Background: Every year huge numbers of compounds are screened for cancer cells proliferation inhibition. However, the mechanism of action and/or the molecular target as decisive factors in drug development often remain unknown. We recently developed a proteomic profiling technique to predict the mode of action of new drug leads. This method is based on recording the comprehensive patterns of variation in proteins in HeLa cells that are treated with compounds. The primary in vitro screening of the candidate anticancer compounds from the chemical library of RIKEN Natural Products Depository (NPDepo) revealed potentially active compounds with diverse chemical structures (NPD6689, NPD8617, and NPD8969). Here, we report on a rapid identification of a molecular target for these compounds by using the proteomic profiling system.

Materials and Methods: HeLa cells were treated with NPD6689, NPD8617, or NPD8969 for 18 h, and the cell lysates were subjected to 2-dimensional fluorescence differential gel electrophoresis (2D-DIGE). The proteome data were linked to our database, which contained proteome data that were generated by 47 compounds with well-established modes of action. Then, hierarchical cluster analysis was performed.

Results: NPD6689, NPD8617, and NPD8969 inhibited the growth of HeLa cells with the IC50 value of 615, 184, and 240 nM, respectively. The proteomic profiling of the effects induced in HeLa cells by 2D-DIGE revealed the NPD6689, NPD8617, and NPD8969 to belong to the same cluster as tubulin inhibitors, like vinblastine, paclitaxel, and colchicine. The predicted mode of action was confirmed by direct inhibition of tubulin polymerization, disruption of microtubule network in interphase cells, abnormal spindle morphology in mitotic cells, and cell cycle arrest at G2/M phase. The effects were time- and concentration-dependent.

Conclusion: These results indicate that NPD6689, NPD8617, and NPD8969 target tubulin and act as its polymerization inhibitors. Furthermore, our study shows that proteomics-based profiling will be very useful in rapid identification of mechanisms of action of anticancer compounds in future drug development and chemical genetic studies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B199.