A high-throughput model for screening anti-tumor agents capable of promoting polymerization of tubulin in vitro.
OBJECTIVE
To establish a high-throughput model for screening anti-tumor agents capable of promoting the polymerization of tubulin in vitro.


METHODS
Tubulin was prepared in different purity for two screening steps. The first step was a high-throughput screening (HTS) for a set of 1500 samples using the GTP-containing tubulin and the end-reading method. The second step was performed on 119 hits from the first screening by a kinetic assay with GTP-lacking tubulin.


RESULTS
The HTS for 1500 samples was accomplished in less than 3 h. From the screening, 108 samples were identified with >20 % promotion activity at 10 mg/L. Five of 108 were further confirmed by the kinetic assay using the purified tubulin subsequently. Three of the hit compounds were Epothilone A or its analogs, the other two compounds had new structures with a common pharmacophore for cytotoxic natural products that stabilize microtubules. In an MTT test, the five selected samples from the screening showed a minimal IC(50) at 0.28+/-0.06 nmol/L to Hela cells.


CONCLUSIONS
The two-step screening method is a high-throughtput, cost-effective, and efficient approach to identify microtubule-stabilizing agents.
Hu
Wei
W
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Ji-nan 250100, China.
Dong
Hui
H
Li
Yue-Zhong
YZ
Hu
Xi-Tao
XT
Han
Guan-Jun
GJ
Qu
Yin-Bo
YB
D000970:Antineoplastic Agents / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D034261:Epothilones / Q000494:pharmacology; D006367:HeLa Cells / Q000187:drug effects; D006801:Humans; D020128:Inhibitory Concentration 50; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D017239:Paclitaxel / Q000494:pharmacology; D014404:Tubulin / Q000187:drug effects* / Q000378:metabolism
D000970:Antineoplastic Agents; D034261:Epothilones; D014404:Tubulin; C093787:epothilone A; D017239:Paclitaxel