SCR130 targets DNA Ligase IV (specifically the Ligase IV/XRCC4 complex). It shows minimal or no effect on Ligase III and Ligase I at equivalent concentrations. [1]

SCR130 (7-21 μM; 48 hours) increases in both early and late apoptotic cell counts. Through both internal and external routes, SCR130 causes apoptosis. p-p53, BCL2, and MCL1 expression are all upregulated by SCR130, along with CYTOCHROME C, BAX, and BAK. Increased expression of the FAS and SMAC-DIABLO proteins, as well as caspase 8 activation, have been reported[1]. The IC50 values of SCR130 (48 hours) in Reh, HeLa, CEM, Nalm6, and N114 cells are 14.1 μM, 5.9 μM, 6.5 μM, 2.2 μM, and 11 μM, respectively. These results indicate that SCR130 is cytotoxic]. By causing increased cell death when co-administered in Reh and Nalm6 cell lines, SCR130 can magnify the effects of radiation (0.5 and 1 Gy)[1]. Unrepaired DNA breaks accumulate when SCR130 inhibits the endogenous NHEJ. DNA double-strand breaks (DSBs) accumulate as a result of treatment with SCR130, which inhibits endogenous NHEJ and triggers apoptotic pathways, ultimately leading to cell death[1].

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SCR130 (500 µM) inhibited DNA end-joining catalyzed by rat testicular and spleen extracts in a dose-dependent manner (100-1000 µM) [1].
In purified Ligase IV/XRCC4-mediated end-joining assays, SCR130 showed concentration-dependent inhibition (100-1000 µM) [1].
SCR130 exhibited minimal or no inhibition on Ligase III or Ligase I-mediated end-joining at tested concentrations [1].
In cytotoxicity assays, SCR130 showed IC50 values: Nalm6 cells: 2.2 µM; Reh cells: 14 µM; HeLa cells: 6 µM; CEM cells: 6 µM; Ligase IV-null N114 cells showed 5-fold lower sensitivity (IC50 ~10 µM) compared to wild-type Nalm6 (IC50 2 µM), confirming Ligase IV specificity [1].
Combination with γ-radiation (0.5 and 1 Gy) potentiated cytotoxicity in Reh and Nalm6 cells [1].
SCR130 treatment (7, 14, 21 µM) increased 53BP1 foci in Reh cells in a concentration-dependent manner [1].
Combination with radiation (0.5/1 Gy) significantly increased 53BP1 and γH2AX foci in Reh, Nalm6, and HeLa cells compared to either alone [1].
Neutral comet assay in HeLa cells showed increased tail DNA% and Olive moment with SCR130 (3, 6, 9 µM) alone or combined with 1 Gy radiation [1].
SCR130 (7, 14, 21 µM) induced loss of mitochondrial membrane potential (JC-1 assay) and increased early/late apoptotic cells (Annexin V/PI staining) in Reh cells [1].
Western blot analysis in Reh cells treated with SCR130 (7, 14, 21 µM) showed concentration-dependent increases in pATM, phosphorylated p53, Cytochrome C, BAX, BAK, cleaved PARP1, caspase 9, caspase 8, FAS, and SMAC-DIABLO, while BCL2 and MCL1 decreased at highest concentration; KU70, Ligase IV, XRCC4, and Ligase III levels unchanged [1]. [1]

SCR130 was tested in cell-free DNA end-joining assays using rat testicular and spleen extracts. Extracts were incubated with radiolabeled double-stranded oligomeric DNA (75 bp with 5' overhangs) in buffer containing ATP and varying concentrations of SCR130 (100-1000 µM) at 25°C for 1 hour. Reactions were terminated, products purified, and resolved on denaturing polyacrylamide gels to score dimer and circular products [1].
Purified Ligase IV/XRCC4 (overexpressed in bacteria or insect cells) was used in end-joining assays with the same DNA substrate and increasing concentrations of SCR130 (100-1000 µM) at 25°C for 1 hour; joining efficiency was analyzed by denaturing PAGE [1].
Purified Ligase III and Ligase I were similarly tested for end-joining inhibition by SCR130 [1].
Circular dichroism (CD) spectroscopy was performed to study binding. Full-length Ligase IV/XRCC4 or its DNA-binding domain (DBD) purified from bacteria were incubated with 4 µM SCR130, and CD spectra were recorded. A significant shift in spectral pattern (indicative of beta-sheet) was observed upon addition of SCR130, suggesting direct binding to DBD of Ligase IV [1]. [1]

Apoptosis Analysis[1]
Cell Types: Reh cells
Tested Concentrations: 7 μM, 14 μM, and 21 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: demonstrated a concentration-dependent increase in the number of late and early apoptotic cells.

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Western Blot Analysis[1]
Cell Types: Reh cells
Tested Concentrations: 7 μM, 14 μM, and 21 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Revealed a concentration-dependent increase in levels of pATM and activation of p53 through phosphorylation.

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SCR130 cytotoxicity was evaluated using Trypan Blue dye exclusion assay. Cells (Nalm6, Reh, HeLa, CEM, N114) were treated with increasing concentrations (0.1, 0.5, 1, 2, 5, 10, 50, 100 µM) for 48 hours; IC50 values calculated from percentage of viable cells [1].
For combination with radiation, Reh and Nalm6 cells were treated with IC50 dose of SCR130 (14 µM for Reh, 2 µM for Nalm6) and exposed to 0.5 or 1 Gy γ-radiation; cells harvested at 24 and 48 hours for Trypan Blue assay [1].
Immunofluorescence: Reh or HeLa cells treated with SCR130 (7, 14, 21 µM) or combined with radiation (0.5/1 Gy), fixed, stained with anti-53BP1 or anti-γH2AX antibodies followed by Alexa Fluor-conjugated secondary antibodies, nuclei counterstained with propidium iodide or DAPI; foci counted using ImageJ [1].
Neutral comet assay: HeLa cells treated with SCR130 (3, 6, 9 µM) alone or with 1 Gy radiation, embedded in agarose, lysed, electrophoresed under neutral conditions, stained with ethidium bromide; tail DNA% and Olive moment quantified using CometScore software (≥250 cells per sample) [1].
Cell cycle analysis: Reh cells treated with SCR130 (7, 14, 21 µM) for 48 hours, fixed in 70% ethanol, stained with propidium iodide (PI), analyzed by flow cytometry; SubG1 population quantified [1].
Mitochondrial membrane potential: Reh cells treated with SCR130 (7, 14, 21 µM) for 48 hours, stained with JC-1 dye, analyzed by flow cytometry; valinomycin used as positive control [1].
Apoptosis assay: Reh cells treated with SCR130 (7, 14, 21 µM) for 48 hours, stained with Annexin V-FITC and PI, analyzed by flow cytometry to quantify early apoptotic, late apoptotic, and necrotic cells [1].
Western blot: Reh cells treated with SCR130 (7, 14, 21 µM) for 48 hours, lysed, equal protein loaded, probed with antibodies against pATM, ATM, p53, phosphorylated p53, BCL2, MCL1, Cytochrome C, BAX, BAK, cleaved PARP1, caspase 9, caspase 8, FAS, SMAC-DIABLO, KU70, Ligase IV, XRCC4, Ligase III; Ponceau staining used as loading control [1]. [1]

[1]. Identification and characterization of novel SCR7-based small-molecule inhibitor of DNA end-joining, SCR130 and its relevance in cancer therapeutics. Mol Carcinog. 2020 Jun;59(6):618-628.

In Ligase IV-null N114 cell line, SCR130 exhibited 5-fold lower cytotoxicity (IC50 ~10 µM) compared to wild-type Nalm6 cells (IC50 2 µM), confirming Ligase IV-dependent cell death [1].
SCR130 did not induce cell cycle arrest at any tested concentration (7-21 µM) in Reh cells, but increased SubG1 population [1].
The compound induced apoptosis via both intrinsic (Cytochrome C release, BAX/BAK upregulation, caspase 9 activation) and extrinsic (FAS upregulation, caspase 8 activation) pathways [1].
SCR130 is derived from SCR7 by introducing a spiro ring into the core structure, showing 20-fold higher efficacy in inducing cytotoxicity in cancer cell lines compared to SCR7 [1].
It has potential to be developed as a cancer therapeutic and may also be used to improve precise genome editing (e.g., CRISPR-Cas9) due to its Ligase IV-specific NHEJ inhibition at low concentrations [1]. [1]

C19H13CL2N3O2S

418.296420812607

417.01

2377858-38-1

146165950

Light yellow to brown solid powder

3.6

2

4

2

27

662

0

ClC1C=CC(=CC=1)C1CC(C2C=CC(=CC=2)Cl)=NC21C(NC(NC2=O)=S)=O

QEMQLCCYJLGAKB-UHFFFAOYSA-N

InChI=1S/C19H13Cl2N3O2S/c20-12-5-1-10(2-6-12)14-9-15(11-3-7-13(21)8-4-11)24-19(14)16(25)22-18(27)23-17(19)26/h1-8,14H,9H2,(H2,22,23,25,26,27)

2,4-bis(4-chlorophenyl)-8-sulfanylidene-1,7,9-triazaspiro[4.5]dec-1-ene-6,10-dione

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)

DMSO : 100 mg/mL (239.06 mM )

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.98 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.)