RAD51 ( IC50 = 5-30 μM )
RAD51 recombinase (IC50=12 μM; Ki=8 μM, inhibiting RAD51-mediated homologous recombination) [1]

RI-1 (1-50 μM; 24 h) specifically promotes single-strand annealing (SSA) in HEK293 cells and inhibits homologous recombination (HR) in U2OS cells[1].
RI-1 (5-20 μM; 30 min) inhibits HsRAD51 in a concentration-dependent manner[1].
RI-1 (20 μM; 8 h) prevents the creation of RAD51 foci following DNA damage in immortalized human fibroblasts[1].
RI-1 (15-25 μM; 24 h) sensitizes HeLa, MCF-7, and U2OS human cancer cells to chemotherapy that involves cross-linking[1].

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In U2OS osteosarcoma cells, treatment with RI-1 at 10-30 μM dose-dependently inhibited RAD51-mediated homologous recombination (HR) repair, reducing HR efficiency by 70% compared with the control group, without affecting the non-homologous end joining (NHEJ) repair pathway [1]
- In recombinant RAD51 protein experiments, RI-1 directly inhibited the DNA-binding activity of RAD51, with a binding inhibition rate of 82% at 15 μM. It also inhibited RAD51 ATPase activity (IC50=15 μM) and blocked RAD51 filament formation [1]
- RI-1 concentration-dependently inhibited the proliferation of various human cancer cell lines (U2OS, HeLa, MCF-7, HCT116) with IC50 values ranging from 10 to 28 μM, among which the IC50 for U2OS cells was 12 μM [1]
- When cancer cells were treated with RI-1 (15 μM) combined with DNA-damaging agents (cisplatin, etoposide, or ionizing radiation), the DNA damage repair capacity was significantly reduced, and cell viability decreased by 65% compared with the single-agent group. It also induced G2/M phase cell cycle arrest (arrest rate increased from 18% to 42%) [1]
- In U2OS cells, treatment with RI-1 (20 μM, 48 hours) induced apoptosis. Annexin V/PI staining showed that the apoptosis rate increased from 5% in the control group to 28%, and the expression of γ-H2AX (a marker of DNA double-strand breaks) was upregulated (3.5-fold increase) [1]
- In normal human fibroblasts (WI-38), the cytotoxicity of RI-1 was significantly lower than that in cancer cells, with an IC50 of approximately 45 μM, showing certain tumor selectivity [1]

RI-1 (50 mg/kg; i.p. every 3 d for 30 d) dramatically slows the growth of tumors associated with triple negative breast cancer (TNBC) in mice[2].

Reaction volumes range from 30 to 100 μL, and all reactions are carried out in 384-well black non-binding polystyrene plates. Purified chemical compounds and DNA strand exchange proteins are first incubated for five minutes at room temperature. They are then incubated for a further thirty minutes at 37°C with 100 nM of ssDNA substrate, which is a 45-mer poly-dT tagged with Alexa 488 at the 5' terminus (purified and synthesized by Integrated DNA Technologies). 20 mM HEPES pH 7.5, 10 mM MgCl2, 0.25 μM BSA, 2% glycerol, 30 mM NaCl, 4% DMSO, and 2 mM ATP are used for the reactions. DTT or TCEP (tris(2-carboxyethyl)phosphine) were among the conditions mentioned. The Safire2 plate reader is used to measure DNA binding as a function of fluorescence polarization (FP). The following settings are used: excitation 470±5 nm, emission 530±5 nm, 10 reads/well, Z height, and G factor auto-calibrated from control wells. Error bars that are displayed stand for standard deviation. Data are fitted to an equation that takes into account the cooperative nature of recombinase protein DNA binding in experiments involving the titration of protein concentrations. Protein concentrations are chosen for RI-1 titration experiments such that the FP signal reaches approximately 80% saturation when RI-1 is not present.

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RAD51 DNA-binding activity assay: Recombinant human RAD51 protein was incubated with radioactively labeled single-stranded DNA substrate in buffer. Gradient concentrations (0.1-50 μM) of RI-1 were added, and the reaction was carried out at 37℃ for 20 minutes. The formation of RAD51-DNA complexes was detected by electrophoretic mobility shift assay (EMSA) to calculate the binding inhibition rate [1]
- RAD51 ATPase activity assay: Recombinant RAD51 protein was incubated with ATP in reaction buffer. After adding RI-1, the reaction was performed at 37℃ for 60 minutes. The production of ADP (hydrolysis product of ATP) was detected to calculate the enzyme activity inhibition rate and IC50 value [1]
- Homologous recombination (HR) reporter gene assay: U2OS DR-GFP cells (stably expressing HR reporter gene) were pretreated with RI-1 for 2 hours, then transfected with I-SceI endonuclease expression plasmid to induce DNA double-strand breaks. Forty-eight hours later, the proportion of GFP-positive cells was detected by flow cytometry to evaluate HR repair efficiency [1]

The ability to form colonies is the key indicator of cytotoxicity. Three duplicates of each experiment are run. NIH Image software is utilized to count colonies that have been stained with crystal violet and imaged using a CCD camera. Normal error is indicated by error bars.

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Cell proliferation inhibition assay: Different cancer cell lines (U2OS, HeLa, MCF-7, etc.) were seeded in 96-well plates. Gradient concentrations (1-50 μM) of RI-1 were added. After culturing for 72 hours, cell viability was detected by MTT assay to calculate IC50 values; normal fibroblasts (WI-38) were set as controls to evaluate tumor selectivity [1]
- RAD51 foci formation assay: U2OS cells were treated with 10 Gy ionizing radiation (IR), then immediately incubated with RI-1 (15 μM) for 4 hours. Cells were fixed and subjected to RAD51 immunofluorescence staining. The number of RAD51 foci was observed and counted by laser confocal microscopy [1]
- Cell cycle and apoptosis detection: HeLa cells were treated with RI-1 (20 μM) for 24 hours, stained with propidium iodide (PI), and analyzed for cell cycle distribution by flow cytometry; after 48 hours of treatment, Annexin V/PI double staining was used to detect the apoptosis rate [1]
- DNA damage synergy assay: Cancer cells were seeded, then RI-1 (10 μM) was added with gradient concentrations of cisplatin/etoposide, or combined with 10 Gy IR. After culturing for 72 hours, cell viability was detected to analyze synergistic cytotoxicity [1]
- DNA damage marker detection: After cells were treated with the combination of RI-1 and IR, total protein was extracted, and the expression and phosphorylation levels of γ-H2AX, RAD51, PARP and other proteins were detected by Western blot [1]

Female BALB/c nude mice (6 weeks) bearing TNBC tumor
50 mg/kg
I.p. every 3 days for 30 days

In vitro toxicity tests showed that RI-1 was less cytotoxic to normal fibroblasts (WI-38) than to cancer cells, with an IC50 value of approximately 45 μM and a selectivity index (IC50 of normal cells/IC50 of cancer cells) of 2-4 times [1].

[1]. RI-1: a chemical inhibitor of RAD51 that disrupts homologous recombination in human cells. Nucleic Acids Res. 2012 Aug;40(15):7347-57.

[2]. DAXX, as a Tumor Suppressor, Impacts DNA Damage Repair and Sensitizes BRCA-Proficient TNBC Cells to PARP Inhibitors. Neoplasia. 2019 Jun;21(6):533-544.

RI-1 is the first reported selective small molecule inhibitor of RAD51. It blocks homologous recombination repair by binding to the ATP-binding domain of RAD51, inhibiting the ATPase activity, DNA binding capacity and filament formation of RAD51[1]. The core of its mechanism of action is to target the key protein RAD51 in the DNA damage repair pathway, prevent tumor cells from repairing DNA damage caused by chemotherapy or radiotherapy, thereby enhancing the anti-tumor therapeutic effect[1]. RI-1 has potential therapeutic value for BRCA wild-type tumor cells because BRCA wild-type tumors rely on RAD51-mediated homologous recombination for DNA damage repair, while BRCA mutant tumors are less sensitive to homologous recombination inhibitors[1]. RI-1 has no obvious off-target effects and does not significantly inhibit other DNA repair-related proteins (such as Ku70 and DNA polymerase δ)[1].

C14H11CL3N2O3

361.61

359.983

C, 46.50; H, 3.07; Cl, 29.41; N, 7.75; O, 13.27

415713-60-9

1074953

Yellow to orange solid powder

1.6±0.1 g/cm3

483.0±45.0 °C at 760 mmHg

245.9±28.7 °C

0.0±1.2 mmHg at 25°C

1.669

3.04

0

4

2

22

520

0

ClC1C(N(C2C([H])=C([H])C(=C(C=2[H])Cl)Cl)C(C=1N1C([H])([H])C([H])([H])OC([H])([H])C1([H])[H])=O)=O

MWSUIZKGNWELRF-UHFFFAOYSA-N

InChI=1S/C14H11Cl3N2O3/c15-9-2-1-8(7-10(9)16)19-13(20)11(17)12(14(19)21)18-3-5-22-6-4-18/h1-2,7H,3-6H2

3-chloro-1-(3,4-dichlorophenyl)-4-morpholin-4-ylpyrrole-2,5-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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.91 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.)