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RI-2 is a reversible RAD51 inhibitor, with an IC50 of 44.17 μM, and specifically inhibits homologous recombination repair in human cells.
| Targets |
RAD51 ( IC50 = 44.2 μM )
Inhibitor of human RAD51 protein, a key protein in the homologous recombination (HR) DNA repair pathway. It binds reversibly to the same site on RAD51 as the irreversible inhibitor RI-1 (Compound 1). The IC50 for inhibiting RAD51 binding to single-stranded DNA (ssDNA) in a fluorescence polarization assay is 44.17 ± 6.75 μM. [1] |
|---|---|
| ln Vitro |
RI-2 (7a) is a RAD51 inhibitor that is reversible, with an IC50 of 44.17 μM. In human cells, homologous recombination repair is specifically inhibited by RI-2. Cells become significantly more sensitive to RI-2 (150 μM)[1].
In a biochemical fluorescence polarization assay, RI-2 inhibited the binding of purified human RAD51 protein to ssDNA with an IC50 of 44.17 ± 6.75 μM. [1] RI-2 showed no time-dependent increase in inhibitory activity against RAD51, in contrast to the irreversible inhibitor RI-1, indicating its reversible binding nature. [1] Immobilized RAD51 protein treated with up to 240 μM RI-2, followed by extensive washing, exhibited no measurable loss of DNA binding activity, further confirming fully reversible inhibition. In competition experiments, RI-2 protected RAD51 from inactivation by RI-1 in a concentration-dependent manner, demonstrating competition for the same binding site. [1] |
| Enzyme Assay |
For the primary RAD51 DNA binding assay, human RAD51 protein was incubated with a 45-mer oligo-dT substrate that was 5‘ end-labeled with Alexa 488. Binding was measured as a function of fluorescence polarization (FP) of the Alexa 488 tag. Reactions were performed in a buffer containing HEPES (pH 7.5), ATP, MgCl2, NaCl, glycerol, BSA, and DMSO in a 384-well plate. The plate was read using a plate reader equipped with appropriate FP filters. The RAD51 concentration was selected to generate an FP signal of 50–80% maximum in the absence of inhibitor for IC50 determination. [1]
To assess reversible vs. irreversible binding, biotin-tagged RAD51 was immobilized on streptavidin-conjugated polyacrylamide beads. The beads were incubated with the compound candidate, then washed extensively with ice-cold buffers containing and then without DMSO to remove unbound compound. The RAD51 protein was subsequently eluted from the beads using Tobacco Etch Virus (TEV) protease cleavage and then analyzed for DNA binding activity using the standard FP-based assay described above. [1] |
| Cell Assay |
For a duration of 24 hours at 37°C and 5% CO2, 300 HEK293 cells are plated into 96-well tissue culture plates at a density of one cell per well, with or without the addition of 50 nM mitomycin C (MMC). After that, the media is swapped out for new media that has 0.5% DMSO plus RI-2 for a further 24 hours. After that, RI-2 is eliminated, and cultures are allowed to develop to a confluence of 50–70%. Using the CellGlo reagentor, the average survival from a minimum of three replicates is determined. If RI-2 causes cells to produce noticeably more toxicity when MMC is present as opposed to when MMC is absent, then the cells have been successfully sensitized to MMC. Sensitization is specifically graded as a "+" for at least two pairs of compound doses when non-overlapping standard errors are noted[1].
In human HEK293 cells containing a DR-GFP reporter for homologous recombination (HR) repair efficiency, treatment with 60 μM RI-2 for 24 hours after induction of a DNA double-strand break significantly inhibited HR. Conversely, in cells containing an SA-GFP reporter for single-strand annealing (SSA) repair (a pathway upregulated when HR is impaired), treatment with 60 μM RI-2 stimulated SSA efficiency. This specific modulation of DNA repair pathway choice indicates on-target inhibition of RAD51/HR within cells. [1] In a cell toxicity assay, HEK293 cells were first treated with varying concentrations of the DNA cross-linking agent mitomycin C (MMC) for 24 hours, followed by treatment with 150 μM RI-2 (or DMSO vehicle) for an additional 24 hours. RI-2 treatment sensitized the cells to MMC, resulting in decreased cell survival compared to treatment with MMC alone. The LD50 of RI-2 in HEK293 cells was determined to be 70.16 ± 3.96 μM. [1] |
| ADME/Pharmacokinetics |
The stability of compound RI-2 against nucleophilic attack was assessed by incubating it in DMSO containing a five-fold molar excess of glutathione at 37 °C. HPLC analysis showed that no reaction with glutathione was detected in RI-2 after 24 hours of incubation, indicating its high stability and lack of Michael receptor reactivity. [1]
The physicochemical properties of RI-2 are as follows: molecular weight of 433.28 g/mol, CLogP value of 4.25, with 6 hydrogen bond acceptors and 0 hydrogen bond donors. It meets the drug similarity requirements of Lipinski's "five rules". [1] |
| Toxicity/Toxicokinetics |
In cell viability assays, the LD50 of RI-2 in HEK293 cells was determined to be 70.16 ± 3.96 μM.
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| References | |
| Additional Infomation |
RI-2 (1-(3,4-dichlorophenyl)-3-(4-methoxyphenyl)-4-morpholino-1H-pyrrole-2,5-dione) is an optimized analogue of the earlier RAD51 inhibitor RI-1 (compound 1). Its key improvement is the substitution of the chloromaleimide core (a Michael receptor that can irreversibly react with cysteine 319 of RAD51) with 4-methoxyphenyl. This modification eliminates the reactivity of the compound as a Michael receptor, making it a reversible binder of RAD51 while retaining its ability to specifically inhibit the homologous recombination repair pathway in human cells. [1] Compared with the more active RI-1, RI-2 was developed to reduce potential off-target effects and improve the stability of the compound in biological systems. RI-2 competes with RI-1 for binding to the same pocket on the surface of the RAD51 protein, possibly through non-covalent interactions. [1]
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| Molecular Formula |
C21H18CL2N2O4
|
|---|---|
| Molecular Weight |
126.904
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| Exact Mass |
432.064
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| CAS # |
1417162-36-7
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| PubChem CID |
71547138
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| Appearance |
Yellow to orange solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
605.6±55.0 °C at 760 mmHg
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| Flash Point |
320.1±31.5 °C
|
| Vapour Pressure |
0.0±1.7 mmHg at 25°C
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| Index of Refraction |
1.645
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| LogP |
4.25
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
4
|
| Heavy Atom Count |
29
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| Complexity |
672
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
JMPJNNOSVCHYOU-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C21H18Cl2N2O4/c1-28-15-5-2-13(3-6-15)18-19(24-8-10-29-11-9-24)21(27)25(20(18)26)14-4-7-16(22)17(23)12-14/h2-7,12H,8-11H2,1H3
|
| Chemical Name |
1-(3,4-dichlorophenyl)-3-(4-methoxyphenyl)-4-morpholin-4-ylpyrrole-2,5-dione
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| Synonyms |
RI-2
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: ~130 mg/mL (~300.0 mM）
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|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 7.8802 mL | 39.4011 mL | 78.8022 mL | |
| 5 mM | 1.5760 mL | 7.8802 mL | 15.7604 mL | |
| 10 mM | 0.7880 mL | 3.9401 mL | 7.8802 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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