Ras-Raf interaction; H-Ras·GTP (Ki = 46±13 μM)

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].

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.

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.

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

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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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.

[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]

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.)