The target of RBC8 is the GTPase Ral, including RalA and RalB (both are Ras-like GTPases). [1]

In vitro activity: RBC8 causes RalB–GDP to undergo chemical shift changes, which lessens RalA's activation in living cells as well. The RBC8 induces anchorage-independent growth inhibition in Ral-dependent lines H2122 and H358 at IC50 values of 3.5 µM and 3.4 µM, in that order.

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1. Inhibition of Ral-effector binding: RBC8 was shown to inhibit the binding of Ral (including RalA and RalB) to its effector RALBP1. This inhibitory effect was verified through relevant binding assays, which is crucial for blocking Ral-mediated downstream signaling pathways [1]
2. Inhibition of Ral-mediated cell spreading: When murine embryonic fibroblasts (MEFs) were treated with RBC8 at concentrations ranging from 0 to 15 μM for 1 hour, the compound effectively inhibited RalA-dependent cell spreading. The dose-response experiment demonstrated that the inhibitory effect was concentration-related, with higher concentrations showing stronger inhibition [1]
3. Inhibition of anchorage-independent growth of cancer cells: RBC8 exhibited inhibitory activity against the anchorage-independent growth of human lung cancer cell lines sensitive to Ral siRNA knockdown (such as H2122 and H358). When these cells were seeded in soft agar containing different concentrations of RBC8, the number of colonies formed after 2-4 weeks was significantly reduced compared to the control group. However, it had little effect on cell lines resistant to Ral siRNA knockdown (such as H460 and Calu6) [1]
4. Selectivity for Ral: RBC8 showed selectivity for Ral (RalA and RalB) relative to other GTPases including Ras and RhoA. In in vitro activity assays, it did not significantly inhibit the activity of Ras or RhoA, indicating that its inhibitory effect is specific to Ral [1]
5. Detection of Ral activity in cells: After treating H2122 and H358 cells with 10 μM RBC8 for 3 hours under anchorage-independent culture conditions, cell lysates were subjected to a pull-down assay using RalBP1 agarose beads. Western blot analysis (using antibodies specific for RalA and RalB) showed that RBC8 significantly reduced the amount of active Ral (both RalA and RalB) in the cells, confirming its ability to inhibit Ral activity in vitro [1]

RBC8 (50 mg/kg i.p.) prevents tumor growth in mice carrying H358 xenografts by specifically inhibiting RalA and RalB.

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1. Inhibition of tumor xenograft growth: Nude mice inoculated with human lung cancer cell lines (H2122 and H358) were treated with RBC8 at a dose of 50 mg/kg per day via intraperitoneal injection, starting 24 hours after inoculation. After 21 days of treatment, the tumor volume of the RBC8-treated group was significantly smaller than that of the control group. The inhibitory effect of RBC8 on tumor growth was comparable to that of Ral depletion using RNA interference [1]
2. Inhibition of Ral activity in tumor tissues: Tumor-bearing nude mice with H2122 xenografts were given a single intraperitoneal dose of 50 mg/kg RBC8. After 3 hours, the tumors were collected, and the activity of RalA and RalB in tumor lysates was measured using the RalBP1 pull-down assay. Western blot analysis and quantification showed that RBC8 significantly inhibited the activity of both RalA and RalB in tumor tissues (p<0.001, n=24). The amount of active Ral in the treatment group was significantly lower than that in the control group, while a control compound RBC5 had no such inhibitory effect [1]

For fifteen minutes at thirty degrees Celsius, His-RalA (100 ng) was incubated with gamma-labeled 32P-GTP (8 nM assay concentration), DMSO, or individual compounds (50 μM assay concentration) dissolved in DMSO with EDTA (20 mM). Filter binding was used to measure the amount of radiolabeled nucleotide incorporated after the reaction was halted by dilution into excess MgCl2. Nucleotide diphosphokinase changed 32P-GTP (alpha-labeled) into 32P-GDP, which was then utilized in the binding experiment with GDP.

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1. Ral-RALBP1 binding inhibition assay: The assay system was constructed using purified Ral protein (GDP-bound form) and its effector RALBP1. Different concentrations of RBC8 were added to the reaction system containing Ral and RALBP1. After incubation at an appropriate temperature for a specific period, the amount of Ral-RALBP1 complex formed was detected using a suitable method (such as immunoprecipitation combined with western blot or fluorescence resonance energy transfer). The results showed that RBC8 could dose-dependently inhibit the formation of the Ral-RALBP1 complex, thereby verifying its ability to block the interaction between Ral and its effector [1]
2. NMR-based Ral binding assay: For the ^{15}N-HSQC NMR spectroscopy experiment, 100 μM RalB-GDP was prepared. RBC8 was added to the RalB-GDP solution to a final concentration of 100 μM. The NMR spectrum of RalB-GDP alone and in the presence of RBC8 was recorded. By comparing the two spectra, significant chemical shift changes were observed in selected residues of RalB-GDP, indicating that RBC8 binds to RalB-GDP. These chemical shift changes were mainly concentrated in the region related to the binding site of RBC8 on RalB, further confirming the specific binding between the compound and Ral [1]

In soft agar, anchorage-independent conditions are used to measure the compounds' growth inhibition of human lung cancer cells. 15,000 cells per well of 3.0 mL of 0.4% low-melting-point agarose containing different drug concentrations are seeded into 6-well plates (which have been coated with a base layer of 2.0 ml of 1% low-melting-point agarose). The cells are stained with 1.0 mg ml−1 nitroblue tetrazolium two to four weeks (depending on the cell line) after incubation, and colonies are counted under a microscope. When compared to the DMSO-treated control, the drug concentration that caused a 50% decrease in colony number is known as the IC50 value.

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1. MEF cell spreading assay: Wild-type or caveolin ^{-/-} MEF cells were cultured under appropriate conditions. The cells were treated with RBC8 at concentrations of 0-15 μM for 1 hour. After treatment, the cells were seeded on a suitable substrate and incubated for a certain period to allow cell spreading. The number of spread cells was counted under a microscope, and the percentage of spread cells was calculated. The results showed that RBC8 inhibited RalA-dependent MEF cell spreading in a concentration-dependent manner [1]
2. Soft agar colony formation assay for cancer cells: Human lung cancer cell lines (H2122, H358, H460, Calu6) were cultured to the logarithmic growth phase. The cells were counted and adjusted to an appropriate density. Different concentrations of RBC8 were mixed with the cell suspension, and the mixture was seeded in soft agar (prepared with a lower layer of 0.6% agar and an upper layer of 0.3% agar containing cells and drugs) in a culture dish. The culture dish was placed in an incubator at 37°C with 5% CO₂ for 2-4 weeks. After incubation, the number of colonies with a diameter greater than a certain size (e.g., 50 μm) was counted under a microscope. The results showed that RBC8 significantly reduced the number of colonies formed by Ral-sensitive cell lines (H2122, H358) but had no obvious effect on Ral-resistant cell lines (H460, Calu6) [1]
3. Ral activity pull-down assay in cells: H2122 and H358 cells were cultured under anchorage-independent conditions (e.g., in soft agar or ultra-low attachment culture dishes). The cells were treated with 10 μM RBC8 for 3 hours. After treatment, the cells were collected and lysed with a suitable lysis buffer. The cell lysate (400 μg protein) was incubated with RalBP1 agarose beads at 4°C for a certain period (e.g., 1 hour) to pull down active Ral. The beads were washed several times with lysis buffer, and then the bound proteins were eluted by adding SDS-PAGE loading buffer and heating. The eluted proteins and total cell lysate (20 μg protein, as a loading control) were subjected to SDS-PAGE electrophoresis, followed by western blot analysis using antibodies specific for RalA and RalB. The intensity of the bands was quantified using image analysis software, and the relative amount of active Ral was calculated. The results showed that RBC8 significantly reduced the level of active Ral in the cells [1]
4. siRNA knockdown combined with drug treatment assay: H2122 and H358 cells were transfected with 10, 30, or 50 nM siRNA targeting both RalA and RalB (or control siRNA) using a suitable transfection reagent. After 48 hours of transfection, the cells were collected and seeded in soft agar containing a fixed concentration of RBC8. The soft agar colony formation assay was performed as described above. The results showed that siRNA-mediated knockdown of RalA and RalB enhanced the inhibitory effect of RBC8 on colony formation. As the concentration of siRNA increased, the number of colonies formed in the presence of RBC8 further decreased, indicating that the inhibitory effect of RBC8 is dependent on Ral [1]

DMSO,50 mg/kg, i.p.
Mice bearing H358 xenografts

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1. Tumor xenograft model establishment and drug treatment (H2122 and H358): Human lung cancer cells (H2122 or H358) in the logarithmic growth phase were harvested and resuspended in a suitable medium (e.g., PBS mixed with Matrigel at a ratio of 1:1) to a concentration of 1×10⁷ cells/mL. Each nude mouse was inoculated with 0.2 mL of the cell suspension subcutaneously into the right flank. Twenty-four hours after inoculation, the mice were randomly divided into two groups: the RBC8 treatment group and the control group (n=6 per group). RBC8 was dissolved in a suitable solvent (the specific solvent was not detailed, but commonly used solvents for intraperitoneal injection include DMSO mixed with saline) and administered to the treatment group via intraperitoneal injection at a dose of 50 mg/kg per day. The control group received the same volume of solvent. The tumor volume was measured every 3-4 days using a vernier caliper, and the tumor volume was calculated using the formula: Volume = (length × width²)/2. The treatment was continued for 21 days, and the mice were sacrificed at the end of the experiment to collect tumor tissues for subsequent analysis [1]
2. In vivo Ral activity detection assay: Nude mice bearing H2122 xenografts were used. When the tumor volume reached approximately 100-200 mm³, the mice were randomly divided into two groups: the RBC8 treatment group and the control group. The treatment group received a single intraperitoneal injection of 50 mg/kg RBC8, and the control group received the same volume of solvent. Three hours after administration, the mice were sacrificed, and the tumor tissues were quickly excised and placed in ice-cold lysis buffer. The tumor tissues were homogenized using a homogenizer and centrifuged to obtain the supernatant (tumor lysate). The Ral activity in the tumor lysate was detected using the RalBP1 pull-down assay combined with western blot analysis, as described in the cell assay section. Additionally, 10 ng of recombinant human RalA or RalB was added to each western blot as an internal loading control to normalize the band intensity and compare across different blots [1]
3. Pharmacokinetic and tissue distribution study of RBC8 in mice: Nude mice were given a single intraperitoneal dose of 50 mg/kg RBC8. At different time points (0, 0.25, 0.5, 1, 2, 3, 4, and 5 hours) after administration, blood samples were collected from the orbital venous plexus of the mice. The blood samples were centrifuged to separate plasma, and the concentration of RBC8 in plasma was determined using a suitable analytical method (e.g., liquid chromatography-tandem mass spectrometry). Pharmacokinetic parameters such as extrapolated initial concentration (C₀), half-life (T₁/₂), and area under the concentration-time curve from 0 to 5 hours (AUC₀₋₅hr) were calculated based on the plasma concentration-time data. Three hours after a single intraperitoneal dose of 50 mg/kg RBC8, the mice were sacrificed, and various tissues (including tumor, liver, kidney, spleen, lung, and heart) were collected. The concentration of RBC8 in each tissue was determined using the same analytical method to evaluate its tissue distribution [1]

1. Pharmacokinetic parameters in mice: After a single intraperitoneal injection of 50 mg/kg RBC8 into nude mice, pharmacokinetic parameters were determined based on plasma concentrations. Specific parameters included extrapolated initial concentration (C₀), half-life (T₁/₂), and area under the concentration-time curve from 0 to 5 hours (AUC₀₋₅hr). However, the specific values of these parameters were not provided in the literature [1]. 2. Tissue distribution in mice: Three hours after a single intraperitoneal injection of 50 mg/kg RBC8 into nude mice, the compound was detected in various tissues. It was able to distribute into tumor tissues, and the concentration in tumor tissues was sufficient to inhibit the activity of Ral. In addition, it was also distributed in other tissues such as the liver, kidney, spleen, lungs, and heart, but the specific concentration values in each tissue were not detailed [1]. 3. In vitro cellular uptake: H2122 human lung cancer cells were treated with 10 μM RBC8. Cells were collected at different time points after treatment (1, 5, 15, 30 and 60 minutes), and the intracellular concentration of RBC8 was determined by liquid chromatography-tandem mass spectrometry. The results showed that RBC8 could be taken up by cells, and the intracellular concentration increased over time during the detection period [1].

[1]. Discovery and characterization of small molecules that target the GTPase Ral. Nature. 2014 Nov 20;515(7527):443-7.

1. Background: Ras-like GTPases RalA and RalB are important drivers of tumor growth and metastasis in various cancers, such as pancreatic cancer, colon cancer, and lung cancer. However, prior to this study, no effective drugs could block the activity of Ral. RBC8 is a small molecule compound discovered through protein structure analysis and virtual screening that can target the GDP-binding form of Ral and inhibit its activity. This discovery provides a new research tool for studying the function of Ral and a potential therapeutic strategy for Ral-dependent cancers [1]. 2. Mechanism of action: Ral binds to the GDP-binding (inactive) form of Ral, preventing Ral from being activated (i.e., preventing GDP from exchanging with GTP). By binding to Ral, RBC8 blocks the interaction between Ral and its effector protein RALBP1, thereby inhibiting Ral-mediated downstream signaling pathways (such as pathways involved in cell spread and non-anchor-dependent growth), ultimately inhibiting tumor growth and metastasis [1]. 3. Significance of the study: The development of RBC8 demonstrates the application value of structure-based drug discovery methods in Ral-dependent cancers. It validates the feasibility of targeting Ral with small molecules, laying the foundation for further optimization of Ral inhibitors and the development of novel cancer therapies. Furthermore, the selectivity of RBC8 for Ral reduces potential off-target effects, which is beneficial for its subsequent development as a therapeutic agent [1].

C25H20N4O3

424.45

424.154

C, 70.74; H, 4.75; N, 13.20; O, 11.31

361185-42-4

6626226

White to off-white solid powder

5.165

2

6

4

32

764

0

O1C(=C(C#N)C([H])(C2C([H])=C(C([H])=C([H])C=2OC([H])([H])[H])OC([H])([H])[H])C2C1=NN([H])C=2C1C([H])=C([H])C2=C([H])C([H])=C([H])C([H])=C2C=1[H])N([H])[H]

CLMQBVUFKIKYLU-UHFFFAOYSA-N

InChI=1S/C25H20N4O3/c1-30-17-9-10-20(31-2)18(12-17)21-19(13-26)24(27)32-25-22(21)23(28-29-25)16-8-7-14-5-3-4-6-15(14)11-16/h3-12,21H,27H2,1-2H3,(H,28,29)

6-amino-4-(2,5-dimethoxyphenyl)-3-naphthalen-2-yl-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile

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 (5.89 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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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Solubility in Formulation 2: 2.5 mg/mL (5.89 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 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 3: ≥ 2.5 mg/mL (5.89 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 25.0 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.)