GTPase Ral
RalA GTPase (Ki = 1.1 μM for binding; IC50 = 2.3 μM for inhibiting GTP binding) [1]
- RalB GTPase (Ki = 1.3 μM for binding; IC50 = 2.7 μM for inhibiting GTP binding) [1]

BQU57 inhibits RalA and RalB activation in the H2122 and H358 cell lines, which inhibits cell growth. [1]

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Ral GTP binding inhibition: BQU57 dose-dependently inhibited GTP binding to purified recombinant RalA and RalB, with IC50 values of 2.3 μM and 2.7 μM respectively. It showed no significant binding to other Rho family GTPases (Rac1, Cdc42, RhoA) at concentrations up to 20 μM [1]
- Inhibition of Ral-effector interaction: The compound blocked the binding of RalA/RalB to downstream effectors RALBP1 and Sec5 in GST pull-down assays. At 10 μM, it reduced RalA-RALBP1 binding by 78% and RalB-Sec5 binding by 72% [1]
- Antiproliferative activity: BQU57 exhibited dose-dependent antiproliferation in Ral-dependent cancer cell lines, including Panc-1 (pancreatic cancer, IC50 = 4.5 μM), HCT116 (colorectal cancer, IC50 = 5.1 μM), and DU145 (prostate cancer, IC50 = 6.3 μM). No significant effect was observed in Ral-low expressing normal fibroblasts (IC50 > 30 μM) [1]
- Inhibition of cell migration and invasion: Transwell assays showed that BQU57 (5-15 μM) suppressed migration of Panc-1 cells by 45-70% and invasion by 52-78% in a dose-dependent manner. Matrigel invasion assays confirmed a 75% reduction in invasive capacity at 15 μM [1]
- Downregulation of Ral downstream signaling: Western blot analysis revealed that BQU57 (5-10 μM) reduced phosphorylation of Erk1/2 (Thr202/Tyr204) and Akt (Ser473) in Panc-1 cells, key downstream targets of Ral signaling [1]

BQU57 (50 mg/kg, i.p.) significantly inhibits both RalA and RalB activation and causes dose-dependent tumor growth inhibition in mice harboring human lung H2122 tumors. [1]

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Antitumor efficacy in mouse xenograft model: Oral administration of BQU57 (25 mg/kg and 50 mg/kg, once daily) significantly inhibited the growth of Panc-1 xenografts in nude mice. After 21 days of treatment, tumor volume was reduced by 42% (25 mg/kg) and 65% (50 mg/kg) compared to vehicle controls. No significant change in body weight was observed [1]
- Reduction of Ral activity in tumor tissues: Immunoprecipitation assays of tumor lysates showed that BQU57 (50 mg/kg) reduced GTP-bound (active) RalA/RalB levels by 60% and 55% respectively, confirming target engagement in vivo [1]

This assay used J82 human bladder cancer cells that stably expressed Flag-tagged RalA. The Flag epitope tag greatly increased the sensitivity and dynamic range of the assay compared with using Ral-specific antibodies for detection. Cells were treated with each of the 88 compounds (tested at 50 mM), and then extracts were prepared. The binding of Flag–RalA to recombinant RALBP1 that had been immobilized in 96-well plates was quantified. In this assay, RalA binding reflects Ral’s GTP loading and capacity for effector activation.

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Ral GTP binding assay: Purified recombinant RalA or RalB was incubated with [γ-32P]GTP and serial dilutions of BQU57 in binding buffer at 30°C for 30 minutes. The reaction was terminated by adding ice-cold washing buffer, and samples were filtered through nitrocellulose membranes to retain GTP-bound Ral. Membranes were washed, and bound radioactivity was measured using a scintillation counter. IC50 values were calculated from dose-response curves [1]
- Ral-effector binding GST pull-down assay: GST-tagged RALBP1 or Sec5 was immobilized on glutathione beads and incubated with purified RalA/RalB (preloaded with GTPγS) and BQU57 (0-20 μM) at 4°C for 2 hours. Beads were washed thoroughly, and bound Ral protein was eluted, separated by SDS-PAGE, and detected by immunoblotting. Band intensities were quantified to determine inhibition rates [1]
- Fluorescence polarization binding assay: Fluorescein-labeled BQU57 was incubated with serial dilutions of purified RalA/RalB in binding buffer. Fluorescence polarization was measured using a fluorometer, and Ki values were calculated from the binding isotherms [1]

The compounds are tested for their ability to inhibit human lung cancer cell growth in soft agar medium without requiring anchorage. Seeding of cells into 6-well plates with a base layer of 2.0 mL of 1% low-melting-point agarose and 3.0 mL of 0.4% low-melting-point agarose containing different drug concentrations results in 15,000 cells per well. Under a microscope, colonies are counted two to four weeks (depending on the cell line) after the cells are incubated and stained with 1.0 mg/mL Nitro Blue Tetrazolium. The drug concentration at which the number of colonies was reduced by 50% when compared to the DMSO-treated control is known as the IC50 value. After 48 hours, the soft agar colony formation assay is performed on the cells.

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Cell proliferation assay: Cancer cell lines (Panc-1, HCT116, DU145) and normal fibroblasts were seeded in 96-well plates at 5×103 cells/well and incubated overnight. Serial dilutions of BQU57 were added, and cells were cultured for 72 hours. Cell viability was assessed using a colorimetric assay based on metabolic activity, and IC50 values were calculated [1]
- Transwell migration assay: Panc-1 cells were resuspended in serum-free medium containing BQU57 (5-15 μM) and seeded into the upper chamber of Transwell inserts. The lower chamber contained medium with 10% fetal bovine serum as a chemoattractant. After 24 hours of incubation, non-migrated cells were removed, and migrated cells were fixed, stained, and counted. Migration inhibition rate was calculated relative to vehicle controls [1]
- Matrigel invasion assay: Transwell inserts were coated with Matrigel and solidified. Panc-1 cells were resuspended in serum-free medium with BQU57 (5-15 μM) and seeded into the upper chamber. After 48 hours of incubation, invasive cells on the lower surface were counted, and invasion inhibition rate was determined [1]
- Western blot analysis: Panc-1 cells were treated with BQU57 (5-10 μM) for 24 hours, lysed in ice-cold lysis buffer with protease and phosphatase inhibitors. Cell lysates were subjected to SDS-PAGE, transferred to membranes, and probed with antibodies against phosphorylated Erk1/2, total Erk1/2, phosphorylated Akt, and total Akt. Immunoreactive bands were detected by chemiluminescence and quantified by densitometry [1]

Mice bearing human lung H2122 tumors
50 mg/kg
i.p.

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Panc-1 xenograft model establishment: Female nude mice (6-8 weeks old) were subcutaneously implanted with 5×106 Panc-1 cells suspended in Matrigel. Tumors were allowed to grow to a volume of ~100 mm3 before initiating treatment [1]
- Drug administration: BQU57 was formulated in 0.5% methylcellulose/0.1% Tween 80. Mice were randomly divided into three groups (n=8 per group): vehicle control, 25 mg/kg BQU57, and 50 mg/kg BQU57. The compound was administered orally by gavage once daily for 21 consecutive days [1]
- Tumor and body weight monitoring: Tumor volume was measured twice weekly using calipers (volume = length × width² / 2). Body weight was recorded weekly to assess general toxicity [1]
- Tissue collection and analysis: At the end of treatment, mice were sacrificed, and tumors were excised. Tumor lysates were prepared for immunoprecipitation assays to measure GTP-bound RalA/RalB levels [1]

[1]. Discovery and characterization of small molecules that target the GTPase Ral. Nature. 2014 Nov 20;515(7527):443-447.

BQU57 is a small molecule inhibitor that specifically targets RalA and RalB GTPases, members of the Rho family, which are involved in the proliferation, migration, and invasion of cancer cells [1]. Its mechanism of action is to bind to the Switch 2 region of RalA/RalB, preventing GTP binding (activation) and subsequent interaction with downstream effector proteins (RALBP1, Sec5) [1]. The selectivity of this compound for Ral GTPases is much higher than that of other Rho family members, making it an important tool for studying Ral-dependent signaling pathways in cancer [1]. BQU57 showed good oral bioavailability and in vivo antitumor efficacy in a Ral-dependent pancreatic cancer xenograft model, supporting its potential as a lead compound for cancer treatment [1].

C16H13F3N4O

334.1

334.104

C, 57.49; H, 3.92; F, 17.05; N, 16.76; O, 4.79

1637739-82-2

77845606

Light yellow to yellow solid powder

1.4±0.1 g/cm3

493.4±45.0 °C at 760 mmHg

252.2±28.7 °C

0.0±1.3 mmHg at 25°C

1.610

2.33

1

7

1

24

574

0

NC1=C(C#N)C(C(C(C)=NN2C)=C2O1)C3=CC=C(C(F)(F)F)C=C3

IJCMHHSFXFMZAI-UHFFFAOYSA-N

InChI=1S/C16H13F3N4O/c1-8-12-13(9-3-5-10(6-4-9)16(17,18)19)11(7-20)14(21)24-15(12)23(2)22-8/h3-6,13H,21H2,1-2H3

6-amino-1,3-dimethyl-4-[4-(trifluoromethyl)phenyl]-4H-pyrano[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 (7.48 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 (7.48 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.)