EPOXOMICIN (BU 4061T)   中文名称: 艾泼米辛

20S proteasome

体外活性：Epoxomicin 与蛋白酶体的 LMP7、X、MECL1 和 Z 催化亚基共价结合。 Epoxomicin (100 nM) 导致 HUVEC 中 p53 蛋白（蛋白酶体的已知靶标）的水平增加 30 倍。 Epoxomicin (10 μM) 会导致 HeLa 细胞中泛素化蛋白的积累。 Epoxomicin (10 μM) 在 HeLa 细胞中抑制 IκBα 降解 10 倍。 Epoxomicin (10 μM) 可显着降低 HeLa 细胞中 TNF-α 刺激的 NF-κB DNA 结合活性，且呈剂量依赖性。 Epoxomicin 通过生物素化嵌合体抑制 EL4 淋巴瘤细胞的增殖，IC50 为 4 nM。 Epoxomicin (1 μM) 导致 LCMV GP33 呈递减少并增强 GP276 呈递。

相对于仅用媒介物治疗的对照组小鼠，Epoxomicin（每天 0.58 mg/kg）使 CS 反应降低 44%。当对小鼠进行攻击后 24 小时进行耳部水肿测量时，Epoxomicin (2.9 mg/kg) 可有效抑制 95% 的刺激相关炎症反应。

---

Epoxomicin在体内能有效减少CS。[1]
CS是对某些类别的化合物和半抗原的炎症反应（30）。基于细胞培养中NF-κB活化的抑制，我们假设Epoxomicin/环氧霉素在体内具有抗炎活性。为了验证这一假设，在氯化苦小鼠CS模型中评估了环氧霉素。用苦酰氯免疫的小鼠在6天后通过在耳朵上施用苦酰氯进行攻击。在苦酰氯耳部激发后0和24小时进行耳部厚度测量。如图5所示，与仅用赋形剂治疗的对照组小鼠相比，每天用无毒剂量为0.58mg/kg的环氧霉素治疗，CS反应降低了44%。由于半抗原可以引发非特异性、刺激性相关的炎症反应（31），我们通过使用非免疫小鼠探索了环氧霉素对皮肤刺激介导的炎症的影响。在第二个实验中，用环氧霉素对小鼠进行预处理，其剂量比以前使用的剂量高五倍，以测试单次注射该药物可以减少对苦氯乙烯耳部刺激的炎症反应的想法。当在攻击后24小时进行耳水肿测量时，以2.9 mg/kg剂量给药的环氧霉素有效地抑制了95%的刺激性相关炎症反应（图5）。

---

疟疾仍然是一个主要的全球健康问题，但目前可用的有效药物库有限。临床上常用的抗疟疾化合物都不能杀死成熟的配子体，配子体是导致疟疾传播的寄生虫形式。导致最致命的人类疟疾的寄生虫恶性疟原虫有一个48小时的无性周期，而完全的性分化需要10到12天。一旦成熟，V期配子体在外周血中循环，可以传播一周以上。因此，如果化疗不能消除配子体，个体在清除无性寄生虫后几周内仍具有传染性。这里报道的工作表明，纳摩尔浓度的蛋白酶体抑制剂环氧霉素有效地杀死了红细胞内寄生虫的所有阶段，但不影响人类或小鼠细胞系的存活率。用100nM环氧霉素治疗24小时后，总寄生虫血症减少了78%，无性寄生虫减少了86%，配子体减少了77%。治疗72小时后，100或10nM治疗组中没有存活的寄生虫。环氧霉素还阻断了蚊子中肠中卵囊的产生。相比之下，半胱氨酸蛋白酶抑制剂环氧琥珀酰-L-亮氨酸氨基-3-甲基丁烷乙酯和N-乙酰-L-亮氨酸-L-亮氨酸-L蛋氨酸阻断了早期配子体中的血红蛋白消化，但对后期没有影响。此外，一旦半胱氨酸蛋白酶抑制剂被移除，性分化就恢复了。这些发现为蛋白酶体抑制剂作为抗疟疾药物的进一步发展提供了强有力的支持，这些药物可有效对抗恶性疟原虫的无性期、性期和蚊子中肠期[5]。

在用于蛋白酶体抑制测定的测定溶液中，添加肽-AMC 底物（5 μM Suc-LLVY-AMC、5 μM Z-LLE-AMC 和 5 μM Boc-LRR-AMC）和依泊昔明的 DMSO 溶液，最终 DMSO 浓度为1%。使用的测定缓冲液如下：pH 8.0/0.5 mM EDTA 和 20 mM Tris-HCl（加上用于 Z-LLE-AMC 和 Suc-LLVY-AMC 测定的 0.035% SDS）。在室温 (23 °C) 下将牛红细胞蛋白酶体添加到含有底物和多索米星的测定缓冲液中，最终体积为 100 μL，然后立即使用 Cytoflor 在 460 nm (λex, 360 nM) 处测量荧光发射荧光板读数器 50 分钟。

---

环氧霉素结合蛋白的纯化。[1]
收获10升EL4细胞（106个细胞/ml），并将其重新悬浮在含有10%FBS的50毫升RPMI 1640培养基中。将环氧霉素-生物素添加至8μM的终浓度，并在37°C下孵育细胞4小时。在裂解缓冲液（25 mM Hepes，pH 7.4/5 mM EGTA/50 mM NaF）和蛋白酶抑制剂（10μg/ml亮肽、胃蛋白酶抑制剂和大豆胰蛋白酶抑制剂以及1 mM PMSF）中使用Powergen均质器收获细胞并均质化。将高速（100000×g）上清液装载到1ml链霉抗生物素蛋白琼脂糖柱上，以去除内源性生物素化蛋白。然后，将流通部分与用裂解缓冲液预平衡的50ml DE52珠一起孵育10分钟，用50ml含有0.1M NaCl的裂解缓冲液洗涤两次，并用50ml含有0.3M NaCl的水解缓冲液洗脱。将SDS以0.5%的终浓度加入洗脱液中，煮沸10分钟，并用裂解缓冲液稀释2.5倍。将稀释的溶液装载到0.4ml NeutrAvidin琼脂糖柱上。收集流通部分，并将其重新装载到同一柱上三次。经过大量洗涤后，通过在0.4 ml 1×SDS样品缓冲液中煮沸NeutrAvidin琼脂糖来洗脱环氧素-生物素结合蛋白。纯化的蛋白质复合物通过SDS-PAGE分离，切除，并由W.M.Keck基金会生物技术资源实验室（耶鲁大学）使用LCQ（MS/MS）和内部胰蛋白酶肽的自动Edman降解进行鉴定。

---

酶动力学分析。[1]
对于蛋白酶体抑制试验，将肽AMC底物（5μM Suc LLVY AMC、5μM Z-LLE-AMC和5μM Boc LRR AMC）和DMSO中的抑制剂添加到试验溶液中，使DMSO的最终浓度为1%。使用以下测定缓冲液：20 mM Tris⋅HCl，pH 8.0/0.5 mM EDTA（加0.035%SDS用于Suc LLVY AMC和Z-LLE-AMC测定）。在室温（23°）下，在Dynex Microfluor II 96孔板中将牛红细胞蛋白酶体添加到含有底物和抑制剂的测定缓冲液中，最终体积为100μl，并使用Cytofluor荧光板读数器在460 nm（λex，360 nm）下立即测量荧光发射50分钟。kobs/[I]值是通过将数据非线性最小二乘拟合到以下方程中，使用激肽图获得的：荧光=vst+[（vo−vs）/kobs][1−exp（−kobst）]，其中vo和vs分别是初始和最终速度，kobs是反应速率常数。牛红细胞20S蛋白酶体（2.5mg/ml）的稀释如下：Suc-LLVY-AMC活性的最终稀释度为1:1200，Z-LLE-AMC为1:3000，Boc-LRR AMC为1:800。抑制反应如前所述进行。对于钙蛋白酶抑制试验，酶以1单位/ml的浓度使用，Suc-LLVY-AMC在含有20 mM Tris、pH 8.0/1 mM CaCl2/2 mM DTT的试验缓冲液中以10μM的终浓度使用。组织蛋白酶B在100 mM乙酸钠/5 mM EDTA中以0.005单位/ml的浓度使用，pH 5.5，组织蛋白酶底物III用作40μM的底物。按照蛋白酶体的描述进行动力学测定。

---

EMSAs。[1]
如所述进行了电磁兼容性分析。简而言之，转录因子NF-κB的共识DNA结合寡核苷酸序列用[γ-32P]ATP标记，并与等量的核裂解物一起孵育。在非变性和非还原条件下，蛋白质-DNA复合物在4%聚丙烯酰胺凝胶上分离。将凝胶干燥并暴露于磷光成像屏幕上，以定量延迟带中的放射性。结果代表了至少进行两次的实验。

细胞毒性试验。[5]
来自小鼠成纤维细胞系NIH-3T3或人肺泡基底上皮细胞系A549（ATCC）的细胞（96孔板中每孔1×104至5×104个细胞）在添加了10%胎牛血清的Dulbecco改良Eagle培养基（DMEM）中与一系列稀释的环氧素（32至2000 nM）或载体（DMSO）单独孵育。DMSO的浓度保持在1%以下，这不影响细胞存活率。添加药物48小时后，根据制造商的说明，使用cell Titer 96水性非放射性细胞增殖测定法测定细胞存活率(http://www.promega.com/enotes/applications/0004/ap0017.htm). 该测定使用可溶性四氮唑盐3-（4,5-二甲基噻唑-2-基）-5-（3-羧甲氧基苯基）-2-（4-磺基苯基）-2H-四氮唑内盐（MTS），其被活细胞还原为弗罗曼，可以通过492 nM的吸光度进行测量

---

对于IκB和泛素蛋白质印迹分析，将60%融合的HeLa单层用10μM的环氧霉素或Z-LLL-H处理2小时。将肿瘤坏死因子α（TNF-α）（10ng/ml）加入一组药物处理的平板中，单独加入单独的细胞培养皿中。15分钟后收获细胞。通过用DMSO、100nM环氧霉素或5μM Z-LLL-H处理的HUVEC的小鼠抗p53抗血清免疫沉淀6小时，然后用兔抗p53抗血清进行免疫印迹分析，分析p53的稳定性。对于电泳迁移率变化测定（EMSA），将环氧霉素重复添加到HeLa细胞中，将TNF-α（10ng/ml）添加到一组药物处理的平板中，也单独添加到单独的细胞培养皿中。1小时后收获细胞，并如上所述制备核裂解物。

---

在用载体、氯沙坦 (10μM)、环氧霉素 (10μM) 或载体对照预处理后，用盐水或 Ang II (100μM) 对 LEC 进行一整天的处理。

BALB/c mice
2.9 mg/kg
intraperitoneal injection

---

Assay for Contact Sensitivity (CS). [1]
CS and irritant-response assays to picrylchloride (a generous gift of P. Asekanse, Yale University) challenge were performed essentially as described, with slight modifications. In brief, for the CS assay, mice were injected i.p. daily for 6 days with vehicle or epoxomicin (0.58 mg/kg body weight) solubilized in 10% DMSO/PBS. Six days after abdomen immunization with picrylchloride, ear thickness measurements (0 hr) of both ears were made in triplicate with an engineer’s micrometer (Peacock dial thickness gauge; Ozaki Manufacturing, Tokyo). Mice subsequently were challenged on both ear lobes by application of 15 μl of a 0.8% solution of picrylchloride (solubilized in high-grade extra virgin olive oil). Ear swelling measurements again were made 24 hr post-ear challenge. In a second assay, elicitation of inflammatory response to the nonspecific vascular activation and permeability effects of picrylchloride (irritant response) were determined by using two groups of four nonimmunized mice. The 0-hr ear thickness measurements were made, a single high-dose injection of epoxomicin (2.9 mg/kg) was delivered i.p. to one group, and the control group was treated with vehicle. Ear thickness was measured 24 hr post-ear challenge.

---

Plasmodium falciparum gametogenesis and mosquito feed assay. [1]
Cultures of P. falciparum parasites (strain 3D7) containing mature gametocytes were incubated for 1 h in the presence of ALLN, epoxomicin, or DMSO alone. To assay gametogenesis and exflagellation, an aliquot (0.5 ml) of the test cultures was pelleted and resuspended in emergence medium (10 μM xanthurenic acid, 1.67 mg ml−1 glucose, 8 mg ml−1 NaCl, 8 mM Tris-Cl [pH 8.2]). After a 10-min incubation at room temperature, parasite morphology and the number of exflagellating males was evaluated at a ×400 magnification. To evaluate oocyst production, an aliquot (1.2 ml) of the test cultures was pelleted onto 125-μl packed erythrocytes. The supernatant was replaced with 120 μl of normal human serum containing active complement, mixed, and introduced into a water-jacketed membrane feeder maintained at 37°C. Anopheles stephensi (SxK Nij.) mosquitoes were allowed to gorge for 10 min and then were grown for 7 more days at 25 ± 1°C and 80% ± 10% humidity. The midguts then were dissected and stained in 1.0% mercurochrome to visualize the P. falciparum oocysts.

[1]. Proc Natl Acad Sci U S A . 1999 Aug 31;96(18):10403-8.

[2]. Bioorg Med Chem Lett . 1999 Dec 6;9(23):3335-40.

[3]. J Immunol . 2000 Jun 15;164(12):6147-57.

[4]. Vet Parasitol . 2010 Jan 20;167(1):19-27.

[5]. Antimicrob Agents Chemother . 2009 Oct;53(10):4080-5.

Epoxomicin is a tripeptide consisting of an Ile-Ile-Thr-NH2 sequence N-substituted on the threonamide amidic nitrogen with a (2S)-4-methyl-1-[(2R)-2-methyloxiran-2-yl]-1-oxopentan-2-yl group and with acetyl and methyl groups on the nitrogen of the isoleucine residue distal to the threonamide; a naturally occurring selective proteasome inhibitor with anti-inflammatory activity. It has a role as a proteasome inhibitor. It is a member of morpholines and a tripeptide.

---

The proteasome regulates cellular processes as diverse as cell cycle progression and NF-kappaB activation. In this study, we show that the potent antitumor natural product epoxomicin specifically targets the proteasome. Utilizing biotinylated-epoxomicin as a molecular probe, we demonstrate that epoxomicin covalently binds to the LMP7, X, MECL1, and Z catalytic subunits of the proteasome. Enzymatic analyses with purified bovine erythrocyte proteasome reveal that epoxomicin potently inhibits primarily the chymotrypsin-like activity. The trypsin-like and peptidyl-glutamyl peptide hydrolyzing catalytic activities also are inhibited at 100- and 1,000-fold slower rates, respectively. In contrast to peptide aldehyde proteasome inhibitors, epoxomicin does not inhibit nonproteasomal proteases such trypsin, chymotrypsin, papain, calpain, and cathepsin B at concentrations of up to 50 microM. In addition, epoxomicin is a more potent inhibitor of the chymotrypsin-like activity than lactacystin and the peptide vinyl sulfone NLVS. Epoxomicin also effectively inhibits NF-kappaB activation in vitro and potently blocks in vivo inflammation in the murine ear edema assay. These results thus define epoxomicin as a novel proteasome inhibitor that likely will prove useful in exploring the role of the proteasome in various in vivo and in vitro systems. [1]

---

While two structurally related epoxyketone-containing antitumor natural products, epoxomicin and eponemycin, share the proteasome as a common intracellular target, they differ in their antiproliferative activity, proteasome subunit binding specificity, and rates of proteasome inhibition. As a first step towards understanding such differences and developing novel proteasome subunit-specific inhibitors, we report here the synthesis and characterization of epoxomicin/dihydroeponemycin chimerae. [2]

---

The complete inhibition of proteasome activities interferes with the production of most MHC class I peptide ligands as well as with cellular proliferation and survival. In this study we have investigated how partial and selective inhibition of the chymotrypsin-like activity of the proteasome by the proteasome inhibitors lactacystin or epoxomicin would affect Ag presentation. At 0.5-1 microM lactacystin, the presentation of the lymphocytic choriomeningitis virus-derived epitopes NP118 and GP33 and the mouse CMV epitope pp89-168 were reduced and were further diminished in a dose-dependent manner with increasing concentrations. Presentation of the lymphocytic choriomeningitis virus-derived epitope GP276, in contrast, was markedly enhanced at low, but abrogated at higher, concentrations of either lactacystin or epoxomicin. The inhibitor-mediated effects were thus epitope specific and did not correlate with the degradation rates of the involved viral proteins. Although neither apoptosis induction nor interference with cellular proliferation was observed at 0.5-1 microM lactacystin in vivo, this concentration was sufficient to alter the fragmentation of polypeptides by the 20S proteasome in vitro. Our results indicate that partial and selective inhibition of proteasome activity in vivo is a valid approach to modulate Ag presentation, with potential applications for the treatment of autoimmune diseases and the prevention of transplant rejection.[3]

---

Epoxomicin potently and irreversibly inhibits the catalytic activity of proteasomal subunits. Treatment of proliferating cells with epoxomicin results in cell death through accumulation of ubiquinated proteins. Thus, epoxomicin has been proposed as a potential anti-cancer drug. In the present study, the inhibitory effects of epoxomicin on the in vitro growth of bovine and equine Babesia parasites were evaluated. The inhibitory effect of epoxomicin on the in vivo growth of Babesia microti was also assessed. The in vitro growth of five Babesia species that were tested was significantly inhibited (P<0.05) by nanomolar concentrations of epoxomicin (IC(50) values=21.4+/-0.2, 4+/-0.1, 39.5+/-0.1, 9.7+/-0.3, and 21.1+/-0.1nM for Babesia bovis, Babesia bigemina, Babesia ovata, Babesia caballi, and Babesia equi, respectively). Epoxomicin IC(50) values for Babesia parasites were low when compared with diminazene aceturate and tetracycline hydrochloride. Combinations of epoxomicin with diminazene aceturate synergistically potentiated its inhibitory effects in vitro on B. bovis, B. bigemina, and B. caballi. In B. microti-infected mice, epoxomicin caused significant (P<0.05) inhibition of the growth of B. microti at the non-toxic doses of 0.05 and 0.5mg/kg BW relative to control groups. Therefore, epoxomicin might be used for treatment of babesiosis.[4]

C28H50N4O7

554.72

554.367

C, 60.63; H, 9.09; N, 10.10; O, 20.19

134381-21-8

11226684

White to off-white solid powder

1.1±0.1 g/cm3

795.7±60.0 °C at 760 mmHg

107-109ºC

435.0±32.9 °C

0.0±6.3 mmHg at 25°C

1.504

3.32

157.44

4

7

16

39

895

8

O=C([C@@H](NC([C@H]([C@@H](C)O)NC([C@@H](NC([C@H]([C@@H](C)CC)N(C)C(C)=O)=O)[C@@H](C)CC)=O)=O)CC(C)C)[C@@]1(C)CO1

DOGIDQKFVLKMLQ-JTHVHQAWSA-N

InChI=1S/C28H50N4O7/c1-11-16(5)21(30-27(38)23(17(6)12-2)32(10)19(8)34)25(36)31-22(18(7)33)26(37)29-20(13-15(3)4)24(35)28(9)14-39-28/h15-18,20-23,33H,11-14H2,1-10H3,(H,29,37)(H,30,38)(H,31,36)/t16-,17-,18+,20-,21-,22-,23-,28+/m0/s1

(2S,3S)-2-[[(2S,3S)-2-[acetyl(methyl)amino]-3-methylpentanoyl]amino]-N-[(2S,3R)-3-hydroxy-1-[[(2S)-4-methyl-1-[(2R)-2-methyloxiran-2-yl]-1-oxopentan-2-yl]amino]-1-oxobutan-2-yl]-3-methylpentanamide

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.51 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

---

配方 2 中的溶解度: ≥ 2.5 mg/mL (4.51 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

---

View More

配方 3 中的溶解度: ≥ 2.5 mg/mL (4.51 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

---

请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。