl (pH 7.4), 50 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 20 mM imidazol]. The radioactivity remaining on the resin after an intensive washing was quantified by liquid scintillation counting. Varying concentrations of compounds were added to the reaction mixtures to observe their inhibitory effect.

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In Vitro Enzyme/Receptor Binding Protocol: For in vitro binding inhibition assays, H-Ras(1-166) preloaded with [γ-S]GTPγS is incubated with GST-c-Raf-1-RBD(50-131) at 25°C for 30 minutes in the presence of the compound. The amount of bound H-Ras is quantified as the radioactivity pulled down by glutathione-sepharose resin. Competition binding experiments with varying concentrations of Kobe-2602 determine Ki values. Migration assays are conducted in wells of a 24-well, 8 μm pore size Transwell plate.

Cell Line: H-ras^G12V-transformed NIH 3T3 cells
Concentration: 20 μM
Incubation Time: 24 hours , 48 hours, 72 hours
Result: Efficiently inhibited colony formation in soft agar in a dose-dependent manner.

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Colony Formation Assays.[1]
Cells (103 to 104 ) were inoculated in 2 mL of DMEM containing 10% (vol/vol) FBS, 0.33% SeaPlaque agarose, and one of the compounds and overlaid onto bottom agar consisting of 4 mL of DMEM containing 10% (vol/vol) FBS, 0.6% SeaPlaque agarose, and the same concentration of the compound in a six-well culture plate. After incubation at 37 °C for 14–21 d, the number of colonies >200 μm in diameter was counted under a dissecting microscope.

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Cell Proliferation Assays.[1]
Cells (2 × 103 ) were seeded in a 96-well plate and cultured in DMEM containing 2% (vol/vol) FBS in the presence of one of the compounds. Viable cell numbers were measured by formazan formation using a Cell Counting Kit 8. Apoptotic cells were detected by a standard TUNEL assay using an In Situ Cell Detection kit.

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In Vitro Cell-Based Assay Protocol: NIH 3T3 cells transiently expressing H-RasG12V are treated with Kobe-2602 at concentrations ranging from 2 to 20 μM for 1 hour. After incubation, cells are lysed and protein phosphorylation is assessed by Western blotting using antibodies against phospho-MEK and phospho-ERK. Cell proliferation is measured using anchorage-dependent and independent growth assays. Apoptosis is evaluated by standard methods such as Annexin V/PI staining or caspase activity assays.

Female athymic nude mice (6-8 wk old), with SW480 cells xenograft
80 mg/kg
Oral administration, five consecutive days per week, for 17 days

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Tumor Xenografts. [1]
Cells (5 × 106 ) were implanted into the right flanks of female athymic nude mice (6–8 wk old). After tumor sizes reached ∼50 mm3 on average, compounds (e.g. Kobe0065) suspended in Cremophor:ethanol:water (1:1:6) were administered orally for five consecutive days per week for 17 d. Tumor volumes (V) were calculated with the following formula: V = A × B2 /2, where A is the largest diameter and B is the perpendicular diameter. Dissected tumors after 17-d administration of the 80 mg/ kg compounds were fixed in 4% (wt/vol) paraformaldehyde and embedded in paraffin. Their sections were subjected to immunohistochemistry with an anti-ERK1/2 antibody or an anti-CD31 antibody using a HISTMOUSE-PLUS kit. Apoptotic cells were detected by a TUNEL assay. Statistical significance for groups of three or more was determined by one-way ANOVA with Tukey’s test for post hoc analysis.

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In Vivo Animal Assay Protocol: Xenograft models using human colon carcinoma SW480 cells carrying the K-ras(G12V) gene are employed. Tumor-bearing mice receive Kobe-2602 at 80 mg/kg via oral administration, five consecutive days per week for 17 days. Tumor growth inhibition is monitored by caliper measurement. Body weight and general health are monitored for toxicity. At study end, tumors are excised for histology and pharmacodynamic markers.

Pharmacokinetics: Detailed pharmacokinetic properties of Kobe-2602 have not been fully characterized in publicly available sources. As a small molecule with molecular weight 419.31, it is expected to have reasonable oral bioavailability given that oral administration at 80 mg/kg demonstrated antitumor activity. The compound is soluble in DMSO at 79 mg/mL (188.4 mM). Further ADME studies would be needed for comprehensive pharmacokinetic profiling.

Toxicity: No detailed toxicity data are publicly available for Kobe-2602. In xenograft studies at 80 mg/kg oral administration, no significant adverse effects were reported. As a research compound not intended for human use, comprehensive toxicological profiling has not been extensively published. Standard preclinical safety assessments would be required before any clinical development. The compound is for research use only.

[1]. In silico discovery of small-molecule Ras inhibitors that display antitumor activity by blocking the Ras-effector interaction. Proceedings of the National Academy of Sciences of the United States of America (2013), 110(20), 8182-8187,

Mutational activation of Ras oncogene products (H-Ras, K-Ras, and N-Ras) is common in human cancers, making them highly promising targets for anticancer drugs. However, due to the lack of well-defined surface pockets suitable for drug binding, there are currently no effective strategies for developing Ras inhibitors. Until recently, such pockets were discovered in the crystal structure of Ras·GTP with its unique conformation. This article reports the successful development of a small-molecule Ras inhibitor through computer screening targeting a pocket found in the crystal structure of M-Ras·GTP carrying an H-Ras-type P40D substitution. The screened compound Kobe0065 and its analogue Kobe2602 exhibited inhibitory activity against H-Ras·GTP-c-Raf-1 binding both in vivo and in vitro. They can effectively inhibit anchorage-dependent and anchorage-independent growth and induce apoptosis in H-ras(G12V)-transformed NIH 3T3 cells, accompanied by downregulation of downstream molecules such as MEK/ERK, Akt and RalA, as well as upstream molecule Son of sevenless. In addition, after oral administration, they also showed antitumor activity against human colon cancer SW480 cell xenografts carrying the K-ras(G12V) gene. The NMR structure of the compound and the H-Ras⋅GTP(T35S) complex showed that it has a unique conformation, confirming that it is inserted into one of the surface pockets and providing a molecular basis for inhibiting its binding with a variety of Ras⋅GTP interacting molecules. This study demonstrates the effectiveness of our structure-based drug design strategy for Ras⋅GTP, and the resulting Kobe0065 family compounds can serve as a scaffold for developing Ras inhibitors with higher potency and specificity. [1]

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Additional Information: Kobe-2602 has CAS number 454453-49-7 and is also known as Kobe2602, 2-[2,6-dinitro-4-(trifluoromethyl)phenyl]-N-(4-fluorophenyl)hydrazinecarbothioamide, and CHEMBL5280849. It is an analog of Kobe0065 that efficiently suppresses H-Ras(G12V)-transformed cell growth. The compound was identified by structure-based drug design and acts as a Ras-Raf interaction inhibitor. It has anticancer chemotherapeutic activities. Kobe-2602 might serve as a building block for the creation of more potent and selective Ras inhibitors. For research use only, not for human therapy.

C14H9N5O4F4S

419.31096

419.031

C, 40.10; H, 2.16; F, 18.12; N, 16.70; O, 15.26; S, 7.65

454453-49-7

3827738

Light yellow solid powder

1.7±0.1 g/cm3

450.8±55.0 °C at 760 mmHg

226.5±31.5 °C

0.0±1.1 mmHg at 25°C

1.683

5.12

3

10

3

28

568

0

FC(C1=CC([N+]([O-])=O)=C(NNC(NC2=CC=C(F)C=C2)=S)C([N+]([O-])=O)=C1)(F)F

NNPBSITXCGPXJC-UHFFFAOYSA-N

InChI=1S/C14H9F4N5O4S/c15-8-1-3-9(4-2-8)19-13(28)21-20-12-10(22(24)25)5-7(14(16,17)18)6-11(12)23(26)27/h1-6,20H,(H2,19,21,28)

1-[2,6-dinitro-4-(trifluoromethyl)anilino]-3-(4-fluorophenyl)thiourea

Kobe 2602; Kobe2602; kobe2602; 454453-49-7; 2-[2,6-Dinitro-4-(trifluoromethyl)phenyl]-N-(4-fluorophenyl)hydrazinecarbothioamide; CHEMBL5280849; 1-[2,6-dinitro-4-(trifluoromethyl)anilino]-3-(4-fluorophenyl)thiourea; 3-{[2,6-dinitro-4-(trifluoromethyl)phenyl]amino}-1-(4-fluorophenyl)thiourea; 2-(2,6-dinitro-4-(trifluoromethyl)phenyl)-N-(4-fluorophenyl)hydrazinecarbothioamide; Kobe-2602

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 14.3~84 mg/mL (34.1~200.3 mM)
Ethanol: ~14 mg/mL

Solubility in Formulation 1: ≥ 5 mg/mL (11.92 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 50.0 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 2: ≥ 5 mg/mL (11.92 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 50.0 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.)