proTAME
(Synonyms: Pro-N-4-tosyl-L-arginine methyl ester) 目录号 : GC44730A prodrug form of TAME
Cas No.:1362911-19-0
Sample solution is provided at 25 µL, 10mM.
Quality Control & SDS
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- Purity: >90.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
proTAME is a cell-permeable prodrug form of N-4-tosyl-L-arginine methyl ester , an inhibitor of the anaphase-promoting complex/cyclosome (APC/C), that is converted to TAME by intracellular esterases. proTAME (12 μM) blocks association of APC/C with the activator CDH1 and inhibits degradation of APC/C substrates in HeLa cells. It induces mitotic arrest in metaphase followed by cell death in synchronized HeLa H2B-GFP cells when used at a concentration of 12 μM. It also increases mitotic duration in asynchronous HeLa H2B-GFP cells at a concentration of 4 μM, an effect that is enhanced by knockdown of the APC/C co-activator CDC20. proTAME decreases the viability of several laboratory and primary patient-derived human multiple myeloma (MM) cell lines (IC50s = 2.8-20.3 μM) and increases apoptosis in RPMI-8226, LP-1, NCI-H929, and U266 MM cells when used at a concentration of 12 μM.
| Cas No. | 1362911-19-0 | SDF | |
| 别名 | Pro-N-4-tosyl-L-arginine methyl ester | ||
| Canonical SMILES | CC1=CC=C(S(N[C@H](C(OC)=O)CCC/N=C(NC(OCOC(CC2=CC=CC=C2)=O)=O)/NC(OCOC(CC3=CC=CC=C3)=O)=O)(=O)=O)C=C1 | ||
| 分子式 | C34H38N4O12S | 分子量 | 726.8 |
| 溶解度 | Soluble in DMSO | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 1.3759 mL | 6.8795 mL | 13.7589 mL |
| 5 mM | 0.2752 mL | 1.3759 mL | 2.7518 mL |
| 10 mM | 0.1376 mL | 0.6879 mL | 1.3759 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
proTAME Arrest in Mammalian Oocytes and Embryos Does Not Require Spindle Assembly Checkpoint Activity
Int J Mol Sci 2019 Sep 13;20(18):4537.PMID:31540287DOI:10.3390/ijms20184537.
In both mitosis and meiosis, metaphase to anaphase transition requires the activity of a ubiquitin ligase known as anaphase promoting complex/cyclosome (APC/C). The activation of APC/C in metaphase is under the control of the checkpoint mechanism, called the spindle assembly checkpoint (SAC), which monitors the correct attachment of all kinetochores to the spindle. It has been shown previously in somatic cells that exposure to a small molecule inhibitor, prodrug tosyl-l-arginine methyl ester (proTAME), resulted in cell cycle arrest in metaphase, with low APC/C activity. Interestingly, some reports have also suggested that the activity of SAC is required for this arrest. We focused on the characterization of proTAME inhibition of cell cycle progression in mammalian oocytes and embryos. Our results show that mammalian oocytes and early cleavage embryos show dose-dependent metaphase arrest after exposure to proTAME. However, in comparison to the somatic cells, we show here that the proTAME-induced arrest in these cells does not require SAC activity. Our results revealed important differences between mammalian oocytes and early embryos and somatic cells in their requirements of SAC for APC/C inhibition. In comparison to the somatic cells, oocytes and embryos show much higher frequency of aneuploidy. Our results are therefore important for understanding chromosome segregation control mechanisms, which might contribute to the premature termination of development or severe developmental and mental disorders of newborns.
Nocodazole-Induced Expression and Phosphorylation of Anillin and Other Mitotic Proteins Are Decreased in DNA-Dependent Protein Kinase Catalytic Subunit-Deficient Cells and Rescued by Inhibition of the Anaphase-Promoting Complex/Cyclosome with proTAME but Not Apcin
Mol Cell Biol 2020 Jun 15;40(13):e00191-19.PMID:32284347DOI:10.1128/MCB.00191-19.
The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has well-established roles in DNA double-strand break repair, and recently, nonrepair functions have also been reported. To better understand its cellular functions, we deleted DNA-PKcs from HeLa and A549 cells using CRISPR/Cas9. The resulting cells were radiation sensitive, had reduced expression of ataxia-telangiectasia mutated (ATM), and exhibited multiple mitotic defects. Mechanistically, nocodazole-induced upregulation of cyclin B1, anillin, and securin was decreased in DNA-PKcs-deficient cells, as were phosphorylation of Aurora A on threonine 288, phosphorylation of Polo-like kinase 1 (PLK1) on threonine 210, and phosphorylation of targeting protein for Xenopus Klp2 (TPX2) on serine 121. Moreover, reduced nocodazole-induced expression of anillin, securin, and cyclin B1 and phosphorylation of PLK1, Aurora A, and TPX2 were rescued by inhibition of the anaphase-promoting complex/cyclosome (APC/C) by proTAME, which prevents binding of the APC/C-activating proteins Cdc20 and Cdh1 to the APC/C. Altogether, our studies suggest that loss of DNA-PKcs prevents inactivation of the APC/C in nocodazole-treated cells.
Identification of the APC/C co-factor FZR1 as a novel therapeutic target for multiple myeloma
Oncotarget 2016 Oct 25;7(43):70481-70493.PMID:27655696DOI:10.18632/oncotarget.12026.
Multiple Myeloma (MM) is a haematological neoplasm characterised by the clonal proliferation of malignant plasma cells in the bone marrow. The success of proteasome inhibitors in the treatment of MM has highlighted the importance of the ubiquitin proteasome system (UPS) in the pathogenesis of this disease. In this study, we analysed gene expression of UPS components to identify novel therapeutic targets within this pathway in MM. Here we demonstrate how this approach identified previously validated and novel therapeutic targets. In addition we show that FZR1 (Fzr), a cofactor of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C), represents a novel therapeutic target in myeloma. The APC/C associates independently with two cofactors, Fzr and Cdc20, to control cell cycle progression. We found high levels of FZR1 in MM primary cells and cell lines and demonstrate that expression is further increased on adhesion to bone marrow stromal cells (BMSCs). Specific knockdown of either FZR1 or CDC20 reduced viability and induced growth arrest of MM cell lines, and resulted in accumulation of APC/CFzr substrate Topoisomerase IIα (TOPIIα) or APC/CCdc20 substrate Cyclin B. Similar effects were observed following treatment with proTAME, an inhibitor of both APC/CFzr and APC/CCdc20. Combinations of proTAME with topoisomerase inhibitors, etoposide and doxorubicin, significantly increased cell death in MM cell lines and primary cells, particularly if TOPIIα levels were first increased through pre-treatment with proTAME. Similarly, combinations of proTAME with the microtubule inhibitor vincristine resulted in enhanced cell death. This study demonstrates the potential of targeting the APC/C and its cofactors as a therapeutic approach in MM.
Cohesion fatigue explains why pharmacological inhibition of the APC/C induces a spindle checkpoint-dependent mitotic arrest
PLoS One 2012;7(11):e49041.PMID:23145059DOI:10.1371/journal.pone.0049041.
The Spindle Assembly Checkpoint (SAC) delays the onset of anaphase in response to unattached kinetochores by inhibiting the activity of the Anaphase-Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase. Once all the chromosomes have bioriented, SAC signalling is somehow silenced, which allows progression through mitosis. Recent studies suggest that the APC/C itself participates in SAC silencing by targeting an unknown factor for proteolytic degradation. Key evidence in favour of this model comes from the use of proTAME, a small molecule inhibitor of the APC/C. In cells, proTAME causes a mitotic arrest that is SAC-dependent. Even though this observation comes at odds with the current view that the APC/C acts downstream of the SAC, it was nonetheless argued that these results revealed a role for APC/C activity in SAC silencing. However, we show here that the mitotic arrest induced by proTAME is due to the induction of cohesion fatigue, a phenotype that is caused by the loss of sister chromatid cohesion following a prolonged metaphase. Under these conditions, the SAC is re-activated and APC/C inhibition is maintained independently of proTAME. Therefore, these results provide a simpler explanation for why the proTAME-induced mitotic arrest is also dependent on the SAC. While these observations question the notion that the APC/C is required for SAC silencing, we nevertheless show that APC/C activity does partially contribute to its own release from inhibitory complexes, and importantly, this does not depend on proteasome-mediated degradation.
CDC20 in and out of mitosis: a prognostic factor and therapeutic target in hematological malignancies
J Exp Clin Cancer Res 2022 Apr 30;41(1):159.PMID:35490245DOI:10.1186/s13046-022-02363-9.
Cell division cycle 20 homologue (CDC20) is a well-known regulator of cell cycle, as it controls the correct segregation of chromosomes during mitosis. Many studies have focused on the biological role of CDC20 in cancer development, as alterations of its functionality have been linked to genomic instability and evidence demonstrated that high CDC20 expression levels are associated with poor overall survival in solid cancers. More recently, novel CDC20 functions have been demonstrated or suggested, including the regulation of apoptosis and stemness properties and a correlation with immune cell infiltration. Here, we here summarize and discuss the role of CDC20 inside and outside mitosis, starting from its network of interacting proteins. In the last years, CDC20 has also attracted more interest in the blood cancer field, being overexpressed and showing an association with prognosis both in myeloid and lymphoid malignancies. Preclinical findings showed that selective CDC20 and APC/CCDC20/APC/CCDH1 inhibitors, namely Apcin and proTAME, are effective against lymphoma and multiple myeloma cells, resulting in mitotic arrest and apoptosis and synergizing with clinically-relevant drugs. The evidence and hypothesis presented in this review provide the input for further biological and chemical studies aiming to dissect novel potential CDC20 roles and targeting strategies in hematological malignancies.