BCPP compounds have been developed as PET imaging probes for neurodegenerative diseases in the living brain. 18F-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 of 3H-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.

Homotypic fusion of early endosomes is important for efficient protein trafficking and sorting. The key controller of this process is Rab5 which regulates several effectors and PtdInsPs levels, but whose mechanisms are largely unknown. Here, we report that vicenistatin, a natural product, enhanced homotypic fusion of early endosomes and induced the formation of large vacuole-like structures in mammalian cells. Unlike YM201636, another early endosome vacuolating compound, vicenistatin did not inhibit PIKfyve activity in vitro but activated Rab5-PAS pathway in cells. Furthermore, vicenistatin increased the membrane surface fluidity of cholesterol-containing liposomes in vitro, and cholesterol deprivation from the plasma membrane stimulated vicenistatin-induced vacuolation in cells. These results suggest that vicenistatin is a novel compound that induces the formation of vacuole-like structures by activating Rab5-PAS pathway and increasing membrane fluidity.

Iejimalides (IEJLs) A-D are 24-membered macrolides isolated from a tunicate Eudistoma cf. rigida, and exhibit potent cytotoxicity in vitro and antitumor activity in vivo. We previously reported that the molecular target of IEJL-A and -B was the vacuolar-type H(+)-ATPases (V-ATPases). However IEJL-C and -D, which are sulfonylated IEJL-A and -B, respectively, show more potent antitumor activity, and their molecular targets remain to be discovered. Here, we report that IEJL-C is also a potent V-ATPase inhibitor by binding in a site similar to the bafilomycin-binding site. Two-hour treatment with IEJL-C resulted in the complete disappearance of acidic organelles in HeLa cells. Interestingly, after 24-h treatment, small actin aggregates were observed instead of actin fibers. The same actin reorganization was also observed in cells treated with another V-ATPase inhibitor, bafilomycin A1. Because IEJLs did not inhibit actin polymerization in vitro, these results suggest that the primary target of IEJL-C, as well as IEJL-A and -B, is V-ATPase, and actin reorganizations are probably caused by the disruption of pH homeostasis via V-ATPase inhibition.

Optical vortex (OV) is light twisted around the propagation axis. Optical trap by OV exerts constant torque on a dielectric object in the same direction of angular momentum at any moment because OV has an orbital angular momentum. Recently, we have succeeded in manipulating beads three dimensionally using OV (Otsu et.al, Sci. Rep., 2014). In this study, we tried to demonstrate that OV trapping can be used for an analysis tool for biological rotary motors. As a result, DNA and F1-ATPase was manipulated by OV via beads in a desired direction and speed. Escherichia coli cells tethered on a coverslip by a single flagellum was accelerated and decelerated by OV. These results show that OV has a potential to reveal the torque generation mechanism of molecular motors.

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.

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.

F-actin-stabilizing drugs induce actin aggresome formation. In this study, we found that an actin-depolymerizing drug, latrunculin A (LatA), induced actin aggresomes. Actin stress fibers were retracted and disappeared in minutes, but a large aggresome formed in consequence of LatA treatment. Because cytochalasin D and mycalolide also induced aggresome formation, these results suggest that actin aggresome formation is a common cellular response to actin toxins.

Abstract 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.

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.

We propose a novel approach to 3D image registration of intracellular volumes. The approach extends a standard image registration framework to the curved cell geometry. An intracellular volume is mapped onto another intracellular domain by using two pairs of point set surfaces approximating their nuclear and plasma membranes. The mapping function consists of the affine transformation, tetrahedral barycentric interpolation, and least-squares formulation of radial basis functions for extracted cell geometry features. An interactive volume registration system is also developed based on our approach. We demonstrate that our approach is capable of creating cell models containing multiple organelles from observed data of living cells.

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.

The paper proposes a novel 3D image registration approach to calculating intracellular volumes. The approach extends a standard image registration framework to the curved cell geometry. An intracellular volume is mapped onto another intracellular domain by using two pairs of point set surfaces approximating their nuclear and plasma membranes. The approach is implemented to an interactive volume registration system. We demonstrate that our approach can create models of cells with multiple organelles from data collected from living cells.