Sayaka Kazami

Application of Proteomic Profiling Based on 2D-DIGE for Classification of Compounds According to the Mechanism of Action

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.

Morphobase, an Encyclopedic Cell Morphology Database, and Its Use for Drug Target Identification

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.

Iejimalides Show Anti-Osteoclast Activity via V-ATPase Inhibition

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+-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.

Structure-activity relationship study of glaziovianin A against cell cycle progression and spindle formation of HeLa S3 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.

Glaziovianin a prevents endosome maturation via inhibiting microtubule dynamics

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.

Actin Stress Fiber Retraction and Aggresome Formation Is a Common Cellular Response to Actin Toxins

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.

Vicenistatin induces early endosome-derived vacuole formation in mammalian cells

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. Graphical abstract Vicenistatin is a novel compound that induces the formation of vacuole-like structures by activating Rab5-PAS pathway and increasing membrane fluidity.

INTERACTIVE REGISTRATION OF INTRACELLULAR VOLUMES WITH RADIAL BASIS FUNCTIONS

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.

BCPP compounds, PET probes for early therapeutic evaluations, specifically bind to mitochondrial complex I

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.

Intracellular Volume Registration

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.