Maho Morita

Fluorescent Aplyronine A: Intracellular Accumulation and Disassembly of Actin Cytoskeleton in Tumor Cells

Actin is one of the abundant proteins in the cytoskeleton and is essential for the regulation of various functions, such as muscle contraction, cell motility, and cell division. Various actin-depolymerizing agents have been found in marine invertebrates, and some show extremely strong cytotoxicity. Among them, aplyronine A (ApA, 1, Figure 1), which was isolated from the sea hare Aplysia kurodai, has been shown to exhibit remarkable antitumor activities in vivo against P388 murine leukemia cells (T/C 545 %, 0.08 mg kg ) and several cancers. It depolymerizes fibrous actin (F-actin) and inhibits the polymerization of actin by forming a 1:1 complex with the monomeric globular molecule (G-actin, Kd 100 nm). [3] Studies on structure–activity relationships, X-ray analysis of the actin– aplyronine A complex, and photoaffinity labeling experiments have established the specific interactions of 1 with actin. However, the modes of action of ApA and related actintargeting natural products in tumor cells have not been well investigated, despite their great potential as preclinical candidates for use in cancer chemotherapy. We have recently developed a biotin derivative of 1 that exhibits potent cytotoxicity and causes actin disassembly in tumor cells, and have identified actin-related proteins 2/3 (Arp2 and Arp3) as presumed targets of 1. It was suggested that ApA might inhibit the ability of the Arp2/3 complex to bind to and branch F-actin. Also, it was shown that ApA (1) caused prominent caspase-dependent apoptosis in human leukemia HL-60 cells and human epithelial carcinoma HeLa S3 cells at sub-nanomolar concentrations. To explain the potent antitumor and apoptogenic effects of 1, we prepared its fluorescent derivatives and observed its dynamic behavior in living cell systems. Using these derivatives as molecular probes, we show here that ApA (1) caused the rapid disassembly of actin cytoskeleton, the malfunction of cell adhesions, and the dephosphorylation of focal adhesion kinase in tumor cells with apoptosis. Based on the finding that the C34 N-formyl enamide moiety of 1 can be replaced with hydrogen bond acceptors without a significant loss of activity, natural ApA (1) was hydrolyzed to give the C34 aldehyde, which was condensed with an oxyamine to afford tetramethylrhodamine-conjugated (TAMRAconjugated) ApA (ApA-FL, 2, Figure 1 and Figures S1 and S2 in the Supporting Information). Similarly, a TAMRA-conjugated derivative of aplyronine C (ApC-FL, 4) was prepared from ApC (3), an extremely minor congener of 1 in A. kurodai that lacks the C7 trimethylserine moiety, as well as a TAMRA-conjugated form of mycalolide B (MyB-FL, 6) from mycalolide B (5), an actin-depolymerizing tris-oxazole macrolide isolated from a Japanese marine sponge (Mycale sp.). For comparison, model TAMRA analogue 7 was also synthesized from 3-phenylpropionaldehyde. ApA-FL (2) showed a potent cytotoxicity against HeLa S3 cells (IC50 370 pm), whereas fluorescent derivatives 4 and 6 exhibited activities ca. 30 and 840 times weaker than that of 2. In an in vitro actin-depolymerizing assay, ApA-FL (2) significantly reduced the fluorescence of pyrene-labeled (pyrenyl) Factin (EC50 0.8 mm against 3 mm actin), and was more effective than 1 (EC50 1.3 mm), whereas model compound 7 scarcely exhibited this activity (Figure 2). Also, inhibition of F-actin sedimentation caused by 2 was directly detected by an SDS-PAGE analysis. On treatment with 1 or 2, the protein bands were observed almost entirely in the supernatant, as with G-actin (far left), which established that they had potent actin-depolymerizing properties. In contrast, 7 did not increase the amount in the supernatant, as with the control (second from left), which suggests that it does not depolymerize F-actin. Fluorescence microscopy observations revealed that the TAMRA derivatives 2, 4, 6, and 7 were all readily (less than 15 min) incorporated into HeLa S3 cells. Notably, ApA-FL (2) and ApC-FL (4) were retained well and distributed through the cytoplasm even after cells were washed with culture medium and incubated for an additional hour (Figure 1). We observed that ApC-FL (4) also accumulated through the nucleus, but that ApA-FL (2) did not at all. In contrast, model 7 was almost completely excluded from the cells under the same treatments. These significant differences in intracellular accumulation were established more clearly by flow cytometry analyses. The initial cellular incorporations of MyB-FL (6) and model 7 were less than that of 2 by factors of ca. 4–5. Furthermore, ApA-FL (2) was recovered as a unique TAMRA-containing component in HeLa S3 cells that had been treated for 1.5 h (Figure S3), which suggested that it exhibits strong bioavailability as well as highlighting the stability of the oxime bond. To visualize actin depolymerization in tumor cells, ApA-FL (2, 3 mm) was added to HEp-2 cells that expressed a green fluores[a] Prof. Dr. M. Kita, K. Yoneda, Y. Hirayama, K. Yamagishi, Y. Saito, Prof. Dr. H. Kigoshi Graduate School of Pure and Applied Sciences, University of Tsukuba 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571 (Japan) E-mail : mkita@chem.tsukuba.ac.jp kigoshi@chem.tsukuba.ac.jp [b] Y. Sugiyama, Dr. Y. Miwa Graduate School of Comprehensive Human Sciences, University of Tsukuba 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575 (Japan) [c] Dr. O. Ohno, M. Morita, Prof. Dr. K. Suenaga Faculty of Science and Technology, Keio University 3-14-1, Hiyoshi, Yokohama 223-8522 (Japan) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201200385.

Apoptosis-inducing activity of the actin-depolymerizing agent aplyronine A and its side-chain derivatives.

Aplyronine A (1) and mycalolide B (2), which are cytotoxic actin-depolymerizing marine macrolides, were revealed to induce apoptosis in human leukemia HL60 cells and human epithelial carcinoma HeLa S(3) cells. Based on these results, actin-depolymerizing compounds were expected to exhibit apoptosis-inducing activity in cancer cells. Compounds 3-6, which were synthesized based on the side-chain structure of aplyronine A, were evaluated for their actin-depolymerizing activities in vitro and cytotoxicities against HL60 cells. The growth-inhibitory activities of 3-6 were well correlated with their actin-depolymerizing activities, and derivative 6 was shown to induce the disruption of actin filaments and apoptosis in HL60 cells. These results suggested that actin-depolymerizing agents 1, 2, and 6-induced apoptosis in HL60 cells may have been due to their actin-depolymerizing activity.

Biselyngbyasides, cytotoxic marine macrolides, are novel and potent inhibitors of the Ca2+ pumps with a unique mode of binding

Biselyngbyasides (BLSs), macrolides from a marine cyanobacterium, are cytotoxic natural products whose target molecule is unknown. Here we report that BLSs are high affinity (K i ∼ 10 nM) inhibitors of Ca2+‐pumps with a unique binding mode. The crystal structures of the Ca2+‐pump in complex with BLSs at 3.2–3.5 Å‐resolution show that BLSs bind to the pump near the cytoplasmic surface of the transmembrane region. The crystal structures and activity measurement of BLS analogs allow us to identify the structural features that confer high potency to BLSs as inhibitors of the pump.

A novel allelopathic substance, 13-epi-orthosiphol N, in Orthosiphon stamineus.

Orthosiphon stamineus (Java tea) has been widely used as traditional herb and several bioactive compounds against animal cells have been isolated. However, no bioactive compound against plants has been reported. Therefore, we investigated possible allelopathic properties and substances in O. stamineus. Aqueous methanol extracts of O. stamineus inhibited root and hypocotyl growth of cress (Lepidium sativum) and lettuce (Lactuca sativa) seedlings. Increasing the extract concentration increased the inhibition, which suggests that O. stamineus may have allelopathic properties. When the extract was divided into an ethyl acetate and an aqueous fraction, the ethyl acetate fraction showed the stronger inhibitory effect. Thus, the ethyl acetate phase was further purified, and the main allelopathic substance was isolated and identified as 13-epi-orthosiphol N, a novel compound, by spectral data. 13-epi-Orthosiphol N inhibited root and hypocotyl growth of cress and lettuce at concentrations greater than 10 μmol/L. The concentrations required for 50% inhibition ranged from 41 to 102 μmol/L. These results suggest that 13-epi-orthosiphol N may be an allelochemical and main contributor to the growth inhibitory effect of O. stamineus and may have potential as a template for the development of new plant control substances.

Leptolyngbyolides, Cytotoxic Macrolides from the Marine Cyanobacterium Leptolyngbya sp.: Isolation, Biological Activity, and Catalytic Asymmetric Total Synthesis

Four new macrolactones, leptolyngbyolides A-D, were isolated from the cyanobacterium Leptolyngbya sp. collected in Okinawa, Japan. The planar structures of leptolyngbyolides were determined by extensive NMR studies, although complete assignment of the absolute configuration awaited the catalytic asymmetric total synthesis of leptolyngbyolide C. The synthesis took advantage of the catalytic asymmetric thioamide-aldol reaction using copper(I) complexed with a chiral bidentate phosphine ligand to regulate two key stereochemistries of the molecule at the outset. The present total synthesis demonstrates the utility of this reaction for the construction of complex chemical entities. In addition to the total synthesis, this work reports that leptolyngbyolides depolymerize filamentous actin (F-actin) both in vitro and in cells. Detailed biological studies suggest the probable order of F-actin depolymerization and apoptosis caused by leptolyngbyolides.

Identification of a molecular target of kurahyne, an apoptosis-inducing lipopeptide from marine cyanobacterial assemblages.

In 2014, we isolated kurahyne, an acetylene-containing lipopeptide, from a marine cyanobacterial assemblage of Lyngbya sp. Kurahyne exhibited growth-inhibitory activity against human cancer cells, and induced apoptosis in HeLa cells. However, its mode of action is not yet clear. To elucidate its mode of action, we carried out several cell-based assays, and identified the intracellular target molecule of kurahyne as sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA). In addition, we found that kurahyne inhibited the differentiation of macrophages into osteoclasts.

Design, synthesis and anti-malarial activities of synthetic analogs of biselyngbyolide B, a Ca2+ pump inhibitor from marine cyanobacteria

Biselyngbyaside, an 18-membered macrolide glycoside from marine cyanobacteria, and its derivatives are known to be sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors. Recently, a SERCA orthologue of the malaria parasite, PfATP6, has attracted attention as a malarial drug target. To provide a novel drug lead, we designed new synthetic analogs of biselyngbyolide B, the aglycone of biselyngbyaside, based on the co-crystal structure of SERCA with biselyngbyolide B, and synthesized them using the established synthetic route for biselyngbyolide B. Their biological activities against malarial parasites were evaluated.