Cloning, enzyme characterization of recombinant human Eg5 and the development of a new inhibitor.
The microtubule-dependent motor protein Eg5 is essential for the development and function of the mitotic spindle. Now it has become an anti-mitotic drug target in high throughput screening for anticancer dugs in vitro. Here is a protocol for cloning, expression and purification of a human Eg5 that codes for motor and linker domain in Escherichia coli BL21 (DE3) cells. The effects of temperature, pH, metal ions and DMSO on ATPase activity were investigated. A new compound CPUYL064 showed good inhibitory effect against Eg5 (IC(50) value, 100 nM). It inhibited the proliferation of human hepatocellular liver carcinoma cell line HepG2 in a dose- and time-dependent manner. CPUYL064 induced a clear G(2)/M phase arrest and caused the monastral spindle in HepG2 cells. Induction of apoptosis was further confirmed by changes in membrane phospholipids, changes in mitochondrial membrane potential and by detection of DNA fragmentation. These results indicate that CPUYL064 could be developed as a new, potent mitotic arrest compound.
Yang
Lei
L
School of Life Science and Technology, China Pharmaceutical University, Nanjing, China.
Jiang
Cheng
C
Liu
Fei
F
You
Qi-Dong
QD
Wu
Wu-Tong
WT
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000813:Anilides / Q000494:pharmacology*; D017304:Annexin A5 / Q000096:biosynthesis / Q000235:genetics; D017209:Apoptosis / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005434:Flow Cytometry; D016195:G2 Phase / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016547:Kinesins / Q000037:antagonists & inhibitors* / Q000235:genetics*; D008670:Metals / Q000494:pharmacology; D051336:Mitochondrial Membranes / Q000187:drug effects; D015363:Quinolones / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D008941:Spindle Apparatus / Q000187:drug effects; D013696:Temperature
C531024:2,2,7-trimethyl-4-(3-nitrophenyl)-5-oxo-N-(4-chlorophenyl)-1,4,5,6,7,8-hexahydroquinoline-3-carboxamide; D000813:Anilides; D017304:Annexin A5; C470243:KIF11 protein, human; D008670:Metals; D015363:Quinolones; D011994:Recombinant Proteins; D004247:DNA; D000251:Adenosine Triphosphatases; D016547:Kinesins; D004121:Dimethyl Sulfoxide