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Purity: ≥98%
Spastazoline is a first-in-class, potent and selective spastin (a microtubule-severing AAA protein) inhibitor, with an IC50 of 99 nM for Human spastin. Spastazoline shows no effect on ATPase activity of a recombinant human VPS4. Spastin is a microtubule-severing AAA (ATPases associated with diverse cellular activities) protein needed for cell division and intracellular vesicle transport.
| Targets |
Human spastin (IC50 = 99 nM)[1]
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| ln Vitro |
HeLa cell division is influenced by spastazoline (10 μM; 4.5 h) by increasing the amount of cells with intercellular bridges [1].
These data, along with computational docking, guided improvements in compound potency and selectivity and led to spastazoline, a pyrazolyl-pyrrolopyrimidine-based cell-permeable probe for spastin. These studies also identified spastazoline resistance-conferring point mutations in spastin. Spastazoline, along with matched inhibitor-sensitive and inhibitor-resistant cell lines we generated, were used in parallel experiments to dissect spastin-specific phenotypes in dividing cells. Together, our findings suggest how chemical probes for AAA proteins, along with inhibitor resistance-conferring mutations, can be designed and used to dissect dynamic cellular processes[1]. |
| ln Vivo |
Mice with SCI in all groups exhibited paralysis in the right hindlimb after hemisection which confirmed the successful establishment of the lateral hemisection SCI model. For mice in the SCI control group, the BMS scores gradually improved to ~4 by 16 DPI. In contrast, the scores of mice in the FC-A group indicated an improvement in locomotor function which was impaired in the spastazoline group. Moreover, the improvement in locomotor function following the administration of FC-A diminished when spastazoline was administered (Figure 7E). We further analyzed the footprint of mice with SCI and calculated the length or width of strides (Figure 7—figure supplement 3). We found that stride length improved in mice of the FC-A group but it was reduced in the spastazoline group although not significantly. The improvement induced by FC-A diminished when co-treated with spastazoline (Figure 7G). Notably, there was no significant difference in stride width between the groups (Figure 7H). Similar effects were observed in the foot fault scan experiment. The faults of the right hindlimb significantly decreased in the FC-A group and increased in the spastazoline group (Figure 7F). The improvement of hindlimb fault in mice administered with FC-A diminished following the application of spastazoline. We also explored whether the stability of MTs was altered by the administration of FC-A and spastazoline in the spinal cord injury model. We found that the ratio of acetylated tubulin to total tubulin was significantly increased in the SCI group, decreased in the FC-A group, and upregulated in the spastazoline group, which further confirmed the successful delivery of drugs to the lesion site (Figure 7—figure supplement 4). These findings suggested that FC-A was effectively delivered to the spinal cord tissue and improved the locomotor function of mice with SCI, and spastin activation is the prerequisite the repair after spinal cord injury. Reference: Elife. 2024 Jan 17;12:RP90184.
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| Enzyme Assay |
Spastin is a microtubule-severing AAA (ATPases associated with diverse cellular activities) protein needed for cell division and intracellular vesicle transport. Currently, we lack chemical inhibitors to probe spastin function in such dynamic cellular processes. To design a chemical inhibitor of spastin, we tested selected heterocyclic scaffolds against wild-type protein and constructs with engineered mutations in the nucleotide-binding site that do not substantially disrupt ATPase activity. These data, along with computational docking, guided improvements in compound potency and selectivity and led to spastazoline, a pyrazolyl-pyrrolopyrimidine-based cell-permeable probe for spastin. These studies also identified spastazoline-resistance-conferring point mutations in spastin. Spastazoline, along with the matched inhibitor-sensitive and inhibitor-resistant cell lines we generated, were used in parallel experiments to dissect spastin-specific phenotypes in dividing cells. Together, our findings suggest how chemical probes for AAA proteins, along with inhibitor resistance-conferring mutations, can be designed and used to dissect dynamic cellular processes[1].
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| Cell Assay |
Rat cortical neurons were seeded in 96-well plates (30,000 cells per well), and then a plastic P10 pipet tip was used to scratch across the center of the well. Half the media was aspirated out and replaced with fresh ones every three days until the 7th DIV. The wells were immediately filled with chemicals after scratching. The cultures were fixed and stained with anti-βIII tubulin (1:1000, ab18207) the following day. Images of axons stained with βIII tubulin were collected, imported in ImageJ, and the proportion of neurites stained by βIII tubulin in the central 70% of the scratch was calculated.Reference: Elife. 2024 Jan 17;12:RP90184.
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| Animal Protocol |
After the establishment of the spinal cord injury model, the mice within the injury group underwent random allocation into cages following a double-blind methodology. Another experimenter, blinded to the groupings, then administered the indicated drug treatment randomly to the mice within the injury group. An equal volume of normal saline was intraperitoneally injected into SCI control mice. Mice were intraperitoneally injected with FC-A at a dose of 15 mg/kg starting immediately after SCI and every other day until the end of the experiment. Mice were intraperitoneally injected with spastazoline at a dose of 20 mg/kg, immediately after SCI then every other day until the end of experiments.
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| References | |
| Additional Infomation |
Axon regeneration is abortive in the central nervous system following injury. Orchestrating microtubule dynamics has emerged as a promising approach to improve axonal regeneration. The microtubule severing enzyme spastin is essential for axonal development and regeneration through remodeling of microtubule arrangement. To date, however, little is known regarding the mechanisms underlying spastin action in neural regeneration after spinal cord injury. Here, we use glutathione transferase pulldown and immunoprecipitation assays to demonstrate that 14-3-3 interacts with spastin, both in vivo and in vitro, via spastin Ser233 phosphorylation. Moreover, we show that 14-3-3 protects spastin from degradation by inhibiting the ubiquitination pathway and upregulates the spastin-dependent severing ability. Furthermore, the 14-3-3 agonist Fusicoccin (FC-A) promotes neurite outgrowth and regeneration in vitro which needs spastin activation. Western blot and immunofluorescence results revealed that 14-3-3 protein is upregulated in the neuronal compartment after spinal cord injury in vivo. In addition, administration of FC-A not only promotes locomotor recovery, but also nerve regeneration following spinal cord injury in both contusion and lateral hemisection models; however, the application of spastin inhibitor spastazoline successfully reverses these phenomena. Taken together, these results indicate that 14-3-3 is a molecular switch that regulates spastin protein levels, and the small molecule 14-3-3 agonist FC-A effectively mediates the recovery of spinal cord injury in mice which requires spastin participation. Reference: Elife. 2024 Jan 17;12:RP90184.
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| Molecular Formula |
C20H30N8
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|---|---|
| Molecular Weight |
382.505802631378
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| Exact Mass |
382.259
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| Elemental Analysis |
C, 62.80; H, 7.91; N, 29.29
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| CAS # |
2351882-11-4
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| PubChem CID |
135397891
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| Appearance |
Typically exists as Off-white to gray solid at room temperature
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| LogP |
4.1
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
28
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| Complexity |
521
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| Defined Atom Stereocenter Count |
1
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| SMILES |
CC(C)[C@@H]1CN(CCN1)C2=NC3=C(C=CN3)C(=N2)NC4=NNC(=C4)C(C)(C)C
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| InChi Key |
HNOLTAKAPDKZRY-AWEZNQCLSA-N
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| InChi Code |
InChI=1S/C20H30N8/c1-12(2)14-11-28(9-8-21-14)19-24-17-13(6-7-22-17)18(25-19)23-16-10-15(26-27-16)20(3,4)5/h6-7,10,12,14,21H,8-9,11H2,1-5H3,(H3,22,23,24,25,26,27)/t14-/m0/s1
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| Chemical Name |
(R)-N-(5-(tert-Butyl)-1H-pyrazol-3-yl)-2-(3-isopropylpiperazin-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine
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| Synonyms |
Spastazoline; 2351882-11-4; (R)-N-(5-(tert-butyl)-1H-pyrazol-3-yl)-2-(3-isopropylpiperazin-1-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine; CHEMBL5190401; Spastazoline?; SCHEMBL22035095; EX-A3095; BDBM50587865;
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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 (~261.43 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 5.25 mg/mL (13.73 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 52.5 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: ≥ 5.25 mg/mL (13.73 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 52.5 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: ≥ 5.25 mg/mL (13.73 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 | 2.6143 mL | 13.0716 mL | 26.1431 mL | |
| 5 mM | 0.5229 mL | 2.6143 mL | 5.2286 mL | |
| 10 mM | 0.2614 mL | 1.3072 mL | 2.6143 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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