Cell migration is a fundamental step for embryonic development, wound repair, immune responses, and tumor cell invasion and metastasis. It is well known that protrusive structures, namely filopodia and lamellipodia, can be observed at the leading edge of migrating cells. The formation of these structures is necessary for cell migration; however, the molecular mechanisms behind the formation of these structures remain largely unclear. Therefore, bioactive compounds that modulate protrusive structures are extremely powerful tools for studying the mechanisms behind the formation of these structures and subsequent cell migration. Therefore, we have screened for bioactive compounds that inhibit the formation of filopodia, lamellipodia, or cell migration from natural products, and attempted to identify the target molecules of our isolated compounds. Additionally, autophagy is a bulk, non-specific protein degradation system that is involved in the pathogenesis of cancer and neurodegenerative disorders. Recent extensive studies have revealed the molecular mechanisms of autophagy, however, they also remain largely unclear. Thus, we also have screened for bioactive compounds that modulate autophagy, and identified the target molecules. In the present article, we introduce the phenotypic screening system and target identification of four bioactive compounds.

Cell migration is a fundamental step for embryonic development, wound repair, immune responses, and tumor cell invasion and metastasis. Extensive studies have attempted to reveal the molecular mechanisms behind cell migration; however, they remain largely unclear. Bioactive compounds that modulate cell migration show promise as not only extremely powerful tools for studying the mechanisms behind cell migration but also as drug seeds for chemotherapy against tumor metastasis. Therefore, we have screened cell migration inhibitors and analyzed their mechanisms for the inhibition of cell migration. In this mini-review, we introduce our chemical and biological studies of three cell migration inhibitors: moverastin, UTKO1, and BU-4664L.

In the course of screening for an inhibitor of farnesyl transferase (FTase), we identified two compounds, N-benzyl-aclacinomycin A (ACM) and N-allyl-ACM, which are new derivatives of ACM. N-benzyl-ACM and N-allyl-ACM inhibited FTase activity with IC50 values of 0.86 and 2.93 μM, respectively. Not only ACM but also C-10 epimers of each ACM derivative failed to inhibit FTase. The inhibition of FTase by N-benzyl-ACM and N-allyl-ACM seems to be specific, because these two compounds did not inhibit geranylgeranyltransferase or geranylgeranyl pyrophosphate (GGPP) synthase up to 100 μM. In cultured A431 cells, N-benzyl-ACM and N-allyl-ACM also blocked both the membrane localization of H-Ras and activation of the H-Ras-dependent PI3K/Akt pathway. In addition, they inhibited epidermal growth factor (EGF)-induced migration of A431 cells. Thus, N-benzyl-ACM and N-allyl-ACM inhibited EGF-induced migration of A431 cells by inhibiting the farnesylation of H-Ras and subsequent H-Ras-dependent activation of the PI3K/Akt pathway.

Autophagy is a bulk, nonspecific protein degradation pathway that is involved in the pathogenesis of cancer and neurodegenerative disease. Here, we observed that xanthohumol (XN), a prenylated chalcone present in hops (Humulus lupulus L.) and beer, modulates autophagy. By using XN-immobilized beads, valosin-containing protein (VCP) was identified as a XN-binding protein. VCP has been reported to be an essential protein for autophagosome maturation. Using an in vitro pull down assay, we showed that XN bound directly to the N domain, which is known to mediate cofactor and substrate binding to VCP. These data indicated that XN inhibited the function of VCP, thereby allowing the impairment of autophagosome maturation and resulting in the accumulation of microtubule-associated protein 1 light chain 3-II (LC3-II). This is the first report demonstrating XN as a VCP inhibitor that binds directly to the N domain of VCP. Our finding that XN bound to and inactivated VCP not only reveals the molecular mechanism of XN-modulated autophagy but may also explain how XN exhibits various biological activities that have been reported previously.

To fully understand the regulation of cellular events, functional analysis of each protein involved in the regulatory systems is required. Among a variety of methods to uncover protein function, chemical genetics is a remarkable approach in which small molecular compounds are used as probes to elucidate protein functions within signaling pathways. However, identifying the target of small molecular bioactive compounds isolated by cell-based assays represents a crucial hurdle that must be overcome before chemical genetic studies can commence. A variety of methods and technologies for identifying target proteins have been reported. This review therefore aims to describe approaches for identifying these molecular targets.The target of compound X would be similar with that of compound A.

Natural products have been utilized for drug discovery. To increase the source diversity, we generated a new chemical library consisting of chemically modified microbial metabolites termed ‘Unnatural Natural Products’ by chemical conversion of microbial metabolites in crude broth extracts followed by purification of reaction products with the LC-photo diode array–MS system. Using this library, we discovered an XIAP inhibitor, C38OX6, which restored XIAP-suppressed enzymatic activity of caspase-3 in vitro. Furthermore, C38OX6 sensitized cancer cells to anticancer drugs, whereas the unconverted natural product did not. These findings suggest that our library could be a useful source for drug seeds.

Cell migration of tumor cells is essential for invasion of the extracellular matrix and for cell dissemination. Inhibition of the cell migration involved in the invasion process represents a potential therapeutic approach to the treatment of tumor metastasis; therefore, a novel series of derivatives of moverastins (moverastins A and B), an inhibitor of tumor cell migration, was designed and chemically synthesized. Among these moverastin derivatives, several compounds showed stronger cell migration inhibitory activity than parental moverastins, and UTKO1 was found to have the most potent inhibitory activity against the migration of human esophageal tumor EC17 cells in a chemotaxis cell chamber assay. Interestingly, although moverastins are considered to inhibit tumor cell migration by inhibiting farnesyltransferase (FTase), UTKO1 did not inhibit FTase, indicating that UTKO1 inhibited tumor cell migration by a mechanism other than the inhibition of FTase.

Identifying the targets of bioactive compounds is a major challenge in chemical biological research. Here, we identified the functional target of the natural bioactive compound glucopiericidin A (GPA) through metabolomic analysis. We isolated GPA while screening microbial samples for a filopodia protrusion inhibitor. Interestingly, GPA alone did not inhibit filopodia protrusion, but synergistically inhibit protrusion with the mitochondrial respiration inhibitor, piericidin A (PA). These results suggested that GPA might inhibit glycolysis. Capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) provided strong evidence that GPA suppresses glycolysis by functionally targeting the glucose transporter. GPA may therefore serve as a glucose transporter chemical probe. Simultaneous inhibition of both glycolysis and mitochondrial respiration dramatically decreased intracellular ATP levels, indicating that GPA inhibits ATP-dependent filopodia protrusion with PA. Our results represent a challenge of molecular target identification using metabolomic analysis.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (156 K)Download as PowerPoint slideHighlights► Two chemical inhibitors isolated from one microbial source block filopodia in synergy ► Metabolomic analysis identifies GLUT function as the target of one inhibitor, GPA ► Simultaneous blockade of glycolysis and respiration caused the filopodia inhibition

In the course of screening for a new type of androgen receptor (AR) antagonist, we isolated a novel compound, arabilin, with two structural isomers, spectinabilin and SNF4435C, produced by Streptomyces sp. MK756-CF1. Structure elucidation on the basis of the spectroscopic properties showed that arabilin is a novel polypropionate-derived metabolite with a p-nitrophenyl group and a substituted γ-pyrone ring. Arabilin competitively blocked the binding of androgen to the ligand-binding domain of AR in vitro. In addition, arabilin inhibited androgen-induced prostate-specific antigen mRNA expression in prostate cancer LNCaP cells.

We searched for compounds that affect the cyclin D1/retinoblastoma protein pathway from the in-house natural product library using a recombinant adenovirus with the Cre/loxP-regulated cyclin D1 overexpression system, and we found that oligomycin inhibited cell growth more effectively in cyclin D1-overexpressing SW480 cells than in control SW480 cells. We also found that oligomycin reduced the expression levels of cyclin D1 protein and that this reduction is, at least in part, mediated by Thr-286 phosphorylation-dependent proteasomal degradation.

In the course of screening for an inhibitor of ER stress-induced XBP1 activation, we isolated a new member of the triene-ansamycin group compound, quinotrierixin, from a culture broth of Streptomyces sp. PAE37. Quinotrierixin inhibited thapsigargin-induced XBP1 activation in HeLa cells with an IC50 of 0.067 μM. We found that other triene-ansamycin group compounds such as demethyltrienomycin A and mycotrienin I also inhibited ER stress-induced XBP1 activation. Moreover, we performed SAR study of twelve triene-ansamycin group compounds. The study showed that OH group at C-13 was crucial, and CH3 group at C-14 would be important for the XBP1 inhibitory activity.

Four novel triene-ansamycin group compounds, quinotrierixin, demethyltrienomycin A, demethyltrienomycin B and demethyltrienomycinol, were isolated from the culture broth of Streptomyces sp. PAE37 as inhibitors of ER stress-induced XBP1 activation. The structures of quinotrierixin, demethyltrienomycin A, demethyltrienomycin B and demethyltrienomycinol were determined on the basis of their spectroscopical and chemical properties. All of four possessed 21-membered macrocyclic lactams including triene moieties.

Migrastatin (MGS) is a Streptomyces metabolite that inhibits cancer cell migration. In this study, we found that MGS also enhanced the cytotoxicity of vinblastine, vincristine, and taxol in P-glycoprotein-overexpressing VJ-300 cells and P388/VCR cells. Furthermore, MGS increased the intracellular concentration of labeled vinblastine, vincristine, and taxol in both VJ-300 cells and P388/VCR cells. P-glycoprotein was photolabeled with [3H]azidopine, but this photolabeling was significantly inhibited in the presence of MGS. These results indicated that MGS directly interacts with and inhibits P-glycoprotein, thereby sensitizing drug-resistant cells to anticancer drugs.

Leptomycin B (LMB) is a Streptomyces metabolite that inhibits the chromosomal region maintenance (CRM)1-dependent nuclear export of proteins. It also induces apoptosis in several types of cancer cells, by a yet undefined mechanism. We used a functional proteomics approach to delineate the pathways and mediators involved in LMB-induced apoptosis in HeLa cells. Using two-dimensional gel electrophoresis, we searched for proteins accumulated in the nuclei of HeLa cells upon LMB treatment. Among such proteins we found prohibitin and Heat shock protein (Hsp)27, identified by peptide mass fingerprinting and mass spectrometry. Immunocytochemistry was carried out to confirm the LMB-induced nuclear accumulation of these two proteins in HeLa cells. Furthermore, we found that the cytoplasmic expression of Hsp27, but not prohibitin, partially inhibited LMB-induced apoptosis, indicating that nuclear sequestration of Hsp27 was, at least in part, involved in the apoptosis.

Fibroblast growth factors (FGF) and their receptors play an important role in cell proliferation, angiogenesis and embryonal development. In this study, we show that expression of the FGF receptor-2 (FGFR-2) protein is induced in the mid-to-late G1 phase of the cell cycle in serum-starved mouse NIH3T3 cells released from starvation. Transcription of mouse FGFR-2 was activated by E2F-1. Analysis of various mouse FGFR-2 promoter mutant constructs showed that a sequence located +57/+64 downstream of the transcriptional initiation site, related to the consensus E2F-responsive sequence, is necessary for the activation. The promoter activity of the mouse FGFR-2 gene is also positively regulated by E2F-2 and E2F-3, but not by E2F-4 and E2F-5. Moreover, the E2F-1-induced activation of mouse FGFR-2 gene transcription is suppressed by pRB. Taken together, the results demonstrate that FGFR-2 is a new class of targets for E2F, and expression of mouse FGFR-2 in mid-to-late G1 phase would be mediated, at least in part, by the activation of a pRB/E2F pathway.

Platelet-derived growth factors (PDGFs) and their receptors play an important role in cell proliferation, angiogenesis, and differentiation during normal development, and have also been implicated in tumorigenesis. In this study, we identified a novel variant of human PDGF receptor α mRNA (type II), which contains the same open reading frame as the known PDGF receptor α mRNA (type I) but a different 5′-untranslated region (5′-UTR). The 5′-UTR of the type II transcript was identified as a 363-bp exon located in intron 1 at position + 1210 to + 1572 relative to the transcriptional initiation site of the type I transcript. This type II transcript was expressed in a subset of human cell lines, such as MG-63 and MNNG/HOS cells. Moreover, transcription of the type II, but not the type I, was regulated by E2F-1 through an E2F-1-responsive site located at position + 1086/+ 1093 downstream of the transcriptional initiation site of the type I transcript. Furthermore, epigenetic modulation might be involved in the expression of the type II transcript. Our findings provide new insights into the regulatory mechanism of PDGF receptor α transcription in normal and tumor cells.