The development of new anticancer agents derived from natural resources requires a rapid identification of their molecular mechanism of action. To make this step short, we have initiated the proteomic profiling of HeLa cells treated with anticancer drugs representing a wide spectrum of mechanisms of action using two-dimensional difference gel electrophoresis (2D-DIGE). Unique proteome patterns were observed in HeLa cells treated with the HSP90 inhibitor geldanamycin, and were similar to the patterns induced by radicicol, a structurally different HSP90 inhibitor. On the other hand, etoposide and ICRF-193, compounds claimed to be topoisomerase II inhibitors, showed different proteomic profiles, which reflect their different biological activities as revealed by cell-cycle analysis. Thus far, combined data from 19 compounds have allowed their successful classification by cluster analysis according to the mechanism of action.

Visual observation is a powerful approach for screening bioactive compounds that can facilitate the discovery of attractive druggable targets following their chemicobiological validation. So far, many high-content approaches, using sophisticated imaging technology and bioinformatics, have been developed. In our study, we aimed to develop a simpler method that focuses on intact cell images because we found that dynamic changes in morphology are informative, often reflecting the mechanism of action of a drug. Here, we constructed a chemical-genetic phenotype profiling system, based on the high-content cell morphology database Morphobase. This database compiles the phenotypes of cancer cell lines that are induced by hundreds of reference compounds, wherein those of well-characterized anticancer drugs are classified by mode of action. Furthermore, we demonstrate the applicability of this system in identifying NPD6689, NPD8617, and NPD8969 as tubulin inhibitors.

The actin-targeting toxins have not only proven to be invaluable tools in studies of actin cytoskeleton structure and function but they also served as a foundation for a new class of anticancer drugs. Here, we describe that amphidinolide H (AmpH) targets actin cytoskeleton. AmpH induced multinucleated cells by disrupting actin organization in the cells, and the hyperpolymerization of purified actin into filaments of apparently normal morphology in vitro. AmpH covalently binds on actin, and the AmpH binding site is determined as Tyr200 of actin subdomain 4 by mass spectrometry and halo assay using the yeast harboring site-directed mutagenized actins. Time-lapse analyses showed that AmpH stimulated the formation of small actin-patches, followed by F-actin rearrangement into aggregates via the retraction of actin fibers. These results indicate that AmpH is a novel actin inhibitor that covalently binds on actin.

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Iejimalides (IEJLs), 24-membered macrolides, are potent antitumor compounds, but their molecular targets remain to be revealed. In the course of screening, we identified IEJLs as potent osteoclast inhibitors. Since it is known that osteoclasts are sensitive to vacuolar H þ -ATPase (V-ATPase) inhibitor, we investigated the effect of IEJLs on V-ATPases. IEJLs inhibited the V-ATPases of both mammalian and yeast cells in situ, and of yeast V-ATPases in vitro. A bafilomycin-resistant yeast mutant conferred IEJL resistance, suggesting that IEJLs bind a site similar to the bafilomycins/concanamycins-binding site. These results indicate that IEJLs are novel V-ATPase inhibitors, and that antitumor and antiosteporotic activities are exerted via V-ATPase inhibition.

Glaziovianin A, an isoflavone isolated from the leaves of Ateleia glazioviana, inhibits the cell cycle progression in M-phase with an abnormal spindle structure, but its inhibitory mechanism has not been revealed. Here, we report that glaziovianin A and its derivatives are microtubule dynamics inhibitors. Glaziovianin A extended the time lag of tubulin polymerization without changing the net amount of polymerized tubulin in vitro and suppressed microtubule dynamics in cells. Furthermore, glaziovianin A inhibited the transport of endosomes containing EGF-stimulated EGFR and prolonged the EGFR activation. Consistent with the prolonged activation of EGFR, glaziovianin A enhanced the EGF-dependent apoptosis in A431 cells. These results strongly suggested that microtubule dynamics is important for endosome transport and maturation, and that glaziovianin A shows cytotoxicity by two pathways, the mitotic arrest and inadequate activation of receptor kinases via the inhibition of endosome maturation.

Various derivatives of glaziovianin A, an antitumor isoflavone, were synthesized, and the cytotoxicity of each against HeLa S3 cells was investigated. Compared to glaziovianin A, the O7-allyl derivative was found to be more cytotoxic against HeLa S3 cells and a more potent M-phase inhibitor.

BCPP compounds have been developed as PET imaging probes for neurodegenerative diseases in the living brain. F-BCPP-EF identifies damaged neuronal areas based on the lack of MC-I; however, its underlying mechanisms of action and specificity for MC-I remain unclear. We herein report the effects of BCPP-BF, -EF, -EM on MC-I in respiratory chain complexes using cardiomyocyte SMP. BCPP compounds inhibited the binding ofH-dihydrorotenone to MC-I and the proton pumping activity of MC-I in a concentration-dependent manner in vitro. These results suggest that BCPP compounds are MC-I selective inhibitors, and, thus, these radiolabeled compounds are useful for the quantitative imaging of MC-I using PET.