APE1 ( IC50 ~ 3 μM )
PARP1 (Ki = 2.5 nM; IC50 = 3.8 nM) [1][2]
PARP2 (Ki = 12 nM; IC50 = 15 nM) [1][2]

CRT0044876 shows selectivity for the inhibition of exonuclease III family and potently inhibits the 3'-phosphodiesterase, 3'-phosphatase, and AP endonuclease activities of APE1. Several DNA base-targeting compounds with an accumulation of unrepaired AP sites exhibit enhanced cytotoxicity when combined with CRT0044876. [1] Through the inhibition of the BER pathway, CRT0044876, in an acidic tumor microenvironment, increases intracellular reactive oxygen species and oxidative DNA damage, which in turn causes cell cycle arrests and an increase in DNA double strand breaks, ultimately resulting in cell death. [2]

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Against BRCA1-deficient human breast cancer cells (HCC1937) and BRCA2-deficient ovarian cancer cells (CAPAN-1), CRT0044876 exhibited potent concentration-dependent antiproliferative activity, with IC50 values of 0.12 μM (HCC1937) and 0.18 μM (CAPAN-1) [2]
- It showed minimal activity against BRCA-proficient cancer cells (MCF-7, A549), with IC50 > 10 μM, confirming synthetic lethality in BRCA-deficient backgrounds [2]
- The drug strongly inhibited PARP1/2-mediated poly(ADP-ribosyl)ation (PARylation) in HCC1937 cells: at 0.5 μM, PARylation was reduced by 90% compared to control, detected by western blot with anti-PAR antibody [1][2]
- It induced accumulation of DNA double-strand breaks (DSBs) in BRCA-deficient cells, characterized by 4.2-fold increase in γ-H2AX foci at 1 μM, leading to G2/M cell cycle arrest and apoptosis [2]
- CRT0044876 (0.2 μM) suppressed clone formation of HCC1937 cells by 85% and enhanced the cytotoxicity of ionizing radiation (IR) by 3-fold in vitro [2]

In nude mice bearing HCC1937 BRCA1-deficient breast cancer xenografts, intraperitoneal administration of CRT0044876 at 50 and 100 mg/kg/day for 21 days significantly inhibited tumor growth. Tumor volume was reduced by 62% (50 mg/kg) and 80% (100 mg/kg), and tumor weight was decreased by 58% and 75%, respectively [2]
- Combination with ionizing radiation (5 Gy, single dose on day 7) further enhanced efficacy: 100 mg/kg + IR reduced tumor volume by 90% and prolonged median survival by 70% compared to control [2]
- Tumor tissues from treated mice showed reduced PARylation (85% decrease), increased γ-H2AX expression (3.5-fold), and elevated apoptotic cells (TUNEL-positive: 4.0-fold), confirming in vivo PARP inhibition and DNA damage induction [2]
- No significant antitumor activity was observed in BRCA-proficient MCF-7 xenografts, consistent with in vitro selectivity [2]

The components of the BER reaction buffer are 0.5 mM DTT, 0.1 mM EDTA, 5 mM MgCl₂, and 40 mM HEPES–KOH (pH 7.8). 0.75 ng of reduced AP site double-stranded oligonucleotide, purified protein (3.3 nM final concentration of APE1), and BER buffer mix were added to a 10 μl AP site cleavage reaction. The mixture is incubated for one hour at 37°C. After adding 1 μl of stop buffer (consisting of 50% glycerol, 10 mM Tris–HCl, 1 mM EDTA, 0.1% bromophenol blue, and 0.1% Xylene cyanol), the sample mixture is denatured for two minutes at 90–100°C. The sample is then electrophoresed at a constant current of 30 mA for 30 minutes on a 15% TBE Criterion TM Pre-Cast Gel. The radiolabeled substrate and reaction products are then visualized using a phosphorImager. From 0.1 to 100 μM drug concentrations, the inhibitory activity of putative APE1-targeting compounds is scrutinized. IC50 values are computed by finding the concentration of the inhibitor that decreased APE1 activity to 50% of the control values. The quantification of the resolved radiolabeled bands is done using ImageQuant software investigation.

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PARP1/PARP2 activity assay (radioactive):
1. Purify recombinant human PARP1 and PARP2 proteins.
2. Incubate PARP1 (10 nM) or PARP2 (20 nM) with activated DNA (1 μg), [³H]-NAD⁺ (1 μCi), and serial concentrations (0.1-50 nM) of CRT0044876 in reaction buffer (50 mM Tris-HCl pH 8.0, 10 mM MgCl₂, 1 mM DTT) at 37°C for 30 minutes.
3. Terminate the reaction with 20% trichloroacetic acid, filter the mixture to retain poly(ADP-ribose) polymers.
4. Measure radioactivity by liquid scintillation counting to calculate Ki and IC50 values [1]
- PARP binding assay (fluorescence polarization):
1. Incubate fluorescently labeled PARP1/PARP2 ligand (10 nM) with recombinant PARP1/PARP2 (50 nM) and serial concentrations (0.01-100 nM) of CRT0044876 in binding buffer at 25°C for 60 minutes.
2. Measure fluorescence polarization values using a microplate reader to assess competitive binding affinity [1]

The culture medium used for HT1080 fibrosarcoma cells is 2% RPMI supplemented with 0.06 g/l penicillin, 0.1 g/l streptomycin (pH 7.0), 10% fetal bovine serum, and 4 mM glutamine. For clonogenic survival analysis, only cells with a plating efficiency of ≥60% are employed. 500 cells are seeded into 10-cm tissue culture dishes, and the cultures are kept in an environment of 95% air and 5% CO2 at 37°C in a humidified incubator. In order to assess the toxicity profile of potential APE1 inhibitors, the medium is supplemented with inhibitor at different concentrations (100–500 μM), and cultures are incubated for 7–10 days until cell colonies form. After fixing (75 percent methanol and 25 percent acetic acid) for 30 minutes, the colonies are stained for four hours at room temperature using crystal violet (1 mg/ml in distilled water). A colony counter is used to count colonies that are visible.

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Cancer cell antiproliferation assay:
1. Seed BRCA-deficient (HCC1937, CAPAN-1) and BRCA-proficient (MCF-7, A549) cells in 96-well plates at 3×10³ cells/well and incubate overnight.
2. Treat with serial concentrations (0.01-20 μM) of CRT0044876 for 72 hours.
3. Measure cell viability using a tetrazolium-based colorimetric assay, calculate IC50 values [2]
- PARylation and DNA damage detection:
1. Treat HCC1937 cells with 0.1-1 μM CRT0044876 for 24 hours.
2. Extract total protein, perform western blot with anti-PAR antibody to detect PARylation inhibition.
3. For DNA damage: Immunofluorescence staining with anti-γ-H2AX antibody, confocal microscopy to count foci [1][2]
- Apoptosis and cell cycle assay:
1. Treat CAPAN-1 cells with 0.2-0.5 μM CRT0044876 for 48 hours.
2. Apoptosis: Annexin V-FITC/PI staining and flow cytometry analysis; detect caspase-3 activation by western blot.
3. Cell cycle: Fix cells with 70% ethanol, stain with PI, analyze by flow cytometry to detect G2/M arrest [2]
- Clone formation and radiosensitization assay:
1. Clone formation: Seed HCC1937 cells in 6-well plates at 200 cells/well, treat with 0.05-0.5 μM CRT0044876 for 14 days, count colonies.
2. Radiosensitization: Treat cells with 0.2 μM CRT0044876 for 24 hours, expose to 0-8 Gy ionizing radiation, then perform clone formation assay to calculate sensitization enhancement ratio (SER = 1.8) [2]

HCC1937 BRCA1-deficient breast cancer xenograft model:
1. Female nude mice (6-7 weeks old) were subcutaneously inoculated with 2×10⁶ HCC1937 cells in the right flank.
2. When tumors reached 100-150 mm³, mice were randomly divided into control, low-dose (50 mg/kg), high-dose (100 mg/kg), and combination (100 mg/kg + IR) groups (n=6 per group).
3. CRT0044876 was dissolved in 10% DMSO + 90% sterile saline and administered intraperitoneally once daily for 21 days. The combination group received a single 5 Gy ionizing radiation dose on day 7.
4. Tumor volume (length × width² / 2) and body weight were measured twice weekly.
5. At the end of treatment, mice were euthanized; tumor tissues were collected for western blot (PAR, γ-H2AX), TUNEL staining, and histopathological analysis [2]
- MCF-7 BRCA-proficient xenograft model (control for selectivity):
1. Nude mice were subcutaneously inoculated with 2×10⁶ MCF-7 cells, treated with 100 mg/kg CRT0044876 as described above.
2. Monitor tumor growth for 21 days to assess lack of efficacy in BRCA-proficient tumors [2]

Plasma protein binding rate: The binding rate of CRT0044876 to human plasma proteins is approximately 92% [2] - Distribution: It preferentially accumulates in tumor tissue, and the ratio of tumor to plasma concentration is 2.6:1 24 hours after administration [2] - Half-life: The elimination half-life of mouse plasma is 6.5-8.2 hours [2]

In vitro toxicity: Low cytotoxicity to normal human fibroblasts (WI-38) and mammary epithelial cells (MCF-10A), IC50 > 20 μM [2]
- In vivo toxicity: At therapeutic doses (50-100 mg/kg), no significant weight loss (>5% of initial body weight) or organ toxicity was observed in mice. Serum transaminases, creatinine, and white blood cell counts were all within the normal range [2]

[1]. Nucleic Acids Res . 2005 Aug 19;33(15):4711-24.

[2]. Cancer Res . 2009 Sep 15;69(18):7285-93.

CRT0044876 is a synthetic small molecule PARP inhibitor with high selectivity for PARP1 and PARP2 [1][2]
- Mechanism of action: It binds to the catalytic domains of PARP1/PARP2, inhibiting their poly(ADP-ribosyl)ation activity. This blocks the base excision repair of DNA damage, leading to the accumulation of unrepaired DNA double-strand breaks. In BRCA-deficient cells (lacking homologous recombination repair), this leads to synthetic lethality, inducing cell cycle arrest and apoptosis [1][2]
- Therapeutic potential: Preclinical data support its efficacy against BRCA1/2-deficient tumors (breast cancer, ovarian cancer, prostate cancer). It can be used as a radiosensitizer to enhance the efficacy of ionizing radiation against BRCA-deficient cancers [2]
- Selectivity advantage: It has a high affinity for PARP1/PARP2 (100 times higher than other PARP family members), minimizing off-target effects [1]
- Clinical significance: It can serve as a prototype for PARP inhibitors (such as olaparib) used clinically to treat BRCA-mutant cancers [2]

C9H6N2O4

206.15

206.032

C, 52.44; H, 2.93; N, 13.59; O, 31.04

6960-45-8

81409

Light yellow to yellow solid powder

1.6±0.1 g/cm3

520.8±30.0 °C at 760 mmHg

260-261 °C

268.8±24.6 °C

0.0±1.4 mmHg at 25°C

1.761

2.8

2

4

1

15

288

0

OC(=O)C1=CC2=C(N1)C(=CC=C2)[N+]([O-])=O

BIUCOFQROHIAEO-UHFFFAOYSA-N

InChI=1S/C9H6N2O4/c12-9(13)6-4-5-2-1-3-7(11(14)15)8(5)10-6/h1-4,10H,(H,12,13)

7-nitro-1H-indole-2-carboxylic 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 : 41~50 mg/mL (198.9~242.5 mM)
Ethanol : ~1 mg/mL (~4.9 mM)

Solubility in Formulation 1: 2.5 mg/mL (12.13 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
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 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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