PARP1; MDM2 E3 ligase
PARP1 (poly(ADP-ribose) polymerase 1) for targeted degradation; MDM2 E3 ubiquitin ligase for recruitment. The PROTAC binds to PARP1 and recruits MDM2, leading to PARP1 ubiquitination and subsequent proteasomal degradation. PARP1 is a key enzyme involved in DNA damage repair and is a validated target for cancer therapy.

Poly (ADP-ribose) polymerase-1 (PARP1) is a major member of the PARP superfamily that is involved in DNA damage signalling and other important cellular processes. Here researchers report the development of a small molecule targeting PARP1 based on the PROTAC strategy. In the MDA-MB-231 cell line, the representative compound 3 can induce significant PARP1 cleavage and programmed cell death [1].

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In vitro, this PROTAC degrades PARP1 in cancer cells, leading to PARP1 cleavage and apoptosis. It inhibits the proliferation of MDA-MB-231 breast cancer cells with an IC50 of 6.12 uM at 24 hours. By degrading PARP1 rather than merely inhibiting its enzymatic activity, the PROTAC approach may provide more sustained target inhibition and overcome resistance mechanisms associated with conventional PARP inhibitors. PARP1 is a member of the PARP superfamily involved in DNA damage signaling; inhibiting PARP significantly reduces tumor cell mortality, making it an important target for anti-tumor drug discovery.

In vivo, this PROTAC is expected to have antitumor activity by degrading PARP1, though specific in vivo data are limited. It serves as a tool to validate PARP1 degradation as a therapeutic strategy. PARP inhibitors have synergistic effects with other anti-tumor drugs, such as EZH2 and EGFR inhibitors.

Not applicable as a direct enzyme assay; the PROTAC's activity is assessed via cellular degradation assays. The ternary complex formation between PARP1, the PROTAC, and MDM2 can be confirmed using immunoprecipitation or AlphaLISA: recombinant PARP1, MDM2, and the PROTAC are incubated together, and complex formation is detected using antibodies against PARP1 and MDM2.

Western Blotting [1]
Cells were lysed for 20 min on ice using RIPA buffer with added protease inhibitor cocktail and PMSF. The lysate was spun at 14,000 g for 15 min at 4 °C. Supernatant was collected and protein quantified using BCA Protein Assay. 40μg of protein were loaded into 8% SDS-PAGE gel. Proteins were transferred onto a PVDF methanol activated membrane using a wet transfer method. Membranes were blocked in 5% non-fat milk diluted in 1xTBST for 1 hour at room temperature while gently rocking. Primary antibodies (listed below) were incubated in 5% BSA in 1x TBST and gently rocked overnight at 4°C. HRP-conjugated secondary antibodies diluted at 1: 3,000 were incubated in 1xTBST and rocked for 1 hour at room temperature. ECL chemiluminescence kit was used to detect protein expression. Western blot images were captured by Tanon 5200 chemiluminescent imaging system. Quantification of band intensities has been performed using Tanon Gis software.

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Cell Viability Assay[1]
Cell viability was evaluated using the Cell Counting Kit-8 (CCK-8) assay. 5x10~3 cells/well were seeded in a 96-well plate. After growing for 24 or 48 hours, cells were treated with indicated reagents. Each group had triplicate wells. After incubation for the indicated hours, 10 μL CCK-8 reagent was added to each well. After incubation for 2-4 hour, the Optical density (OD) values at 490 nm were measured with a microplate reader. Data was analyzed using GraphPad Prism 7.0. Error bars represent ± SD for three independent experiments.

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Cellular degradation assay: MDA-MB-231 or other cancer cells are seeded in 6-well plates and treated with the PROTAC PARP1 degrader (0.1-10 uM) for 4-24 hours. Cells are lysed in RIPA buffer with protease inhibitors, and 40 ug protein is resolved by SDS-PAGE (8% gel). Proteins are transferred to PVDF membranes and probed with anti-PARP1 (1:1000) and anti-GAPDH (1:5000) antibodies. PARP1 cleavage is assessed by the appearance of PARP1 cleavage fragments. Band intensity is quantified by densitometry. Cell viability is measured using CCK-8 assay: 5×103 cells/well in 96-well plates are treated with the PROTAC, and OD at 490 nm is measured after CCK-8 addition.

Mouse xenograft models: The PROTAC could be tested in tumor-bearing mice (e.g., MDA-MB-231 xenografts) to assess its antitumor efficacy and pharmacodynamic effects on PARP1 levels in tumors. Dosing would typically be via intraperitoneal injection. Tumor PARP1 levels would be assessed by Western blot and immunohistochemistry.

As a PROTAC (molecular weight approximately 1145.1 g/mol), its pharmacokinetics are influenced by its large size and properties. It may have limited oral bioavailability and require parenteral administration (intravenous or intraperitoneal). PK parameters such as half-life, clearance, and bioavailability are not extensively reported.

No specific toxicity data are reported. As a targeted degrader, it may have off-target effects on other proteins containing similar degron motifs. The MDM2-based recruitment may also lead to degradation of p53 in cells expressing wild-type p53, which could have complex biological effects.

[1]. Induction of apoptosis in MDA-MB-231 breast cancer cells by a PARP1-targeting PROTAC small molecule. Chem Commun (Camb). 2019 Jan 2;55(3):369-372.

ADP-ribose polymerase (PARP) catalyzes the parylation process, which plays a crucial role in various cellular functions. Because inhibiting PARP significantly reduces tumor cell mortality, it has become an important target for discovering novel anti-tumor drugs. Researchers worldwide are dedicated to developing novel and highly effective PARP inhibitors for cancer treatment. Studies have shown that PARP inhibitors have synergistic effects with other anti-tumor drugs, such as EZH2 and EGFR inhibitors, in cancer cells. Combined inhibition of PARP and its synergistic targets may provide a rational strategy to improve the efficacy of existing anti-cancer regimens. This article summarizes the latest advances in PARP-targeted drugs, including single-target inhibitors/degraders and dual-target inhibitors/degraders, explores the fundamental theories behind the development of these dual-target drugs, and analyzes their structure-activity relationships in depth. J Med Chem. Dec 22, 2022; 65(24): 16099-16127.

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This is a research compound, not an approved drug. It is a tool for studying PARP1 degradation and exploring PROTAC technology for cancer therapy. Also known as PARP1-targeting PROTAC. CAS number: 2369022-68-2.

1145.09

1143.413

2369022-68-2

rel-PROTAC PARP1 degrader

138454769

White to off-white solid powder

1.4±0.1 g/cm3

1.661

3.88

2

14

26

81

2030

2

N1=C2C(C=CC=C2C(N)=O)=CN1C1=CC=C(C2=CN(CCOCCOCCOCCOCCNC(=O)CN3CCN(C(N4C(C5=CC=C(OC)C=C5OC(C)C)=N[C@H](C5=CC=C(Cl)C=C5)[C@@H]4C4=CC=C(Cl)C=C4)=O)CC3=O)N=N2)C=C1

RQWKTYKJNCZUJW-OMUYKDLESA-N

InChI=1S/C58H63Cl2N11O10/c1-38(2)81-50-33-46(76-3)19-20-47(50)57-63-54(40-7-13-43(59)14-8-40)55(41-9-15-44(60)16-10-41)71(57)58(75)68-23-22-67(52(73)37-68)36-51(72)62-21-25-77-27-29-79-31-32-80-30-28-78-26-24-69-35-49(64-66-69)39-11-17-45(18-12-39)70-34-42-5-4-6-48(56(61)74)53(42)65-70/h4-20,33-35,38,54-55H,21-32,36-37H2,1-3H3,(H2,61,74)(H,62,72)/t54-,55+/m1/s1

2-[4-[1-[2-[2-[2-[2-[2-[[2-[4-[(4R,5S)-4,5-bis(4-chlorophenyl)-2-(4-methoxy-2-propan-2-yloxyphenyl)-4,5-dihydroimidazole-1-carbonyl]-2-oxopiperazin-1-yl]acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]ethyl]triazol-4-yl]phenyl]indazole-7-carboxamide

PROTAC PARP1 degrader; 2369022-68-2; rel-PROTAC PARP1 degrader; PARP1-targeting PROTAC 3; CHEMBL5284804;

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: ~100 mg/mL (~87.3 mM; with ultrasonication)

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.)