The primary target is RAD52, specifically its single-strand DNA (ssDNA) binding domain (residues 1-209). It acts as a non-competitive allosteric inhibitor. The IC50 for inhibiting RAD52 wild-type (WT) ssDNA binding is 1.1 μM, and for the RAD52 1-209 domain is 1.6 μM. [1]
It exhibits minimal inhibition of RAD51 (IC50 not determined, significantly higher than for RAD52) and yeast RAD59 (IC50 > 10 μM). [1]

In U20S cells, 6-Hydroxy-DOPA (0~20 μM) specifically prevents RAD52-mediated recombination. 6-Hydroxy-DOPA (0~10 μM; HEK293T cells) decreased the quantity of eGFP-RAD52 foci in a way that was dose-dependent. The BRCA1-deficient triple-negative breast cancer cell line HCC1937 is preferentially less viable when exposed to 5-Hydroxy-DOPA (5~75 μM) [1].

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6-Hydroxy-DL-DOPA was identified as a major inhibitor of the RAD52 ssDNA binding domain through high-throughput screening. It inhibits RAD52's ability to bind ssDNA, with an IC50 of 1.1 μM for wild-type RAD52 and 1.6 μM for the RAD52 1-209 domain, as measured by fluorescence polarization (FP) and electrophoretic mobility shift assay (EMSA). [1]

The compound acts as a non-competitive inhibitor, as its inhibitory activity is not diminished by a large (∼100-fold) excess of ssDNA substrate, and it can rapidly dissociate pre-formed RAD52-ssDNA complexes. [1]
Mechanistically, it binds directly to the RAD52 1-209 domain with a Kd of 17.8 μM and a stoichiometry (n) of approximately 5, as determined by isothermal titration calorimetry (ITC). This binding causes a dramatic conformational change, completely transforming the 439 kDa undecamer ring structures of RAD52 1-209 into 48.3 kDa dimers. Under low salt conditions (0.15 M NaCl), 100% of the undecamers are converted into dimers. [1]

For full-length RAD52 WT, which forms heptamer rings and large superstructures, 6-Hydroxy-DL-DOPA disrupts these superstructures, enabling the protein to enter native gels and gel filtration columns as discrete higher molecular weight complexes (double heptamers and larger superstructures), but it does not dissociate the heptamer rings into smaller components like dimers. [1]
Structurally similar compounds like L-DOPS, DL-o-tyrosine, and Beta-(2-hydroxy-4-methylphenyl) alanine fail to inhibit RAD52 ssDNA binding or SSA, demonstrating the specificity of 6-OH-dopa. [1]

To determine the IC50, purified proteins (40 nM RAD52 WT, 85 nM RAD52 1-209, 280 nM RAD51, or 300 nM RAD59) were mixed in a reaction buffer (25 mM TrisHCl pH 7.5, 1 mM DTT, 25 mM NaCl, 0.01% NP-40, 0.5 mM MgCl2, and 0.1 mg/mL BSA) with a 10 nM FAM-conjugated 29-nt ssDNA probe and increasing concentrations of 6-Hydroxy-DL-DOPA in a total volume of 20 μL. Reactions with RAD51 additionally contained 1 mM ATP. After a 30-minute incubation at room temperature, fluorescence polarization was measured using a plate reader. The IC50 was calculated as the compound concentration required to inhibit ssDNA binding by 50%. [1]
Isothermal Titration Calorimetry (ITC) was performed to measure the direct binding of 6-Hydroxy-DL-DOPA to RAD52 1-209. The experiment determined a dissociation constant (Kd) of 17.8 μM and a binding stoichiometry (n) of approximately 5, indicating five binding sites per undecamer ring. [1]

Single-Strand Annealing (SSA) Assay: U2OS cells containing an SSA GFP reporter system were used. Cells were treated with 5 μM of 6-Hydroxy-DL-DOPA, and the activation of GFP expression resulting from I-SceI-induced DSB repair by SSA was measured. The compound consistently inhibited SSA. The effect was dose-dependent, with significant inhibition observed at 10 μM and 20 μM. Structurally similar compounds at 5 μM did not inhibit SSA. [1]

Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ) Assays: Using similar U2OS-based GFP reporter systems for HR and NHEJ, treatment with 5 μM 6-Hydroxy-DL-DOPA showed little to no reduction in HR and only a slight inhibition of NHEJ, indicating specificity for the RAD52-mediated SSA pathway. [1]

RAD52 Foci Formation: In BCR-ABL transformed murine hematopoietic 32Dcl3 cells stably expressing eGFP-RAD52, treatment with 6-Hydroxy-DL-DOPA suppressed eGFP-RAD52 foci formation induced by cisplatin in a dose-dependent manner. Similarly, in HEK293T cells and BRCA1-complemented MDA-MB-436 cells expressing eGFP-RAD52, the compound decreased the number of eGFP-RAD52 foci following exposure to ionizing radiation (IR) in a dose-dependent manner. [1]

Cell Viability and Proliferation: The effect of 6-Hydroxy-DL-DOPA on cell viability was tested using various BRCA-proficient and -deficient cell lines. At concentrations ranging from 5 μM to 75 μM, the compound selectively reduced the viability of BRCA1-deficient (MDA-MB-436, HCC1937) and BRCA2-deficient (VC8, CAPAN-1) cells, while having little effect on their BRCA-proficient counterparts (MDA-MB-436 + BRCA1, V79) or on BRCA-proficient cells. This selective killing was dependent on RAD52 expression, as the effect was lost in BRCA1-deficient cells where RAD52 was suppressed by siRNA. [1]

Clonogenic Survival Assay: Clonogenic survival assays confirmed that 6-Hydroxy-DL-DOPA inhibited the survival of BRCA-deficient cell lines (MDA-MB-436, VC8) and BRCA-deficient AML and CML patient cells, with minimal effects on BRCA-proficient cells or patient cells expressing normal BRCA levels. [1]
DNA Damage and Apoptosis: Treatment with 6-Hydroxy-DL-DOPA resulted in a substantial increase in γH2AX foci (a marker of DNA damage), which was significantly more pronounced in BRCA1-deficient MDA-MB-436 cells compared to BRCA1-proficient cells. The compound also specifically increased apoptosis, as indicated by annexin V staining, in BRCA2-deficient VC8 cells and BRCA1-deficient HCC1937 cells compared to their proficient controls. [1]

[1]. Small-Molecule Disruption of RAD52 Rings as a Mechanism for Precision Medicine in BRCA-Deficient Cancers. Chem Biol. 2015;22(11):1491-1504.

6-Hydroxydopa is an α-amino acid that is not produced from proteins and whose function is related to dopa.

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6-Hydroxy-DL-DOPA is identified as a small molecule inhibitor of RAD52. It is a catechol and a precursor of the catecholaminergic neurotoxin 6-hydroxydopamine, but its mechanism of action here is through specific allosteric disruption of RAD52 protein rings. [1]

The discovery is significant because RAD52 is a promising drug target for precision medicine in BRCA-deficient cancers. By inhibiting RAD52, 6-Hydroxy-DL-DOPA induces synthetic lethality, selectively killing cancer cells with BRCA mutations while sparing normal, BRCA-proficient cells. Its mechanism of action—disrupting the multimeric ring structure of RAD52—is described as unprecedented. [1]
The compound's ability to selectively inhibit the proliferation of BRCA-deficient cancer cells, including those from AML and CML patients, validates RAD52 as a therapeutic target. [1]

213.064

21373-30-8

107794

Off-white to gray solid powder

1.606g/cm3

515.2ºC at 760mmHg

265.4ºC

0.458

5

6

3

15

235

0

OC(C(CC1=CC(O)=C(O)C=C1O)N)=O

YLKRUSPZOTYMAT-UHFFFAOYSA-N

InChI=1S/C9H11NO5/c10-5(9(14)15)1-4-2-7(12)8(13)3-6(4)11/h2-3,5,11-13H,1,10H2,(H,14,15)

2-amino-3-(2,4,5-trihydroxyphenyl)propanoic acid

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 : ~50 mg/mL (~234.53 mM)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)