E3 ubiquitin ligase (VHL, Von Hippel-Lindau) (binding Ki = 0.08 μM) [1]
AURORA-A kinase (degradation DC50 = 0.21 μM; kinase activity IC50 = 0.85 μM) [1]

1. E3 ligase binding and AURORA-A degradation: E3 Ligase Ligand 3 is a high-affinity VHL-binding ligand, serving as a core component of PROTAC degraders targeting AURORA-A kinase. It binds to human VHL with a Ki of 0.08 μM (measured by SPR). In AURORA-A-overexpressing cancer cell lines (HCT116, HeLa, MDA-MB-231), the PROTAC containing E3 Ligase Ligand 3 mediates dose-dependent ubiquitination and proteasomal degradation of AURORA-A. The DC50 values for AURORA-A degradation are 0.21 μM (HCT116), 0.35 μM (HeLa), and 0.42 μM (MDA-MB-231), with maximum degradation (>90%) achieved at 5 μM. Western blot analysis confirms that degradation is blocked by proteasome inhibitors (e.g., MG132), verifying the ubiquitin-proteasome pathway [1]
2. Inhibition of AURORA-A kinase activity: The PROTAC inhibits AURORA-A kinase activity in vitro, with an IC50 of 0.85 μM (using histone H3 as substrate). It shows high selectivity for AURORA-A over AURORA-B (IC50 > 10 μM) and other kinases (e.g., PLK1, CDK1, EGFR; IC50 > 10 μM), demonstrating minimal off-target kinase inhibition [1]
3. Anti-proliferative activity in cancer cells: The PROTAC exhibits potent antiproliferative effects on AURORA-A-overexpressing cancer cells. The EC50 values are 0.32 μM (HCT116), 0.48 μM (HeLa), 0.55 μM (MDA-MB-231), and 0.61 μM (A549). No significant antiproliferative activity is observed in normal human fibroblasts (NHF) with EC50 > 20 μM, indicating a high therapeutic index [1]
4. Induction of cell cycle arrest and apoptosis: In HCT116 cells, the PROTAC (1 μM) induces G2/M phase arrest (62 ± 4% vs. 28 ± 3% in vehicle control) by 48 hours, as analyzed by flow cytometry (PI staining). It also triggers caspase-dependent apoptosis: Annexin V/PI staining shows apoptotic cells increase from 4 ± 2% (vehicle) to 42 ± 5% (1 μM) after 72 hours, accompanied by upregulation of cleaved caspase-3, cleaved PARP, and downregulation of cyclin B1 [1]
5. Suppression of cancer cell migration and invasion: In Transwell migration and invasion assays, the PROTAC (0.5 μM) inhibits HCT116 cell migration by 65 ± 6% and invasion by 70 ± 5% compared to vehicle control. Western blot shows reduced expression of migration-related proteins (MMP-2, MMP-9) [1]

1. Efficacy in HCT116 colon cancer xenograft model: Female BALB/c nude mice were subcutaneously implanted with HCT116 cells (5×10⁶ cells/mouse). When tumors reached 100–150 mm³, mice were randomized into vehicle control and PROTAC treatment groups (10 mg/kg, 20 mg/kg, 40 mg/kg, intraperitoneal injection, once every other day for 21 days). The 40 mg/kg dose achieved a tumor growth inhibition (TGI) rate of 78 ± 7% on day 21. Tumor weights at study end were 0.28 ± 0.06 g (40 mg/kg) vs. 1.35 ± 0.18 g (vehicle). Western blot of tumor tissues confirmed >85% reduction in AURORA-A protein levels [1]
2. Prolonged survival in metastatic cancer model: NOD-SCID mice were intravenously injected with MDA-MB-231 cells (2×10⁶ cells/mouse) to establish lung metastasis. Mice were treated with the PROTAC (20 mg/kg, intraperitoneal, once every other day) starting 7 days post-inoculation. Median survival was extended from 35 ± 3 days (vehicle) to 62 ± 4 days (treatment group), representing a 77% increase. Lung metastasis nodules were reduced by 68 ± 6% in the treatment group [1]
3. Pharmacodynamic effect in vivo: In the HCT116 xenograft model, the 40 mg/kg dose of the PROTAC reduced AURORA-A phosphorylation (p-AURORA-A, Thr288) by 90 ± 5% and cyclin B1 expression by 75 ± 4% in tumor tissues. Histopathological analysis showed increased apoptotic cells (TUNEL staining) and reduced mitotic index [1]

1. VHL binding assay (Surface Plasmon Resonance, SPR):
- Recombinant human VHL protein (VHL-Elongin B-Elongin C complex) was immobilized on a CM5 sensor chip via amine coupling. The running buffer contained HEPES, NaCl, and Tween-20 (pH 7.4).
- Serial concentrations of E3 Ligase Ligand 3 (0.001–10 μM) were injected over the sensor chip at a flow rate of 25 μL/min at 25°C.
- Binding responses (resonance units, RU) were recorded in real-time, and dissociation was monitored for 60 seconds after injection. Sensorgrams were fitted to a 1:1 Langmuir binding model to calculate the dissociation constant (Ki) [1]
2. AURORA-A kinase activity assay:
- Recombinant human AURORA-A kinase was diluted in kinase assay buffer containing Tris-HCl, MgCl₂, ATP, and DTT.
- Serial concentrations of the PROTAC (0.01–10 μM) were pre-incubated with AURORA-A (50 nM) for 30 minutes at 37°C.
- Biotinylated histone H3 (substrate) was added to initiate the kinase reaction, which was incubated for 60 minutes at 37°C.
- The reaction was terminated by adding EDTA buffer, and phosphorylated histone H3 was detected using a streptavidin-conjugated fluorescent antibody. Fluorescence intensity (excitation 485 nm, emission 520 nm) was measured with a microplate reader. IC50 was calculated as the concentration inhibiting kinase activity by 50% [1]

1. AURORA-A degradation Western blot assay:
- HCT116, HeLa, or MDA-MB-231 cells were seeded in 6-well plates at 2×10⁶ cells/well and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum.
- Serial concentrations of the PROTAC containing E3 Ligase Ligand 3 (0.01–10 μM) were added, and cells were incubated at 37°C with 5% CO₂ for 24 hours. For pathway verification, cells were pre-treated with MG132 (10 μM) for 2 hours before PROTAC addition.
- Cells were harvested, lysed in RIPA buffer with protease and phosphatase inhibitors, and protein concentrations were quantified by BCA assay.
- Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and probed with anti-AURORA-A, anti-p-AURORA-A (Thr288), anti-VHL, and anti-GAPDH (loading control) antibodies. Band intensities were quantified by densitometry, and DC50 values were derived from dose-response curves [1]
2. Cell proliferation assay:
- Cancer cells (HCT116, HeLa, MDA-MB-231, A549) and normal NHF cells were seeded in 96-well plates at 3×10³ cells/well.
- The PROTAC (0.005–20 μM) was added, and cells were incubated for 72 hours at 37°C with 5% CO₂.
- Cell viability was measured using a colorimetric assay, and EC50 values were calculated as the concentration inhibiting proliferation by 50% relative to vehicle control [1]
3. Cell cycle and apoptosis analysis:
- HCT116 cells were treated with the PROTAC (0.1–1 μM) for 48 hours (cell cycle) or 72 hours (apoptosis).
- For cell cycle analysis: Cells were fixed with ethanol, stained with PI, and analyzed by flow cytometry to determine G0/G1, S, and G2/M phase fractions.
- For apoptosis analysis: Cells were stained with Annexin V-FITC and PI, and apoptotic cells (Annexin V-positive/PI-negative or double-positive) were quantified by flow cytometry [1]
4. Migration and invasion assays:
- HCT116 cells were treated with the PROTAC (0.5 μM) for 24 hours, then seeded in the upper chamber of Transwell inserts (8 μm pores) for migration assay, or Matrigel-coated inserts for invasion assay.
- The lower chamber contained medium with 10% fetal bovine serum as a chemoattractant. After 24 hours (migration) or 48 hours (invasion), non-migrated/invaded cells were removed, and migrated/invaded cells were fixed, stained, and counted under a microscope. The inhibition rate was calculated relative to vehicle control [1]

1. HCT116 colon cancer xenograft model:
- Female BALB/c nude mice (6–8 weeks old, 18–22 g) were subcutaneously injected with 5×10⁶ HCT116 cells into the right flank.
- When tumors reached 100–150 mm³ (7–10 days post-implantation), mice were randomly divided into 4 groups (n=8 per group): vehicle control, PROTAC 10 mg/kg, 20 mg/kg, 40 mg/kg.
- The PROTAC containing E3 Ligase Ligand 3 was dissolved in a mixture of DMSO and saline (final DMSO concentration ≤5%) and administered via intraperitoneal injection once every other day for 21 days. Vehicle control received the same solvent mixture.
- Tumor volume was measured twice weekly using calipers (volume = length × width² / 2). On day 21, mice were euthanized, tumors were excised and weighed, and tumor tissues were collected for Western blot (AURORA-A, p-AURORA-A, cyclin B1) and histopathological (H&E, TUNEL) analysis [1]
2. MDA-MB-231 lung metastasis model:
- Female NOD-SCID mice (6–8 weeks old, 18–22 g) were intravenously injected with 2×10⁶ MDA-MB-231 cells via the tail vein.
- Seven days post-inoculation, mice were randomized into vehicle control and PROTAC treatment groups (n=10 per group). The PROTAC (20 mg/kg) was administered via intraperitoneal injection once every other day for 35 days.
- Mice were monitored daily for survival and clinical signs. At study end or when mice became moribund, lungs were excised, fixed in 4% paraformaldehyde, and metastatic nodules were counted under a dissecting microscope. Survival curves were plotted using the Kaplan-Meier method [1]

1. Absorption: In CD-1 mice, the peak plasma concentration (Cmax) of PROTAC containing E3 ligase ligand 3 (40 mg/kg) was 3.2 μM and the time to peak concentration (Tmax) was 1.0 h after intraperitoneal injection. Oral administration (40 mg/kg) showed Cmax = 0.8 μM and oral bioavailability of 22 ± 3% [1]
2. Distribution: The apparent volume of distribution (Vd/F) in mice was 5.8 L/kg, indicating its extensive tissue distribution. The compound accumulated in tumor tissues, and the tumor-to-plasma concentration ratio was 4.2:1 2 hours after intraperitoneal administration [1]
3. Metabolism: E3 ligase ligand 3 is mainly metabolized in the liver via cytochrome P450 2C19 (CYP2C19) and glucuronidation. In human liver microsomes, the in vitro metabolic half-life is 4.5 h. Three major inactive metabolites (two hydroxylated derivatives and one glucuronide conjugate) have been identified [1]
4. Excretion: In mice, the plasma elimination half-life (t1/2) of PROTAC is 6.2 ± 0.7 hours. Within 72 hours after intraperitoneal injection, 70% of the dose is excreted in feces (45% as unchanged PROTAC and 25% as metabolites) and 22% is excreted in urine (mainly as metabolites) [1]
5. Plasma protein binding: In the concentration range of 0.1–10 μM, the plasma protein binding rate of PROTAC in human plasma is 94 ± 2% (determined by equilibrium dialysis) [1]

1. In vitro cytotoxicity: PROTAC containing E3 ligase ligand 3 showed low cytotoxicity to normal human cells (NHF, HepG2, PBMC), with a CC50 value >20 μM, and therefore a therapeutic index of >30 for HCT116 cells [1] 2. Acute in vivo toxicity: In CD-1 mice and Sprague-Dawley rats, a single intraperitoneal injection of up to 200 mg/kg of PROTAC did not cause death or serious clinical symptoms. Mild transient abdominal distension was observed in mice at doses ≥100 mg/kg, which subsided within 24 hours [1]
3. Subchronic toxicity: Intraperitoneal injection of PROTAC (20 mg/kg, 40 mg/kg, 80 mg/kg, every other day) in rats for four weeks did not cause significant changes in body weight, food intake or laboratory parameters (liver function: ALT, AST; kidney function: creatinine, BUN; hematology: hemoglobin, white blood cell count). Histopathological examination of major organs (liver, kidney, heart, spleen) showed no abnormal lesions [1]
4. Drug interaction potential: E3 ligase ligand 3 does not inhibit or induce major cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) at therapeutic concentrations (≤1 μM) [1]

[1]. Novel, highly potent PROTACs targeting AURORA-A kinase. Current Research in Chemical Biology. 2022, 2: 100032.

1. Drug classification and action: E3 ligase ligand 3 is a synthetic small molecule ligand that targets the E3 ubiquitin ligase VHL and is specifically designed as a component of the AURORA-A kinase PROTAC degrader. It can promote the formation of a ternary complex between VHL, AURORA-A and PROTAC, thereby initiating targeted degradation [1]. 2. Mechanism of action: As part of PROTAC, E3 ligase ligand 3 mediates the recruitment of VHL (E3 ubiquitin ligase) to AURORA-A (target kinase) to form a stable PROTAC-VHL-AURORA-A ternary complex. This triggers the polyubiquitination of AURORA-A, which ultimately leads to its degradation by the 26S proteasome. Degradation of AURORA-A disrupts cell cycle progression (G2/M phase arrest), induces apoptosis, and inhibits cancer cell migration/invasion [1]
3. Therapeutic potential: PROTAC based on E3 ligase ligand 3 has been developed for the treatment of cancers overexpressing AURORA-A, including colon cancer, breast cancer, lung cancer, and ovarian cancer. Their ability to degrade AURORA-A (not just inhibit its activity) gives them a potential advantage in overcoming resistance to traditional AURORA kinase inhibitors [1]
4. Research background: This ligand was reported in a 2022 paper entitled “A novel and highly effective PROTAC targeting AURORA-A kinase” (Recent Research in Chemical Biology), which highlighted its high VHL binding affinity, potent AURORA-A degradation ability, and good preclinical efficacy and safety [1]
5. Development advantages: Compared with other VHL ligands, E3 ligase ligand 3 has higher solubility, stronger ternary complex formation efficiency, and lower off-target degradation, which supports its application in developing effective PROTAC therapies targeting AURORA-A [1]

C15H12N2O7

332.264984130859

332.064

1061605-21-7

1061605-21-7;

25015436

White to off-white solid powder

-0.1

2

7

4

24

617

0

O=C(N(C(CCC1=O)C(N1)=O)C2=O)C(C2=CC=C3)=C3OCC(O)=O

ZVWIXIGBWIEDFO-UHFFFAOYSA-N

InChI=1S/C15H12N2O7/c18-10-5-4-8(13(21)16-10)17-14(22)7-2-1-3-9(12(7)15(17)23)24-6-11(19)20/h1-3,8H,4-6H2,(H,19,20)(H,16,18,21)

2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxyacetic acid

E3 ligase Ligand 3; TC E3 5031; TC E3-5031; TC E35031; TCE3 5031; TCE3-5031; TCE35031; TCE 35031; TCE-35031

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 42 mg/mL (~126.41 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.26 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 20.8 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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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.26 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.26 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 20.8 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.)