A new structural class of proteasome inhibitors identified by microbial screening using yeast-based assay.
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.
Asai
Akira
A
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co Ltd., 3-6-6 Asahi-machi, Machida-shi, 194-8533, Tokyo, Japan. aasai@kyowa.co.jp
Tsujita
Tetsuya
T
Sharma
Sreenath V
SV
Yamashita
Yoshinori
Y
Akinaga
Shiro
S
Funakoshi
Minoru
M
Kobayashi
Hideki
H
Mizukami
Tamio
T
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000494:pharmacology; D017489:CDC28 Protein Kinase, S cerevisiae / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D003546:Cysteine Endopeptidases; D004151:Dipeptides / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007783:Lactones / Q000494:pharmacology*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D010455:Peptides / Q000302:isolation & purification / Q000494:pharmacology*; D046988:Proteasome Endopeptidase Complex; D012441:Saccharomyces cerevisiae / Q000187:drug effects; D025801:Ubiquitin / Q000378:metabolism
D004151:Dipeptides; D036341:Intercellular Signaling Peptides and Proteins; C481349:KF33955; D007783:Lactones; D009097:Multienzyme Complexes; D010455:Peptides; D025801:Ubiquitin; C406052:belactosin A; C067713:lactacystin; D017489:CDC28 Protein Kinase, S cerevisiae; D018844:Cyclin-Dependent Kinases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D000111:Acetylcysteine