To discover novel drugs for neuroblastoma treatment, we have previously screened a panel of drugs and identified 30 active agents against neuroblastoma cells. Here we performed microarray gene expression analysis to monitor the impact of these agents on a neuroblastoma cell line and used the connectivity map (cMAP) to explore putative mechanism of action of unknown drugs. We first compared the expression profiles of 10 compounds shared in both our dataset and cMAP database and observed the high connectivity scores for 7 of 10 matched drugs regardless of the differences of cell lines utilized. The screen of cMAP for uncharacterized drugs indicated the signature of Epoxy anthraquinone derivative (EAD) matched the profiles of multiple known DNA targeted agents (topoisomerase I/II inhibitors, DNA intercalators, and DNA alkylation agents) as predicted by its structure. Similar result was obtained by querying against our internal NB-cMAP (http://pob.abcc.ncifcrf.gov/cgi-bin/cMAP), a database containing the profiles of 30 active drugs. These results suggest that Epoxy anthraquinone derivative may inhibit neuroblastoma cells by targeting DNA replication inhibition. Experimental data also demonstrate that Epoxy anthraquinone derivative indeed induces DNA double-strand breaks through DNA alkylation and inhibition of topoisomerase activity. Our study indicates that Epoxy anthraquinone derivative may be a novel DNA topoisomerase inhibitor that can be potentially used for treatment of neuroblastoma or other cancer patients.