A new structural class of proteasome inhibitors identified by microbial screening using yeast-based assay
A new structural class of proteasome inhibitors identified by microbial screening using yeast-based assay
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DOI：
10.1016/j.bcp.2003.08.035
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发表时间：
2004-01-15
影响因子：
5.8
通讯作者：
Mizukami, T
中科院分区：
文献类型：
--
作者：
Asai, A;Tsujita, T;Mizukami, T
利用酵母菌株开发了基于酵母的生长干扰测定，其中诱导非洲爪蟾细胞周期蛋白 A1 的表达以提高细胞分裂周期 28 (Cdc28) 激酶活性。由于酵母中 Cdc28 激酶的过度激活会导致生长停滞表型，因此能够挽救细胞周期蛋白 A1 诱导的生长停滞的化合物可能是潜在的新型抗肿瘤候选药物，作用于细胞周期蛋白依赖性、激酶介导的细胞周期调节途径。在我们的微生物筛选计划过程中，鉴定出了新的链霉菌代谢物贝乳素。正如之前报道的，belactosin A 诱导人类癌细胞的细胞周期停滞在 G2/M 期。然而，作用的分子机制尚不清楚。我们在此证明了贝乳糖素 A 的蛋白酶体抑制作用。贝乳糖素 A 在体外不抑制酵母 Cdc28 激酶和人细胞周期蛋白依赖性激酶。另一方面，它抑制兔20S蛋白酶体的胰凝乳蛋白酶样活性。从最初的 SAR 研究中，我们鉴定出了一种疏水性贝乳糖素 A 衍生物 KF33955，它对 HeLa S3 细胞的生长抑制活性比贝乳糖素 A 强 100 倍，这可能是由于其更高的细胞渗透性。使用KF33955的生化分析表明KF33955的蛋白酶体抑制活性是不可逆的并且需要β-内酯部分来抑制蛋白酶体。 KF33955 增加 NIH3T3 细胞中蛋白质泛素化的细胞内水平。此外，KF33955 处理导致 HeLa S3 细胞中已知蛋白酶体底物的积累。这些结果表明，贝乳糖素 A 是一种有用的先导化合物，可靶向蛋白酶体，用于治疗其病因依赖于不受调节的泛素-蛋白酶体途径的疾病。 (C) 2003 Elsevier Inc. 保留所有权利。
A yeast-based growth interference assay was developed utilizing a yeast strain in which expression of Xenopus cyclin A1 was induced to elevate cell division cycle 28 (Cdc28) kinase activity. Since the hyperactivation of Cdc28 kinase in yeast results in a growth-arrest phenotype, compounds which could rescue the cyclin A1-induced growth arrest might be potential new, antitumor drug candidates acting on the cyclin-dependent, kinase-mediated, cell cycle regulation pathway. In the course of our microbial screening program, the new Streptomyces metabolites, belactosins, were identified. As reported previously, belactosin A induced cell cycle arrest at G2/M phase in human cancer cells. However, the molecular mechanism of action was unknown. We herein demonstrate the proteasome inhibition by belactosin A. Belactosin A did not inhibit yeast Cdc28 kinase and human cyclin-dependent kinase in vitro. On the other hand, it inhibited the chymotrypsin-like activity of the rabbit 20S proteasome. From the initial SAR studies, we identified a hydrophobic belactosin A derivative, KF33955, which exhibited a 100-fold greater growth-inhibitory activity against HeLa S3 cells than belactosin A, presumably due to its higher cell permeability. The biochemical analysis using KF33955 suggested that the proteasome inhibitory activity of KF33955 were irreversible and required the beta-lactone moiety to inhibit the proteasome. KF33955 increased the intracellular levels of protein ubiquitination in NIH3T3 cells. In addition, KF33955 treatment resulted in the accumulation of known proteasome substrates in HeLa S3 cells. These results identify belactosin A as a useful lead compound to target proteasome for the treatment of disease whose etiology is dependent on the unregulated ubiquitin-proteasome pathway. (C) 2003 Elsevier Inc. All rights reserved.