Aurora A Kinase (AURKA). In MV4-11 cells, the half-maximal degradation concentration (DC₅₀) of JB170 for AURKA is 28 nM. In HEK293 cells, the half-maximal effective concentration (EC₅₀) for JB170 binding to AURKA, determined by BRET assay, is 193 nM. The dissociation constant (K_d) for JB170 binding to purified AURKA protein, measured by isothermal titration calorimetry (ITC), is 375 nM.
JB170 binds to CEREBLON with lower affinity, with a K_d of 6.88 μM as determined by ITC. However, its affinity for the binary complex of AURKA and CEREBLON is significantly enhanced, with a K_d of 183 nM. [1]
Aurora A (DC50 = 28 nM); Aurora A (Kd = 99 nM); Aurora A (EC50 = 193 nM);
Cereblon

JB170 (1 μM; 24-72 hours; MV4-11 cells) decreases the survival of cancer cells via mediating the depletion of Aurora-A [1]. AURORA-A levels are lowered by JB170 (0.01-10 μM; 6 hours; MV4-11 cells) [1]. JB170 (0.5 μM; 12 hours; MV4-11 cells) inhibits or slows the advancement of the S phase [1]. JB170 (0.5 μM; 0-72 hours; MV4-11 cells) exclusively targets AURORA-A to trigger apoptosis[1]. JB170 (0.1 μM; 0-9 hours; IMR5 cells) exhibits a quick AURORA-A depletion. In comparison to AURORA-A, JB170 (0~1 μM; 6 hours; MV4-11 cells) was significantly diminished in the mutants. In MV4-11 cells, JB170 (0.1 μM; 18 hours) does not cause AURORA-A activation. JB170 (0~1 μM; 24 hours; IMR5 cells) significantly eliminates the depletion of AURORA-AT217D. JB170 (1 μM; 4 days; IMR5 cells) mediates the reduction of Aurora-A, which prevents the survival of cancer cells. By lowering AURORA-A mRNA levels, JB170 (IMR5 cells) lowers AURORA-A levels [1].

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AURORA-A Degradation: In MV4-11 leukemia cells, JB170 (0.1 μM) treatment reduced AURORA-A levels as early as 3 hours, reaching maximal degradation by 6 hours. JB170 also induced rapid AURORA-A degradation in various cancer cell lines, including U2OS, HLE, and IMR5. Following JB170 treatment, the half-life of AURORA-A protein was reduced from 3.8 hours to 1.3 hours. [1]
Degradation Mechanism and Specificity: JB170-induced AURORA-A degradation is dependent on CEREBLON and the proteasome. Co-incubation with alisertib or thalidomide blocked its degradation activity; the proteasome inhibitor MG132 and the neddylation inhibitor MLN4924 completely abrogated degradation. In MV4-11 cells, quantitative SILAC proteomics revealed that out of 4,259 detected proteins, AURORA-A was the only protein significantly downregulated (by 73%), with no effects on AURORA-B or other alisertib-binding proteins. [1]
Effects on Cell Cycle: In contrast to the G2/M arrest induced by alisertib, treatment of MV4-11 cells with JB170 (0.5 μM) for 12 hours primarily caused a reduction in BrdU incorporation in S-phase cells, indicating S-phase delay or arrest. In IMR5 cells, this S-phase arrest phenotype was completely reversed by expressing a JB170-insensitive AURORA-A^T217D mutant, confirming it was specifically mediated by AURORA-A depletion. [1]
Induction of Apoptosis: In MV4-11 cells, JB170 (0.1-1 μM) treatment for 72 hours reduced viable cell count to 32% of control and increased the fraction of apoptotic cells to 56%. In IMR5 cells, JB170 similarly induced apoptosis and inhibited colony formation, effects that were completely reversed by expressing AURORA-A^T217D. The inactive analogue JB211 did not induce apoptosis. [1]

Isothermal titration calorimetry[1]
All titrations were performed on a Nano ITC calorimeter at 25 °C. The titrations of the binary complexes (AURORA-A into JB170 and CEREBLON-TBD into JB170) were performed as reverse titrations. Protein concentrations were determined spectroscopically at 280 nm using calculated extinction coefficients and a Thermo Scientific NanoDrop spectrophotometer and a buffer of 25 mM HEPES pH 7.5, 200 mM NaCl, 0.5 mM TCEP, 5% glycerol was used. For AURORA-A, concentrations in the injector (between 57 and 110 µM) had to be optimized due to protein stability issues matching JB170 concentrations between 1.0 and 10.0 µM. Values were calculated from four titrations. Best conditions were achieved at 110 µM AURORA-A and 10 µM JB170. For the CEREBLON (TBD) titration concentrations between 88 and 100 µM were used for the protein and 2.0 and 3.5 µM for JB170. Dissociation constants were calculated from three independent titrations.
Titrations for the ternary complexes were determined as previously described41. Briefly, CEREBLON(TBD) at 0.1 µM was titrated as described above. The binary complex remained in the calorimeter and the excess of solution after the titration was removed using a syringe. AURORA-A (110 µM) was titrated into the binary complex which had a JB170 concentration of 3.2 µM and 2.8 µM in two independent titration experiments. All data were fitted using a single binding site model in NanoAnalyse software to obtain Kd values and thermodynamic binding parameters.

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Isothermal Titration Calorimetry for Binding Affinity: The binding affinity of JB170 to AURORA-A, CEREBLON, and their complex was determined using an isothermal titration calorimeter. Purified AURORA-A protein (57-110 μM) was titrated into a JB170 solution (1.0-10.0 μM) to determine the dissociation constant (K_d) for the binary complex. Similarly, purified CEREBLON protein (88-100 μM) was titrated into a JB170 solution (2.0-3.5 μM). For the ternary complex, CEREBLON-TBD was pre-incubated with JB170 (3.2 μM) to form a binary complex, followed by titration with AURORA-A (110 μM). All data were fitted using a single binding site model. [1]
Kinobead Selectivity Profiling: Competition binding experiments were performed using immobilized broad-spectrum kinase inhibitors (Kinobeads epsilon) in MV4-11 cell lysates. Lysates were pre-incubated with increasing concentrations of JB170 (3 nM to 30 μM) before incubation with the beads. Bound proteins were eluted, digested, and analyzed by LC-MS/MS. The half-maximal inhibitory concentration (EC₅₀) for each protein was determined and corrected to calculate the apparent binding constant (K_d^app). [1]
Thermal Stability Shift Assay: Purified recombinant AURORA-A protein (2 μM) was incubated with 10 μM JB170 at room temperature for ~10 minutes. Following the addition of SYPRO Orange dye, the temperature-dependent protein unfolding was monitored by measuring fluorescence changes using a real-time PCR instrument to assess the effect of compound binding on protein thermal stability. [1]

Cell Viability Assay[1]
Cell Types: MV4-11 cells
Tested Concentrations: 1 µM
Incubation Duration: 24-72 hrs (hours)
Experimental Results: After 72 hrs (hours), the number of viable cells was 32% of control levels.

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Western Blot Analysis[1]
Cell Types: MV4-11 cells
Tested Concentrations: 0.01~10 μM
Incubation Duration: 6 hrs (hours)
Experimental Results: Substantial degradation was observed at 100 nM and 1 µM.

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Apoptosis Analysis[1]
Cell Types: MV4-11 cells
Tested Concentrations: 0.5 µM
Incubation Duration: 0~72 hrs (hours)
Experimental Results: Apoptosis was exclusively caused by targeting AURORA-A.

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Cell Cycle Analysis[1]
Cell Types: MV4-11 cells
Tested Concentrations: 0.5 µM
Incubation Duration: 12 hrs (hours)
Experimental Results: Delayed or arrested S-phase progression.

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Target Engagement Assay (BRET): HEK293 cells were transfected to express AURORA-A or AURORA-B fused with NanoLuc luciferase. Cells were incubated with various concentrations of JB170 and a fluorescent tracer for 2 hours. BRET signal was measured, and EC₅₀ values for compound competition were calculated. [1]
HiBiT Degradation Assay: MV4-11 cells stably expressing HiBiT-tagged AURORA-A were seeded in 96-well plates and treated with serial dilutions of JB170 for 6 hours. After lysis, the complementary LargeBiT fragment was added, and luminescence was measured to quantify AURORA-A protein levels for DC₅₀ calculation. [1]
Western Blot: Cells treated with compounds were lysed in RIPA buffer containing protease and phosphatase inhibitors. After protein quantification, samples were separated by SDS-PAGE and transferred to PVDF membranes. Immunoblotting was performed using specific antibodies against AURORA-A, AURORA-B, CEREBLON, cleaved caspase-3, cyclin B1, etc., with vinculin as a loading control. [1]
Immunoprecipitation: Lysates from MV4-11 cells stably expressing HA-tagged AURORA-A were incubated with HA-coupled magnetic beads for 3 hours. Bound protein complexes were eluted, and co-precipitated proteins, such as CEREBLON, were detected by immunoblotting. [1]
Flow Cytometry for Cell Cycle Analysis: Cells treated with JB170 were labeled with 10 μM BrdU for 1 hour. After fixation, cells were treated with 2M HCl to denature DNA, stained with anti-BrdU-FITC antibody, and counterstained with propidium iodide (PI) for DNA content. BrdU incorporation and DNA content were analyzed by flow cytometry. [1]
Flow Cytometry for Apoptosis Detection: Harvested cells were resuspended in Annexin V Binding Buffer and stained with Annexin V/Pacific Blue and propidium iodide (PI). Live, early apoptotic, and late apoptotic/necrotic cells were distinguished by flow cytometry. [1]
Cell Viability Assay (AlamarBlue): MV4-11 cells were seeded in 96-well plates and treated with JB170 for various durations (media refreshed every 24 hours). AlamarBlue HS Cell Viability Reagent was added, and fluorescence was measured (excitation 550 nm, emission 600 nm) to assess viability. [1]
Colony Formation Assay: IMR5 cells were treated with JB170 for 4 days (media refreshed every 24 hours), then stained with crystal violet solution for 30 minutes, washed, and dried to visualize colonies. [1]
Real-Time Apoptosis Monitoring (IncuCyte): NCI-H446 cells were seeded in 96-well plates and treated with JB170 along with a Caspase-3/7 reagent. Green fluorescent images were acquired every 2 hours using an IncuCyte Zoom live-cell imaging system to monitor caspase-3/7 activation in real-time. [1]
Quantitative SILAC Mass Spectrometry: Light, medium, and heavy SILAC-labeled MV4-11 cells were treated with DMSO, 100 nM JB170, or 100 nM alisertib for 6 hours, respectively. Equal amounts of cells from each condition were mixed, lysed, and proteins were separated by SDS-PAGE, in-gel digested, and analyzed by LC-MS/MS. MaxQuant software was used for protein identification and quantification. [1]
IP-MS for AURORA-A Interactome: Lysates from MV4-11 cells stably expressing HA-AURORA-A or empty vector were subjected to immunoprecipitation using HA magnetic beads. Eluted protein complexes were digested and analyzed by LC-MS/MS. Proteins were identified and quantified using MaxQuant to determine AURORA-A interacting partners. [1]

[1]. PROTAC-mediated degradation reveals a non-catalytic function of AURORA-A kinase. Nat Chem Biol. 2020;16(11):1179-1188.

Mitotic kinase AURORA-A is crucial for cell cycle progression and is considered an important target for cancer therapy. Although the catalytic activity of AURORA-A is essential for its mitotic function, recent studies have shown that it also has non-catalytic functions that are difficult to target with conventional small molecule drugs. Therefore, we developed a series of chemical degrading agents (PROTACs) by linking clinical kinase inhibitors of AURORA-A with E3 ubiquitin ligase-binding molecules (e.g., thalidomide). One of these degrading agents can rapidly, persistently and highly specifically degrade AURORA-A. In addition, we found that the synergistic binding between AURORA-A and CEREBLON can stabilize the degrading agent complex. Degrading agent-mediated AURORA-A depletion leads to S-phase defects, which is different from the cell cycle effects observed with kinase inhibition, indicating that AURORA-A has an important non-catalytic function in DNA replication. AURORA-A degradation can induce a large number of apoptosis in cancer cell lines, thus providing a multifunctional starting point for developing new therapies to counteract the role of AURORA-A in cancer. [1]

C48H44CLFN8O11

963.361373901367

962.28

C, 59.84; H, 4.60; Cl, 3.68; F, 1.97; N, 11.63; O, 18.27

2705844-82-0

153835264

Light yellow to yellow solid powder

1.49±0.1 g/cm3(Predicted)

3.7

4

16

19

69

1870

0

C(NCCOCCOCCNC(COC1=CC=CC2=C1C(=O)N(C1CCC(=O)NC1=O)C2=O)=O)(=O)C1=CC=C(NC2=NC=C3C(=N2)C2=CC=C(Cl)C=C2C(C2=C(OC)C=CC=C2F)=NC3)C=C1OC

GYKNPXCQINZRLL-UHFFFAOYSA-N

InChI=1S/C48H44ClFN8O11/c1-65-35-7-4-6-33(50)41(35)43-32-21-27(49)9-11-29(32)42-26(23-53-43)24-54-48(57-42)55-28-10-12-30(37(22-28)66-2)44(61)52-16-18-68-20-19-67-17-15-51-39(60)25-69-36-8-3-5-31-40(36)47(64)58(46(31)63)34-13-14-38(59)56-45(34)62/h3-12,21-22,24,34H,13-20,23,25H2,1-2H3,(H,51,60)(H,52,61)(H,54,55,57)(H,56,59,62)

4-[[9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-N-[2-[2-[2-[[2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxyacetyl]amino]ethoxy]ethoxy]ethyl]-2-methoxybenzamide

2705844-82-0; CHEMBL5278315; 4-((9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-benzo[c]pyrimido[4,5-e]azepin-2-yl)amino)-N-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)ethoxy)ethoxy)ethyl)-2-methoxybenzamide; SCHEMBL25163855; EX-A7164; BDBM50609429; 4-[[9-chloro-7-(2-fluoro-6-methoxyphenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-N-[2-[2-[2-[[2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxyacetyl]amino]ethoxy]ethoxy]ethyl]-2-methoxybenzamide;

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 : 100 mg/mL (103.80 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.60 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 25.0 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.)