Description: BAY-524 is a novel and potent Bub1 inhibitor with an IC50 of 450 nM for human Bub1 in the presence of 2 mM ATP. It demonstrated potent Bub1 kinase inhibition both in vitro and in intact cells. The kinase Bub1 functions in the spindle assembly checkpoint (SAC) and in chromosome congression, but the role of its catalytic activity remains controversial. Bub1 inhibition affected chromosome association of Shugoshin and the chromosomal passenger complex (CPC), without abolishing global Aurora B function. Consequently, inhibition of Bub1 kinase impaired chromosome arm resolution but exerted only minor effects on mitotic progression or SAC function. Importantly, BAY-320 and BAY-524 treatment sensitized cells to low doses of Paclitaxel, impairing both chromosome segregation and cell proliferation. These findings are relevant to our understanding of Bub1 kinase function and the prospects of targeting Bub1 for therapeutic applications.
Physicochemical Properties
| Molecular Formula | C24H24F2N6O3 |
| Molecular Weight | 482.48 |
| Exact Mass | 482.187 |
| CAS # | 1445830-39-6 |
| PubChem CID | 71611179 |
| Appearance | Off-white to light yellow solid powder |
| LogP | 3.8 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 10 |
| Rotatable Bond Count | 9 |
| Heavy Atom Count | 35 |
| Complexity | 636 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | FC1C([H])=C(C([H])=C(C=1C([H])([H])N1C(=C(C([H])([H])[H])C(C2=NC([H])=C(C(N([H])C3C([H])=C([H])N=C([H])C=3[H])=N2)OC([H])([H])[H])=N1)OC([H])([H])[H])F)OC([H])([H])C([H])([H])[H] |
| InChi Key | LMRCQVHTZUUHFN-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C24H24F2N6O3/c1-5-35-16-10-18(25)17(19(26)11-16)13-32-24(34-4)14(2)21(31-32)23-28-12-20(33-3)22(30-23)29-15-6-8-27-9-7-15/h6-12H,5,13H2,1-4H3,(H,27,28,29,30) |
| Chemical Name | 2-[1-[(4-ethoxy-2,6-difluorophenyl)methyl]-5-methoxy-4-methylpyrazol-3-yl]-5-methoxy-N-pyridin-4-ylpyrimidin-4-amine |
| Synonyms | BAY-524; BAY 524; BAY524 |
| HS Tariff Code | 2934.99.9001 |
| 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) |
Biological Activity
| Targets | At an IC50 of 450 nM, BAY-524 inhibits the human Bub1 recombinant catalytic domain[1]. BAY-524 selectively inhibits Bub1 kinase at 7–10 μM or 0–30 μM for 14 hours or 1 hour [1]. The effects of BAY-524 (7 μM, 12 hours or 7 μM, 12 μM, 3 hours) on chromatid cohesion and Sgo1 and Sgo2 localization in cells have been reported [1]. BAY-524 (7 μM, 10 μM) modifies CPC's activity and localization [1]. Additive effects on CPC centromere association are observed with BAY-524 (7 μM, 2 h) [1]. There is a slight impact of BAY-524 (7 μM, 48 hours) on SAC signaling [1]. Cells are sensitized to low doses of paclitaxel by BAY-524 (7 μM, 10 μM) [1]. |
| ln Vitro |
At an IC50 of 450 nM, BAY-524 inhibits the human Bub1 recombinant catalytic domain[1]. BAY-524 selectively inhibits Bub1 kinase at 7–10 μM or 0–30 μM for 14 hours or 1 hour [1]. The effects of BAY-524 (7 μM, 12 hours or 7 μM, 12 μM, 3 hours) on chromatid cohesion and Sgo1 and Sgo2 localization in cells have been reported [1]. BAY-524 (7 μM, 10 μM) modifies CPC's activity and localization [1]. Additive effects on CPC centromere association are observed with BAY-524 (7 μM, 2 h) [1]. There is a slight impact of BAY-524 (7 μM, 48 hours) on SAC signaling [1]. Cells are sensitized to low doses of paclitaxel by BAY-524 (7 μM, 10 μM) [1]. BAY-524 inhibits Bub1 kinase activity in vitro, reducing both Bub1 autophosphorylation and phosphorylation of histone H2A at threonine 120 (pT120-H2A). [1] BAY-524 (7–10 µM) significantly reduces centromeric levels of pT120-H2A in HeLa S3 and RPE1 cells, as shown by immunofluorescence and Western blot. [1] BAY-524 treatment (7 µM) leads to persistent sister chromatid arm cohesion and redistribution of Shugoshin proteins (Sgo1 and Sgo2) from centromeres to chromosome arms. [1] BAY-524 reduces centromeric localization of the chromosomal passenger complex (CPC) components Aurora B, Borealin, and INCENP by ~50%, and partially reduces Aurora B activity at centromeres. [1] BAY-524 does not significantly affect kinetochore recruitment of SAC proteins Mad1, Mad2, or BubR1, nor does it dramatically weaken the spindle assembly checkpoint in nocodazole-arrested cells. [1] BAY-524 does not impair chromosome alignment or significantly increase micronucleation when used alone. [1] BAY-524 sensitizes HeLa and RPE1 cells to low doses of Paclitaxel (1–4 nM), leading to increased chromosome segregation errors and reduced cell proliferation in colony formation assays. [1] |
| Enzyme Assay |
The inhibitory activity of BAY-524 against the recombinant catalytic domain of human Bub1 (amino acids 704–1085) was measured using a time-resolved fluorescence energy transfer (TR-FRET) kinase assay. The assay monitored phosphorylation of a biotinylated synthetic peptide substrate in the presence of 2 mM ATP. The IC₅₀ value was determined to be 450 ± 60 nM. [1] In vitro kinase assays were performed using LAP-tagged Bub1 wild-type protein purified from mitotic HEK 293T cells. Reactions contained recombinant histone H2A as substrate, γ-³²P-ATP, and serial dilutions of BAY-524. After incubation at 30°C for 30 minutes, reactions were stopped and analyzed by SDS-PAGE, followed by autoradiography and Western blotting for pT120-H2A. [1] |
| Cell Assay |
Western Blot Analysis[1] Cell Types: hTERT-RPE1 (RPE1) and HeLa cells Tested Concentrations: 7-10 μM Incubation Duration: 14 hrs (hours) Experimental Results: Significant reduction in T120 phosphorylation. Immunofluorescence[1] Cell Types: RPE1 and HeLa cells Tested Concentrations: 0-30 μM Incubation Duration: 1 hour Experimental Results: diminished phosphorylation of histone H2A-T120. For immunofluorescence analysis of pT120-H2A, HeLa S3 or RPE1 cells were treated with the proteasomal inhibitor MG132 for 2 hours, followed by nocodazole and increasing doses of BAY-524 for 1 hour. Cells were fixed and stained with antibodies against pT120-H2A and CREST serum, and analyzed by immunofluorescence microscopy. [1] To assess chromatid cohesion and Sgo localization, HeLa S3 cells were synchronized by thymidine block, released into nocodazole with BAY-524 (7 µM), and chromosome spreads were prepared. Alternatively, cells were fixed and stained for Sgo1, Sgo2, and CREST. [1] For analysis of CPC localization, synchronized HeLa cells treated with BAY-524 were fixed and stained for Aurora B, Borealin, INCENP, and centromere markers. [1] Cell proliferation and colony formation assays were performed by treating HeLa or RPE1 cells with BAY-524 (7 or 10 µM) in the presence or absence of Paclitaxel (1–4 nM) for 7 days. Colonies were fixed, stained, and quantified. [1] Live-cell imaging was used to monitor mitotic progression and chromosome segregation in HeLa or RPE1 cells expressing H2B-GFP treated with BAY-524 and/or Paclitaxel. [1] |
| References |
[1]. Probing the catalytic functions of Bub1 kinase using the small molecule inhibitors BAY-320 and BAY-524. Elife. 2016 Feb 17;5. pii: e12187. |
| Additional Infomation |
BAY-524 is a substituted benzylpyrazole compound and an ATP-competitive inhibitor of Bub1 kinase. [1] It is used as a chemical tool to probe the catalytic versus scaffolding functions of Bub1 in mitosis. [1] Its kinase selectivity profile was not detailed for BAY-524 specifically in this study, though a related compound BAY-320 showed high selectivity for Bub1 in a broad kinome scan. [1] The study suggests that BAY-524 may have potential for therapeutic application in combination with microtubule-targeting agents like Paclitaxel, especially in aneuploid cancer cells. [1] |
Solubility Data
| Solubility (In Vitro) | DMSO : ~166.67 mg/mL (~345.44 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 4.17 mg/mL (8.64 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 41.7 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. Solubility in Formulation 2: ≥ 4.17 mg/mL (8.64 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 41.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0726 mL | 10.3631 mL | 20.7262 mL | |
| 5 mM | 0.4145 mL | 2.0726 mL | 4.1452 mL | |
| 10 mM | 0.2073 mL | 1.0363 mL | 2.0726 mL |