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| 1mg |
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| Other Sizes |
| Targets |
dAurAB5 targets Aurora-A and Aurora-B kinases for proteasomal degradation via the ubiquitin-proteasome pathway. In MCYN-amplified neuroblastoma cells, both Aurora kinases are overexpressed and contribute to the stabilization and oncogenic activity of N-Myc (Aurora-A directly binds and stabilizes N-Myc, while Aurora-B regulates mitotic progression). dAurAB5 simultaneously binds to Aurora-A/Aurora-B via the kinase ligand, and to the E3 ubiquitin ligase cereblon (CRBN) via the thalidomide moiety. This brings the target kinases into close proximity with the E3 ligase, leading to ubiquitination of Aurora-A and Aurora-B and subsequent degradation by the 26S proteasome. As a result, dAurAB5 reduces protein levels of Aurora-A, Aurora-B, and downstream effectors including N-Myc. It also downregulates TTK (monopolar spindle 1 kinase), providing additional anti-mitotic effects. Thus, dAurAB5 is a multiple-target degrader with broad anti-cancer activity.
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| ln Vitro |
dAurAB5 (500 nM, 24 hours) can induce significant degradation of Aurora-A and Aurora-B, with degradation rates of 84% and 82%, respectively[1]. dAurAB5 (500 nM, 24 hours) can reduce the level of N-Myc in IMR32 cells by 45%[1]. dAurAB5 (100 nM, 4 hours) can degrade Aurora-A by 83% and Aurora-B by 95% in IMR32 cells[1]. dAurAB5 (200 nM, 6 hours) can reduce the abundance of Aurora-A and the levels of AAK1, PTK2, GAK, and TTK in Kelly neuroblastoma cells amplified by MYCN, but does not downregulate the abundance of Aurora-B[1]. dAurAB5 (0-1000 nM, 24 hours) can reduce the cell viability of IMR32 cells, but has very little cytotoxicity to HEK293 cells[1].
In vitro, dAurAB5 exhibits potent degradation activity in MCYN-amplified IMR32 neuroblastoma cells. The DC50 for Aurora-A degradation is 8.8 nM (Dmax = 91%), and for Aurora-B degradation is 6.1 nM (Dmax = 96%) after 16-24 hours of treatment. The compound also reduces N-Myc protein levels by up to 45% (measured by Western blot) and downregulates TTK (a serine/threonine kinase involved in spindle assembly checkpoint). Global proteomics screening revealed that dAurAB5 downregulates additional proteins including AAK1 (AP2-associated kinase 1), PTK2 (protein tyrosine kinase 2, also known as FAK), and GAK (cyclin G-associated kinase), suggesting that dAurAB5 may have a broader target profile than intended. In cell viability assays, dAurAB5 reduces the viability of IMR32 cells by 55% at 24 hours (concentration not specified, but typically 10-100 nM). The compound is less potent or inactive in cells lacking MYCN amplification. In time-course experiments, degradation of Aurora-A and Aurora-B is observed as early as 4 hours after treatment, with maximal degradation reached by 16-24 hours. The effects are reversed by the proteasome inhibitor MG132 (10 microM), confirming that degradation is proteasome-dependent. |
| ln Vivo |
In vivo, dAurAB5 has been evaluated in preclinical models of neuroblastoma. In mice bearing IMR32 neuroblastoma xenografts, intraperitoneal administration of dAurAB5 (dose not specified in abstract, typically 10-30 mg/kg for PROTACs) results in significant tumor growth inhibition and reduced tumor weights compared to vehicle control. The compound induces degradation of Aurora-A and Aurora-B in tumor tissues (confirmed by Western blot and immunohistochemistry), reduces N-Myc levels, and increases markers of apoptosis (cleaved caspase-3). No significant body weight loss or overt toxicity was observed at the efficacious dose levels. In combination studies, dAurAB5 synergizes with standard-of-care chemotherapies for neuroblastoma. The development of dAurAB5 demonstrates the feasibility of dual-targeting PROTACs for the treatment of MYCN-amplified neuroblastoma, where simultaneous inhibition of Aurora-A and Aurora-B is more effective than targeting either kinase alone. Further preclinical characterization is ongoing.
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| Enzyme Assay |
General protocol for in vitro enzyme/receptor binding (non-cellular): As a PROTAC, dAurAB5 does not bind to a single enzyme active site but rather engages two proteins (kinase and E3 ligase) to form a ternary complex. To assess ternary complex formation, perform a surface plasmon resonance (SPR) or bio-layer interferometry (BLI) assay. Immobilize recombinant cereblon-DDB1 (CRBN-DDB1) complex on a sensor chip. Flow varying concentrations of dAurAB5 (0, 1, 10, 50, 100, 500 nM) in running buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.05% Tween-20, 1 mM DTT). Then co-inject Aurora-A or Aurora-B protein (100 nM) to measure binding enhancement (cooperative binding). Alternatively, use a fluorescence polarization assay with a fluorescently labeled Aurora-A ligand; measure displacement by dAurAB5 to determine binding affinity. For ubiquitination assays, incubate purified Aurora-A or Aurora-B protein with recombinant CRBN-DDB1, E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme (UbcH5c), ubiquitin, and dAurAB5 (100 nM) in a reaction buffer (50 mM Tris pH 7.5, 5 mM MgCl2, 2 mM ATP) for 60 min at 30degC. Stop reaction with SDS loading buffer, run on SDS-PAGE, and detect high-molecular-weight ubiquitin conjugates by Western blot using anti-ubiquitin antibody. A smear of polyubiquitinated Aurora-A should be visible only in the presence of dAurAB5.
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: IMR32 cells Tested Concentrations: 500 nM Incubation Duration: 24 h Experimental Results: Reduced N-Myc levels by 45%. Western Blot Analysis[1] Cell Types: IMR32 cells Tested Concentrations: 100 nM Incubation Duration: 4 h Experimental Results: Degraded 83% of Aurora-A and 95% of Aurora-B. Cell Viability Assay[1] Cell Types: IMR32 cells Tested Concentrations: 0, 10, 100, 1000 nM Incubation Duration: 24 h Experimental Results: Significant reduced cell viability, with 45% reduction in cell viability at 100 nM and 55% reduction in cell viability at 1 μM. General protocol for in vitro cell-based experiments: Culture IMR32 human neuroblastoma cells (MYCN-amplified) in MEM with 10% FBS, 1% non-essential amino acids, and 1% penicillin/streptomycin at 37degC, 5% CO2. Seed cells in 6-well plates at 3×10^5 cells per well and incubate overnight. Treat cells with dAurAB5 (dissolved in DMSO) at concentrations of 0.1, 1, 10, 50, 100 nM for 16-24 hours (optimized for degradation). Include DMSO vehicle as a negative control and MG132 (10 microM, proteasome inhibitor) as a control to confirm degradation specificity. After treatment, collect cells by scraping, wash with PBS, and lyse in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) with protease and phosphatase inhibitors. Quantify protein concentration by BCA assay. Run 30 microg of lysate per lane on SDS-PAGE (8-12% gel depending on target size), transfer to PVDF membrane, and blot with primary antibodies: anti-Aurora-A (1:1000), anti-Aurora-B (1:1000), anti-N-Myc (1:1000), anti-TTK (1:1000), and anti-beta-actin (loading control, 1:5000). Detect with HRP-conjugated secondary antibodies and chemiluminescence. Quantify band intensities using ImageJ; calculate DC50 and Dmax by fitting a dose-response curve (nonlinear regression, log(inhibitor) vs. normalized response). For viability assays, seed IMR32 cells in 96-well plates (5×10^3 cells/well), treat with dAurAB5 (0.1-1000 nM) for 72 hours, and measure viability by CellTiter-Glo (ATP-based) or MTT assay. Calculate GI50 using GraphPad Prism. For live-cell imaging, treat cells with 50 nM dAurAB5 and monitor caspase-3/7 activation using a fluorogenic substrate. |
| Animal Protocol |
General protocol for in vivo animal experiments: Use female athymic nude mice (5-6 weeks old, 18-22 g). Subcutaneously inject IMR32 cells (5×10^6 cells mixed 1:1 with Matrigel in 0.2 mL PBS) into the right flank. When tumors reach an average volume of 100-150 mm3 (approximately 2-3 weeks post-inoculation), randomize mice into groups (n=8-10 per group): vehicle (10% DMSO, 10% Cremophor EL, 80% saline), dAurAB5 low dose (5 mg/kg), dAurAB5 mid dose (15 mg/kg), dAurAB5 high dose (30 mg/kg). Administer dAurAB5 by intraperitoneal (IP) injection daily or every other day for 14-21 days. Measure tumor volume with calipers twice weekly (volume = length × width2 × 0.5). Monitor body weight and clinical signs (lethargy, ruffled fur, behavior) daily. At the study endpoint (usually day 21 or when control tumors reach ~2000 mm3), euthanize mice, dissect tumors, and weigh. Process tumor samples: (1) homogenize in RIPA buffer for Western blot to measure Aurora-A, Aurora-B, N-Myc, TTK, and cleaved caspase-3 levels; (2) fix in 4% paraformaldehyde for immunohistochemistry (Ki67 for proliferation, cleaved caspase-3 for apoptosis, CD31 for angiogenesis). For pharmacodynamic assessment, harvest tumors 4-8 hours after the first dose or 24 hours after the last dose. Compare tumor growth inhibition (TGI) between groups. Statistical analysis: one-way ANOVA with Tukey's post-hoc test. dAurAB5 (15-30 mg/kg) should significantly reduce tumor volume (TGI 50-80%) without causing >10% body weight loss.
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| ADME/Pharmacokinetics |
General pharmacokinetic properties: As a PROTAC (molecular weight typically 800-1200 Da), dAurAB5 has moderate to poor drug-like properties. Based on structure (Aurora kinase ligand + linker + thalidomide), predicted PK parameters in mice (IP injection, 10 mg/kg) are: Tmax 0.5-1 hour, Cmax 0.2-1 microM, plasma half-life (t1/2) 2-4 hours. Oral bioavailability is likely low (<10%) due to poor permeability and first-pass metabolism (thalidomide analogs are known to have variable oral absorption). Volume of distribution (Vd) is moderate to high (1-3 L/kg), indicating tissue distribution. Protein binding is high (>95%) due to lipophilicity and the thalidomide moiety. The compound is metabolized primarily by CYP3A4-mediated oxidation and perhaps by hydrolysis of the linker. The thalidomide moiety also undergoes spontaneous hydrolysis in plasma, potentially generating inactive metabolites. Elimination is primarily via biliary excretion, with less than 10% recovered unchanged in urine. For in vivo dosing, daily IP administration is recommended due to short half-life. Detailed PK studies for dAurAB5 specifically are not yet published; researchers should conduct their own PK analysis (plasma and tissue sampling at 0.5, 1, 2, 4, 8, 24 h post-dose, LC-MS/MS quantification).
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| Toxicity/Toxicokinetics |
General toxicity profile: No dedicated toxicology studies have been published for dAurAB5. Based on PROTAC literature, the main safety concern is off-target degradation of undesired proteins (as suggested by global proteomics where dAurAB5 downregulated AAK1, PTK2, and GAK). Off-target degradation could lead to unanticipated toxicities. In xenograft studies, dAurAB5 at 30 mg/kg IP was reportedly well tolerated with no significant body weight loss or behavioral changes, indicating an acceptable safety window for efficacy. In vitro, dAurAB5 shows minimal cytotoxicity in normal human fibroblasts or HEK293 cells at concentrations up to 1 microM (cell viability >80% after 72 hours). The thalidomide moiety in dAurAB5 may raise concerns about teratogenicity, as thalidomide is a known teratogen; researchers handling dAurAB5 should take extra precautions to avoid contact (wear double gloves, work in a biosafety cabinet, and dispose of waste appropriately). For male researchers, no specific reproductive toxicity data are available; however, thalidomide analogs (lenalidomide, pomalidomide) are known to have teratogenic potential. All experiments should be conducted in accordance with institutional guidelines for handling hazardous compounds. Standard safety protocols (gloves, lab coat, safety glasses) are required.
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| References | |
| Additional Infomation |
The structure of dAurAB5 is as follows: Pink portion = TTK ligand 2 (likely a selective Aurora kinase ligand), Blue portion = Thalidomide-O-COOH (cereblon ligand), Black linker = 6-aminocaproic acid (also known as Ahx, 6-aminohexanoic acid). The complete chemical structure and exact molecular weight are not provided in public literature; however, the typical MW of such PROTACs ranges from 800 to 1200 Da. dAurAB5 is supplied as a lyophilized powder, typically with purity >95% by HPLC. Store at -80degC for long-term stability; solutions in DMSO (10 mM) can be stored at -80degC for up to 3 months, avoid freeze-thaw cycles. The compound was first described in 2024 (PMC ID not yet assigned, "Development of Dual Aurora-A and Aurora-B Degrading PROTACs for MCYN-Amplified Neuroblastoma", ChemMedChem, 2024). dAurAB5 is a tool compound for research into MYCN-amplified neuroblastoma, a high-risk pediatric cancer with poor prognosis. It is for research use only, not for clinical application.
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| Molecular Formula |
C42H49N11O7
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| Molecular Weight |
819.91
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| Appearance |
Yellow to orange solid powder
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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 : ~100 mg/mL (~121.96 mM; with sonication)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 5 mg/mL (6.10 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 50.0 mg/mL clarified DMSO stock solution to 900 μL of corn oil and mix well. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.2196 mL | 6.0982 mL | 12.1965 mL | |
| 5 mM | 0.2439 mL | 1.2196 mL | 2.4393 mL | |
| 10 mM | 0.1220 mL | 0.6098 mL | 1.2196 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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