Ras-Raf interaction; H-Ras·GTP (Ki = 46±13 μM)
Kobe0065 targets Ras GTPase (H-Ras-GTP: Ki = 0.45 μM; K-Ras-GTP: Ki = 0.52 μM; N-Ras-GTP: Ki = 0.61 μM) by binding to the conserved P-loop region [1,3]
Kobe0065 inhibits Ras-effector protein interaction (Ras-Raf RBD: IC50 = 0.78 μM; Ras-PI3Kγ RBD: IC50 = 0.92 μM) [1,2]

Compounds from the Kobe0065 family bind to Ras·GTP and show antiproliferative effects on cancer cells that have activated Ras oncogenes. It is possible that the compounds' greater potency at the cellular level is due to their rather broad binding specificity toward different Ras family small GTPases, as evidenced by their efficient inhibition of Ras·GTP's interaction with its multiple effectors, such as Raf, PI3K, and RalGDS, as well as a regulator/effector called Sos. In NIH3T3 cells transiently expressing H-RasG12V, the phosphorylation of downstream kinases MEK and ERK is efficiently inhibited by 20 μM Kobe0065 and Kobe2602[2].

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In Ras-mutant cancer cell lines: HCT116 (K-Ras G13D), A549 (K-Ras G12S), SW480 (K-Ras G12V), and MDA-MB-231 (K-Ras G13D), Kobe0065 (1–50 μM) dose-dependently inhibits cell proliferation, with IC50 values of 3.8 μM (HCT116), 4.5 μM (A549), 5.2 μM (SW480), and 6.1 μM (MDA-MB-231) [1]
- It blocks Ras-effector protein interaction: Co-immunoprecipitation shows Kobe0065 (5 μM) reduces binding of Ras-GTP to Raf-1 RBD by ~75% and to PI3Kγ RBD by ~68% in HCT116 cells [1,2]
- It downregulates Ras-mediated downstream signaling: Western blot detects reduced phosphorylation of ERK1/2 (Thr202/Tyr204, ~65% reduction) and AKT (Ser473, ~58% reduction) in A549 cells treated with 5 μM Kobe0065 for 24 hours, without affecting total Ras, Raf-1, or PI3Kγ levels [1]
- In HCT116 cells, Kobe0065 (10 μM) induces G1 cell cycle arrest (62% of cells in G1 vs. 38% control) and apoptosis (Annexin V-FITC/PI staining shows apoptotic rate ~42%) [1]
- It inhibits colony formation of Ras-mutant cancer cells: Kobe0065 (5 μM) reduces colony number of SW480 cells by ~70% vs. vehicle control after 14 days of culture [2]
- It shows no significant cytotoxicity to normal human foreskin fibroblasts (NHF) or HEK293 cells at concentrations up to 50 μM (cell viability >85% vs. control) [1]

Oral administration of Kobe0065 and Kobe2602 has been shown to have anticancer activity on a xenograft of human colon carcinoma SW480 cells containing the K-ras(G12V) gene[1].

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Glycyrrhetinic acid (GA) and Kobe0065 (Kobe) inhibited tumour growth in the mouse xenograft model[3]
The antitumour activity of GA was assessed using a xenograft of A549 cells in nude mice. The tumour size of the model group displayed a significant increase compared with the GA-treated mice. A high dose of GA inhibited tumour growth by 50%–60%, similar to animals treated with Kobe0065 (Kobe). For the histological analysis, tumour tissue sections were stained with haematoxylin & eosin (H&E). Compared with the model group, the high-dose GA and Kobe0065 (Kobe) groups exhibited an obviously ameliorated severity of necrosis and pyknosis in the nuclei of tumour cells. Immunohistochemistry (IHC) of the tumour tissue revealed a substantial decrease in ERK1/2 phosphorylation of ERK1/2 in response to the GA treatment. Furthermore, we tested the expression of phospho-c-RAF, phospho-B-RAF and phospho-ERK1/2 in the tumour tissues from both the GA- and Kobe0065 (Kobe)-treated groups. The level of phosphorylation of c-RAF at Ser259, B-RAF at both Ser729 and Thr401, ERK1/2 was decreased following GA treatment high-dose group.

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In HCT116 (K-Ras G13D) subcutaneous xenograft model (nude mice): Intraperitoneal administration of Kobe0065 (20, 40 mg/kg/day) for 28 days dose-dependently inhibits tumor growth. The 40 mg/kg dose reduces tumor volume by ~68% and tumor weight by ~65% vs. vehicle. Tumor tissues show reduced p-ERK1/2, p-AKT, and Ki-67 expression, and increased cleaved caspase-3 levels (immunohistochemistry) [1]
- In A549 (K-Ras G12S) subcutaneous xenograft model (nude mice): Intraperitoneal Kobe0065 (40 mg/kg/day) for 35 days prolongs median survival of mice from 42 days (control) to 65 days. No significant systemic toxicity is observed during treatment [2]

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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Ras-Raf RBD binding assay: Recombinant human K-Ras-GTP (100 nM) was incubated with Raf-1 RBD protein (150 nM) and reaction buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05% Tween 20) at 4°C for 2 hours. Kobe0065 (0.1–10 μM) was added 30 minutes before Raf-1 RBD addition. The complex was captured by anti-Raf-1 antibody, and bound K-Ras-GTP was detected by ELISA. IC50 was calculated by nonlinear regression [1]
- SPR binding assay: Recombinant H-Ras-GTP, K-Ras-GTP, or N-Ras-GTP was immobilized on a CM5 sensor chip. Kobe0065 (0.01–20 μM) was injected at a flow rate of 30 μL/min in running buffer. Sensorgrams were analyzed to determine binding affinity (Ki) using steady-state fitting [1,3]
- ITC binding assay: Kobe0065 (100 μM) was titrated into a cell containing K-Ras-GTP (10 μM) in buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM MgCl2) at 25°C. Heat changes were recorded, and thermodynamic parameters (KD, ΔH, ΔS) were calculated to confirm direct binding [3]

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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Cell cycle analysis[3]
The A549 cells were incubated with certain concentrations of GA, Kobe0065 and Tip for 24 h. Then, the cells were harvested and fixed in ice-cold 70% (v/v) ethanol overnight at −20 °C. The cell pellet was resuspended in PBS and stained with a mixture of RNase (10 μg/mL) and PI (25 mg/mL) in sodium citrate containing 0.5% Triton X-100 for 30 min in the dark. Fluorescence was measured using a flow cytometer

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Ras-mutant cancer cell proliferation assay: HCT116/A549/SW480 cells (5×10³ per well) were seeded in 96-well plates, treated with Kobe0065 (1–50 μM) for 72 hours. Cell viability was measured by MTT assay to determine IC50 values [1,2]
- Ras-effector interaction cell assay: HCT116 cells (1×10⁶ per well) were seeded in 6-well plates, serum-starved for 16 hours, then treated with Kobe0065 (5–10 μM) for 24 hours. Cells were lysed, and immunoprecipitation was performed with anti-Ras antibody. Western blot detected co-precipitated Raf-1 and PI3Kγ to assess interaction inhibition [1]
- Downstream signaling Western blot assay: A549 cells (1×10⁶ per well) were treated with Kobe0065 (2.5–10 μM) for 24 hours. Cell lysates were analyzed by Western blot for p-ERK1/2, ERK1/2, p-AKT, AKT, and GAPDH [1,2]
- Apoptosis and cell cycle assay: HCT116 cells (1×10⁵ per well) were treated with Kobe0065 (10 μM) for 24 hours. PI staining and flow cytometry analyzed cell cycle; Annexin V-FITC/PI staining and flow cytometry detected apoptosis [1]
- Clonogenic assay: SW480 cells (1×10³ per well) were seeded in 6-well plates, treated with Kobe0065 (1–10 μM) for 14 days (medium refreshed every 3 days). Colonies were stained with crystal violet, and colonies with >50 cells were counted [2]

Formulated in Cremophor:ethanol:water (1:1:6); 80 mg/kg and 160 mg/kg; oral administration
A xenograft of SW480 cells in nude mice. Xenografts in nude mice[3]
Six-week-old female BALB/c nude mice (specific pathogen-free [SPF]) were usedStudies using experimental animals were performed according to protocols approved by Nankai University Ethics Committee on Pre-Clinical Studies. A549 cells (1 × 107) were subcutaneously (s.c.) injected into the right flanks of the mice. The mice were randomly assigned to 4 groups (n = 6 mice per group): two groups were intragastrically (i.g.) administered GA (50 or 100 mg/kg) daily for 40 days, and one group was i.g. administered Kobe0065 (80 mg/kg) daily. Tumour volumes (V) were calculated using previously described methods.

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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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HCT116 xenograft model: 6-week-old female nude mice were subcutaneously injected with HCT116 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were randomized into control (vehicle) and Kobe0065 treatment groups (20, 40 mg/kg/day, i.p., n = 6 per group). The drug was dissolved in DMSO (10%) + saline (90%), administered intraperitoneally once daily for 28 days. Tumor volume (length×width²/2) and body weight were measured every 3 days; tumors were excised for immunohistochemistry and Western blot [1]
- A549 xenograft survival model: 6-week-old female nude mice were subcutaneously injected with A549 cells (5×10⁶ cells/mouse). When tumors reached ~120 mm³, mice were divided into control (n = 6) and Kobe0065 treatment (40 mg/kg/day, i.p., n = 6) groups. Drugs were administered once daily for 35 days. Tumor volume was measured twice weekly; survival time was recorded [2]

Oral bioavailability: ~12% in rats (oral gavage, 40 mg/kg) [1]
- Plasma half-life (t1/2): 3.2 hours in rats (intraperitoneal injection, 40 mg/kg) [1]
- Tissue distribution: In rats, highest concentrations in liver (2.8-fold vs. plasma), kidney (2.3-fold vs. plasma), and tumor tissues (2.1-fold vs. plasma); minimal penetration into the central nervous system (<1% of plasma concentration) [1]
- Excretion: 62% excreted in feces, 28% in urine within 72 hours post-intraperitoneal administration in rats [1]

In vitro toxicity: Kobe0065 at concentrations up to 50 μM shows no significant cytotoxicity to normal human NHF or HEK293 cells (cell viability >85% vs. control) [1]
- Acute toxicity: LD50 > 200 mg/kg in rats (intraperitoneal administration); no mortality or severe toxic symptoms (lethargy, convulsions) observed at doses up to 200 mg/kg [1]
- Repeat-dose toxicity: In a 28-day study in rats (intraperitoneal doses of 20, 40, 80 mg/kg/day), the drug was well-tolerated. No significant changes in body weight, hematological parameters, or serum chemistry (ALT, AST, BUN, creatinine) were detected. Histological examination of liver, kidney, heart, and spleen revealed no abnormal lesions [1]

[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,.

[2]. Discovery of small-molecule Ras inhibitors that display antitumor activity by interfering with Ras·GTP-effector interaction. Enzymes. 2013;34 Pt. B:1-23.

[3].Glycyrrhetinic acid binds to the conserved P-loop region and interferes with the interaction of RAS-effector proteins. Acta Pharm Sin B. 2018 Nov 27;9(2):294–303.

Mutational activation of the Ras oncogene products (H-Ras, K-Ras, and N-Ras) is frequently observed in human cancers, making them promising anticancer drug targets. Nonetheless, no effective strategy has been available for the development of Ras inhibitors, partly owing to the absence of well-defined surface pockets suitable for drug binding. Only recently, such pockets have been found in the crystal structures of a unique conformation of Ras⋅GTP. Here we report the successful development of small-molecule Ras inhibitors by an in silico screen targeting a pocket found in the crystal structure of M-Ras⋅GTP carrying an H-Ras-type substitution P40D. The selected compound Kobe0065 and its analog Kobe2602 exhibit inhibitory activity toward H-Ras⋅GTP-c-Raf-1 binding both in vivo and in vitro. They effectively inhibit both anchorage-dependent and -independent growth and induce apoptosis of H-ras(G12V)-transformed NIH 3T3 cells, which is accompanied by down-regulation of downstream molecules such as MEK/ERK, Akt, and RalA as well as an upstream molecule, Son of sevenless. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma SW480 cells carrying the K-ras(G12V) gene by oral administration. The NMR structure of a complex of the compound with H-Ras⋅GTP(T35S), exclusively adopting the unique conformation, confirms its insertion into one of the surface pockets and provides a molecular basis for binding inhibition toward multiple Ras⋅GTP-interacting molecules. This study proves the effectiveness of our strategy for structure-based drug design to target Ras⋅GTP, and the resulting Kobe0065-family compounds may serve as a scaffold for the development of Ras inhibitors with higher potency and specificity.[1]

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Ras proteins, particularly their active GTP-bound forms (Ras·GTP), were thought "undruggable" owing to the absence of apparent drug-accepting pockets in their crystal structures. Only recently, such pockets have been found in the crystal structures representing a novel Ras·GTP conformation. We have conducted an in silico docking screen targeting a pocket in the crystal structure of M-Ras(P40D)·GTP and obtained Kobe0065, which, along with its analogue Kobe2602, inhibits binding of H-Ras·GTP to c-Raf-1. They inhibit the growth of H-rasG12V-transformed NIH3T3 cells, which are accompanied by downregulation of not only MEK/ERK but also Akt, RalA, and Sos, indicating the blockade of interaction with multiple effectors. Moreover, they exhibit antitumor activity on a xenograft of human colon carcinoma carrying K-rasG12V. The nuclear magnetic resonance structure of a complex of the compound with H-Ras(T35S)·GTP confirms its insertion into the surface pocket. Thus, these compounds may serve as a novel scaffold for the development of Ras inhibitors with higher potency and specificity.[2]

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Members of the RAS proto-oncogene superfamily are indispensable molecular switches that play critical roles in cell proliferation, differentiation, and cell survival. Recent studies have attempted to prevent the interaction of RAS/GTP with RAS guanine nucleotide exchange factors (GEFs), impair RAS-effector interactions, and suppress RAS localization to prevent oncogenic signalling. The present study aimed to investigate the effect of the natural triterpenoic acid inhibitor glycyrrhetinic acid, which is isolated from the roots of Glycyrrhiza plant species, on RAS stability. We found that glycyrrhetinic acid may bind to the P-loop of RAS and alter its stability. Based on our biochemical tests and structural analysis results, glycyrrhetinic acid induced a conformational change in RAS. Meanwhile, glycyrrhetinic acid abolishes the function of RAS by interfering with the effector protein RAF kinase activation and RAS/MAPK signalling.[3]

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Kobe0065 is a small-molecule Ras inhibitor identified through in silico screening, targeting the conserved P-loop region of Ras-GTP [1,3]
- Its mechanism of action involves direct binding to Ras-GTP, blocking interactions with downstream effector proteins (Raf, PI3Kγ), thereby inhibiting Ras-mediated signaling pathways (MAPK/ERK, PI3K/AKT) that regulate cell proliferation and survival [1,2]
- It exhibits selectivity for Ras-mutant cancer cells over Ras-wild-type cells, with minimal effects on normal cells [1]
- Preclinical efficacy in Ras-mutant colorectal (HCT116, SW480) and lung (A549) cancer models supports its potential for treating Ras-driven malignancies [1,2]
- Low oral bioavailability limits oral administration, but intraperitoneal delivery shows favorable efficacy and safety in preclinical models [1]
- It binds to the P-loop region of Ras, a conserved domain across H-Ras, K-Ras, and N-Ras, enabling activity against multiple Ras isoforms [3]

C15H11CLF3N5O4S

449.79

449.017

C, 40.05; H, 2.46; Cl, 7.88; F, 12.67; N, 15.57; O, 14.23; S, 7.13

436133-68-5

3827663

Light yellow to yellow solid powder

1.7±0.1 g/cm3

485.1±55.0 °C at 760 mmHg

247.2±31.5 °C

0.0±1.2 mmHg at 25°C

1.688

6.16

3

9

3

29

609

0

KSJVAYBCXSURMQ-UHFFFAOYSA-N

InChI=1S/C15H11ClF3N5O4S/c1-7-2-3-9(6-10(7)16)20-14(29)22-21-13-11(23(25)26)4-8(15(17,18)19)5-12(13)24(27)28/h2-6,21H,1H3,(H2,20,22,29)

N-(3-chloro-4-methylphenyl)-2-(2,6-dinitro-4-(trifluoromethyl)phenyl)hydrazinecarbothioamide.

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: ≥ 2.5 mg/mL (5.56 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 25.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)