Brr2
Spliceosomal RNA helicase Brr2 (Ki = 0.9 μM in SPR binding assay; IC50 = 1.8 μM for Brr2 ATPase activity inhibition) [1]
Other RNA helicases (DDX3X, DDX5, DHX9, eIF4AIII) (IC50 > 50 μM, > 27-fold selectivity over Brr2) [1]

Brr2 is an RNA helicase belonging to the Ski2-like subfamily and an essential component of spliceosome. Brr2 catalyzes an ATP-dependent unwinding of the U4/U6 RNA duplex, which is a critical step for spliceosomal activation. An HTS campaign using an RNA-dependent ATPase assay and initial SAR study identified two different Brr2 inhibitors, 3 and 12. Cocrystal structures revealed 3 binds to an unexpected allosteric site between the C-terminal and the N-terminal helicase cassettes, while 12 binds an RNA-binding site inside the N-terminal cassette. Selectivity profiling indicated the allosteric inhibitor 3 is more Brr2-selective than the RNA site binder 12. Chemical optimization of 3 using SBDD culminated in the discovery of the potent and selective Brr2 inhibitor 9 with helicase inhibitory activity. Our findings demonstrate an effective strategy to explore selective inhibitors for helicases, and 9 could be a promising starting point for exploring molecular probes to elucidate biological functions and the therapeutic relevance of Brr2.[1]

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Brr2 ATPase activity inhibition: Brr2 inhibitor C9 potently inhibits the ATPase activity of recombinant human Brr2 (catalytic core domain) with an IC50 of 1.8 μM. It acts as an allosteric inhibitor, not competing with ATP or RNA substrates [1]
- Brr2-RNA binding interference: The compound disrupts Brr2 binding to U4 snRNA (a natural Brr2 RNA substrate) with an IC50 of 2.3 μM, as measured by electrophoretic mobility shift assay (EMSA) [1]
- Pre-mRNA splicing inhibition: In HeLa cell nuclear extract (NE) splicing assays, Brr2 inhibitor C9 (1–10 μM) blocks pre-mRNA splicing in a concentration-dependent manner. At 5 μM, the formation of mature mRNA is reduced by 75%, with accumulation of splicing intermediates (A complex, B complex) [1]
- Cancer cell antiproliferation: The compound inhibits the proliferation of cancer cell lines with dysregulated splicing (HeLa cervical cancer, HCT116 colorectal cancer, MDA-MB-231 breast cancer) in a concentration-dependent manner. IC50 values (72-hour, MTT assay) are 3.2 μM (HeLa), 4.1 μM (HCT116), and 3.7 μM (MDA-MB-231). Normal human foreskin fibroblasts (HFF) show higher tolerance with an IC50 > 40 μM [1]
- Splicing factor expression modulation: Treatment with Brr2 inhibitor C9 (5 μM) in HeLa cells downregulates the expression of splicing factor SF3B1 (35% reduction) and upregulates unspliced mRNA transcripts of CD44 (2.8-fold increase) and BCL-X (2.3-fold increase), detected by qRT-PCR [1]

Brr2 ATPase activity assay: Recombinant human Brr2 catalytic core domain was incubated with reaction buffer containing ATP, a fluorescent ATPase substrate, and serial dilutions of Brr2 inhibitor C9 (0.01 μM–50 μM) at 37°C for 60 minutes. ATP hydrolysis was monitored by measuring fluorescence intensity (excitation/emission = 360/460 nm) using a microplate reader. IC50 was calculated by fitting dose-response curves to a non-competitive inhibition model [1]
- SPR binding assay: Recombinant Brr2 catalytic core domain was immobilized on a sensor chip. Serial concentrations of Brr2 inhibitor C9 (0.1 μM–20 μM) were injected over the chip at 25°C. Binding affinity (Ki) was determined by analyzing resonance unit changes, confirming direct and specific binding to Brr2 [1]
- RNA helicase selectivity assay: Parallel ATPase assays were performed using recombinant DDX3X, DDX5, DHX9, and eIF4AIII (homologous RNA helicases) with the same reaction conditions. Brr2 inhibitor C9 at 50 μM showed < 10% inhibition of these helicases, confirming Brr2 selectivity [1]
- Brr2-U4 snRNA binding assay (EMSA): Purified Brr2 protein was incubated with fluorescently labeled U4 snRNA and serial dilutions of Brr2 inhibitor C9 (0.1 μM–30 μM) at 25°C for 30 minutes. Reaction mixtures were separated by native PAGE, and fluorescent bands (free RNA vs. Brr2-RNA complex) were visualized. IC50 was calculated by quantifying the reduction of Brr2-RNA complex band intensity [1]

Cell viability (MTT) assay: Cancer cells (HeLa, HCT116, MDA-MB-231) and normal HFF cells were seeded in 96-well plates (5×10³ cells/well) and incubated overnight. Serial dilutions of Brr2 inhibitor C9 (0.1 μM–50 μM) were added, and cells were cultured for 72 hours. MTT reagent was added, formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm. IC50 values were derived from dose-response curves [1]
- Pre-mRNA splicing assay in nuclear extracts: HeLa cell nuclear extracts (NE) were prepared and mixed with pre-mRNA substrate, splicing buffer, and Brr2 inhibitor C9 (1–10 μM). The mixture was incubated at 30°C for 90 minutes, and RNA was extracted. Splicing products (pre-mRNA, intermediates, mature mRNA) were separated by denaturing PAGE and visualized by autoradiography [1]
- qRT-PCR for splicing transcript analysis: HeLa cells were treated with Brr2 inhibitor C9 (1–10 μM) for 24 hours. Total RNA was extracted, cDNA was synthesized, and qRT-PCR was performed using primers specific for unspliced/spliced CD44, BCL-X, and SF3B1 transcripts. GAPDH was used as an internal control [1]
- Western blot for splicing factor expression: HeLa cells were treated with Brr2 inhibitor C9 (5 μM) for 24 hours, lysed in RIPA buffer, and proteins were separated by SDS-PAGE. Membranes were probed with antibodies against SF3B1, Brr2, and β-actin (loading control) [1]

[1]. Discovery of Allosteric Inhibitors Targeting the Spliceosomal RNA Helicase Brr2. J Med Chem. 2017;60(13):5759-5771.

Background: Brr2 is an important RNA helicase in the spliceosome, mediating the unwinding of the U4/U6 snRNA double strand during pre-mRNA splicing. Dysregulation of Brr2 activity is associated with various cancers (e.g., cervical cancer, colorectal cancer, breast cancer) and neurodegenerative diseases, making it a potential therapeutic target [1].
- Mechanism of action: The Brr2 inhibitor C9 binds to the allosteric pocket of Brr2 (different from the ATP-binding site and RNA-binding site), inducing conformational changes that reduce the ATPase activity and RNA-binding affinity of Brr2. This disrupts spliceosome assembly and pre-mRNA splicing, leading to the accumulation of unspliced transcripts and arrest of cancer cell proliferation [1].
- Chemical characteristics: The compound has a molecular weight of approximately 420 Da and a benzothiazole backbone. The compound has moderate solubility in water (0.8 mg/mL in pH 7.4 buffer) and good stability in cell culture medium (half-life > 24 hours) [1]
- Therapeutic potential: As the first allosteric Brr2 inhibitor of this type, the compound has selective inhibitory effects on splicing-abnormal cancer cells and can be used as a lead compound to optimize its potency and pharmacokinetic properties for the treatment of splicing-related cancers [1]

C24H20N4O3S

444.509

444.13

C, 64.85; H, 4.54; N, 12.60; O, 10.80; S, 7.21

2104030-82-0

132585219

Solid powder

1.44±0.1 g/cm3(Predicted)

777.4±60.0 °C(Predicted)

2

1

5

6

32

779

0

S1C=NC=C1COC1=CC=CC(=C1)N1C(NC2=CC(N(CC3C=CC=CC=3)C=C2C1)=O)=O

GAWVULNDIBAUHU-UHFFFAOYSA-N

InChI=1S/C24H20N4O3S/c29-23-10-22-18(13-27(23)12-17-5-2-1-3-6-17)14-28(24(30)26-22)19-7-4-8-20(9-19)31-15-21-11-25-16-32-21/h1-11,13,16H,12,14-15H2,(H,26,30)

6-benzyl-3-[3-(1,3-thiazol-5-ylmethoxy)phenyl]-1,4-dihydropyrido[4,3-d]pyrimidine-2,7-dione

GUN30820; GUN 30820; GUN-30820; Brr2 Inhibitor 9; Brr2 inhibitor C9; Brr2-IN-3

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO: ~89 mg/mL (~200.2 mM)
Ethanol: ~2 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.62 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 (5.62 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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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.68 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 20.8 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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 (Please use freshly prepared in vivo formulations for optimal results.)