| Size | Price | Stock | Qty |
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| 25mg |
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Purity: ≥98%
BTB-1 is a novel, potent and the first small molecule inhibitor of the mitotic motor protein Kif18A. It exhibits selectivity within the Kif4, Eg5, MKLP-1, MKLP-2, MPP1, CENP-E, and MCAK kinesin subgroups. Antimitotic medications target kinesin motors because they have a variety of functions during mitosis. Kif18A is frequently overexpressed in solid tumors, so drugs such as BTB-1 are not only very interesting for basic research but may also lead to new approaches in the treatment of human diseases. BTB-1 was added during in vitro motility tests, and this reversibly inhibited Kif18A's ability to glide through MT. Surprisingly, BTB-1 traps Kif18A on the microtubule, while its binding site is similar to that of well-studied Kif11 inhibitors that prevent tight microtubule binding.
| Targets |
Kif18A (IC50 = 1.69 μM)
Human mitotic motor protein Kif18A (kinesin Kif18A): Inhibits its ATPase activity, with IC₅₀ value in the low micromolar range [2] |
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| ln Vitro |
BTB-1 blocks the motility of Kif18A in a reversible manner. BTB-1 slows down the progress of cells through mitosis by inhibiting Kif18A in a manner that is competitive with adenosine triphosphate (ATP) but uncompetitive with microtubules. No other tested mitotic kinesin is significantly inhibited by 100 μM BTB-1. Only when the motor protein is connected to its pseudosubstrate microtubules does BTB-1 compete with ATP for Kif18A binding. BTB-1 treatment causes dose-dependent accumulation of HeLa cells during mitosis[1]. BTB-1 demonstrates cytotoxicity, exhibiting an EC50 value of 35.8 δM. When 50 μM BTB-1 is applied to HeLa cells, significant abnormalities in chromosome alignment and spindle morphology are observed. Treatment with high BTB-1 concentrations does not cause spindles to elongate[2]. 1. Inhibition of Kif18A ATPase activity: BTB-1 acts as a small-molecule inhibitor targeting the mitotic motor protein Kif18A. It inhibits the ATPase activity of Kif18A, which is crucial for the protein's function in mitosis [1][2] 2. Selectivity against related kinesins: Compared with its optimized derivatives, BTB-1 shows lower selectivity for Kif18A when tested against a panel of Kif18A-related kinesins in vitro [2] 3. Impact on microtubule polymerization: Structure-activity relationship studies indicate that BTB-1 may have substituents that mediate undesired inhibitory effects on microtubule polymerization [2] |
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| ln Vivo |
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| Enzyme Assay |
In DMSO, BTB-1 is prepared. The His-Kif18Amotor sactivityis observed as ATP concentration increases when 3 μM Mts and increasing BTB-1 concentrations (0.21 μM, 0.42 μM, 0.85 μM, 1.7 μM) or DMSO as a control are present[1].
1. Kif18A ATPase activity inhibition assay: Purified recombinant Kif18A protein is prepared and incubated with different concentrations of BTB-1 in a reaction buffer containing ATP. The reaction mixture is incubated under specific conditions (temperature, time) to allow the ATPase reaction to proceed. The amount of inorganic phosphate (Pi) generated from ATP hydrolysis is detected using a suitable method (e.g., colorimetric assay) to measure ATPase activity. The IC₅₀ value of BTB-1 for inhibiting Kif18A ATPase activity is calculated by fitting the concentration-response curve of the inhibition rate [2] 2. Selectivity assay against related kinesins: Purified recombinant proteins of a panel of Kif18A-related kinesins are prepared. Each kinesin's ATPase activity is measured in the presence of BTB-1 at a specific concentration. The inhibition rate of BTB-1 on each related kinesin is compared with that on Kif18A to evaluate its selectivity [2] |
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| Cell Assay |
In a reversible way, BTB-1 inhibits Kif18A motility. Cell division is slowed down by BTB-1, which inhibits Kif18A in a way that is competitive for adenosine triphosphate (ATP) but uncompetitive for microtubules. It was found that none of the other tested mitotic kinesins are significantly inhibited by 100 μM BTB-1. BTB-1 and ATP are only in competition for Kif18A binding when the motor protein is attached to its pseudosubstrate microtubules. BTB-1 treatment causes dose-dependent accumulation of HeLa cells during mitosis. With an EC50 value of 35.8 μM, BTB-1 exhibits cell toxicity. Severe defects in spindle morphology and chromosome alignment are observed in HeLa cells treated with 50 μM BTB-1. Longitudinal spindles are not produced by treatment with high BTB-1 concentrations.
1. Mitosis-related cell assay: HeLa cells are cultured and treated with BTB-1 at different concentrations. The cells are fixed at specific time points after treatment, and immunofluorescence staining is performed using antibodies against mitosis-related markers (e.g., tubulin for microtubule visualization, histone H3 phosphorylation for mitotic cell identification). The effect of BTB-1 on mitotic progression is observed under a fluorescence microscope, including changes in mitotic spindle structure and the proportion of mitotic cells [1] 2. Microtubule polymerization impact assay: Cells are treated with BTB-1, and then the microtubule cytoskeleton is visualized using immunofluorescence staining with tubulin-specific antibodies. The effect of BTB-1 on microtubule polymerization is evaluated by observing the integrity and distribution of microtubules under a microscope. Additionally, biochemical methods (e.g., microtubule co-sedimentation assay) can be used to directly measure the impact of BTB-1 on microtubule polymerization in vitro [2] |
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| Animal Protocol |
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| References | |||
| Additional Infomation |
1. Role as a lead compound: BTB-1 is the first small molecule Kif18A inhibitor discovered. As a lead compound, it is used to synthesize and optimize Kif18A inhibitors, laying the foundation for the development of more effective and selective Kif18A-targeting compounds [2]. 2. Biological significance of Kif18A: Kif18A is a mitotic kinetic protein that plays a key role in the shape and function of the mitotic spindle, mediating genome segregation during cell division. Errors in this process are associated with tumorigenesis, and Kif18A is often overexpressed in solid tumors. Kif18A inhibitors, such as BTB-1, have potential applications in basic research and the development of novel cancer treatment strategies [2]. 3. Chemical class: BTB-1 belongs to the sulfone class of compounds, and its structure-activity relationship has been studied to guide the optimization of its derivatives [1][2].
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| Molecular Formula |
C12H8CLNO4S
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| Molecular Weight |
297.71
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| Exact Mass |
296.986
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| Elemental Analysis |
C, 48.41; H, 2.71; Cl, 11.91; N, 4.70; O, 21.50; S, 10.77
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| CAS # |
86030-08-2
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| Related CAS # |
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| PubChem CID |
291461
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| Appearance |
White to off-white solid powder
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| Density |
1.476g/cm3
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| Boiling Point |
471.2ºC at 760mmHg
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| Melting Point |
115ºC
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| Flash Point |
238.8ºC
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| Index of Refraction |
1.623
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| LogP |
4.685
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
19
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| Complexity |
422
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| Defined Atom Stereocenter Count |
0
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| SMILES |
[O-][N+](C1C(S(C2C=CC=CC=2)(=O)=O)=CC=C(Cl)C=1)=O
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| InChi Key |
VZDUQPHKUBZMLW-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C12H8ClNO4S/c13-9-6-7-12(11(8-9)14(15)16)19(17,18)10-4-2-1-3-5-10/h1-8H
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| Chemical Name |
1-(benzenesulfonyl)-4-chloro-2-nitrobenzene
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| Synonyms |
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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 |
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| 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) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (8.40 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 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (8.40 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 25.0 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (8.40 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.3590 mL | 16.7949 mL | 33.5897 mL | |
| 5 mM | 0.6718 mL | 3.3590 mL | 6.7179 mL | |
| 10 mM | 0.3359 mL | 1.6795 mL | 3.3590 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.
Inhibition of Kif18A activities by BTB-1.
Modeling of the putative BTB-1 binding site.ACS Chem Biol.2015 Feb 20;10(2):554-60. |
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Conformation of the MT-bound Kif18A motor domain in the presence of BTB-1.ACS Chem Biol.2015 Feb 20;10(2):554-60. |
Effect of point mutations of Kif18A around the putative BTB-1–binding pocket on BTB-1 inhibition of MT gliding activity.ACS Chem Biol.2015 Feb 20;10(2):554-60. |
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