Masaki Kita

Zamamistatin, a significant antibacterial bromotyrosine derivative, from the Okinawan sponge Pseudoceratina purpurea

Abstract Zamamistatin was isolated from the Okinawan sponge Pseudoceratina purpurea. It was determined to be a novel bromotyrosine derivative by careful analysis of 2D NMR spectra and comparison of its 1H NMR spectrum with those of structurally related compounds. The absolute stereostructure of zamamistatin was determined by the modified Moshers method. Zamamistatin exhibited significant antibacterial activity against Rhodospirillum salexigens, which has adhering properties.

Inhibition of microtubule assembly by a complex of actin and antitumor macrolide aplyronine A.

Aplyronine A (ApA) is a marine natural product that shows potent antitumor activity. While both ApA and ApC, a derivative of ApA that lacks a trimethylserine ester moiety, inhibit actin polymerization in vitro to the same extent, only ApA shows potent cytotoxicity. Therefore, the molecular targets and mechanisms of action of ApA in cells have remained unclear. We report that ApA inhibits tubulin polymerization in a hitherto unprecedented way. ApA forms a 1:1:1 heterotrimeric complex with actin and tubulin, in association with actin synergistically binding to tubulin, and inhibits tubulin polymerization. Tubulin-targeting agents have been widely used in cancer chemotherapy, but there are no previous descriptions of microtubule inhibitors that also bind to actin and affect microtubule assembly. ApA inhibits spindle formation and mitosis in HeLa S3 cells at 100 pM, a much lower concentration than is needed for the disassembly of the actin cytoskeleton. The results of the present study indicate that ApA represents a rare type of natural product, which binds to two different cytoplasmic proteins to exert highly potent biological activities.

Nakiterpiosin, a novel cytotoxic C-nor-D-homosteroid from the Okinawan sponge Terpios hoshinota

A novel cytotoxic compound, nakiterpiosin (1), was isolated from the Okinawan sponge Terpios hoshinota. Its structure was determined by spectroscopic analysis. The absolute stereostructure of 1 was also determined by a modified Moshers method. Nakiterpiosin showed potent cytotoxicity against P388 cells.

Development of highly cytotoxic and actin-depolymerizing biotin derivatives of aplyronine A.

Tied up: a PEG-linked biotin derivative of marine macrolide aplyronine A (ApA) is shown to exhibit potent cytotoxicity and cause actin disassembly in tumor cells. This method of introducing a PEG linker at the end of the aliphatic tail should offer perspectives for developing and using versatile actin-targeting molecular probes. PEG=poly(ethylene glycol).

Bioactive secondary metabolites from symbiotic marine dinoflagellates: symbiodinolide and durinskiols.

Symbiotic relationships play critical roles in marine ecosystems. Among symbionts, marine dinoflagellates have attracted the attention of natural products chemists, biologists, and ecologists, since they are rich sources of unique bioactive secondary metabolites. The polyol compound symbiodinolide, which was isolated from the symbiotic dinoflagellate Symbiodinium sp., exhibits significant voltage-dependent N-type Ca(2+) channel-opening activity and may serve as a defense substance to prevent digestion of the host animals. Durinskiols are also unique long carbon-chain polyol compounds that were isolated from the dinoflagellate Durinskia sp. We found a selective cleavage reaction of allylic 1,2-diol using an olefin metathesis catalyst, and developed a fluorescent-labeling method for MS/MS analysis to achieve the structural elucidation of huge polyol compounds. This review highlights recent advances in structural and biological studies on symbiodinolide, durinskiols, and related polyol compounds.

Interactions of the antitumor macrolide aplyronine A with actin and actin-related proteins established by its versatile photoaffinity derivatives.

The antitumor and apoptogenic macrolide aplyronine A (ApA) is a potent actin-depolymerizing agent. We developed an ApA acetylene analog that bears the aryldiazirine group at the C34 terminus, which formed a covalent bond with actin. With the use of the photoaffinity biotin derivatives of aplyronines A and C, Arp2 and Arp3 (actin-related proteins) were specifically purified as binding proteins along with actin from tumor cell lysate. However, Arp2 and Arp3 did not covalently bind to aplyronine photoaffinity derivatives. Thus, actin-related proteins might indirectly bind to ApA as the ternary adducts of the actin/ApA complex or through the oligomeric actin.

Purification and characterisation of blarinasin, a new tissue kallikrein-like protease from the short-tailed shrew Blarina brevicauda: comparative studies with blarina toxin

Abstract A new tissue kallikrein-like protease, blarinasin, has been purified from the salivary glands of the short-tailed shrew Blarina brevicauda. Blarinasin is a 32-kDa N-glycosylated protease with isoelectric values ranging between 5.3 and 5.7, and an optimum pH of 8.5 for enzyme activity. The cloned blarinasin cDNA coded for a pre-pro-sequence and a mature peptide of 252 amino acids with a catalytic triad typical for serine proteases and 43.7–54.0% identity to other mammalian tissue kallikreins. Blarinasin preferentially hydrolysed Pro-Phe-Arg-4-methylcoumaryl-7-amide (MCA) and N-tert-butyloxycarbonyl-Val-Leu-Lys-MCA, and preferentially converted human high-molecular-weight kininogen (HK) to bradykinin. The activity of blarinasin was prominently inhibited by aprotinin (K i=3.4 nM). A similar kallikrein-like protease, the lethal venom blarina toxin, has previously been purified from the salivary glands of the shrew Blarina and shows 67.9% identity to blarinasin. However, blarinasin was not toxic in mice. Blarinasin is a very abundant kallikrein-like protease and represents 70–75% of kallikrein-like enzymes in the salivary gland of B. brevicauda.

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.

Recent aspects of chemical ecology: Natural toxins, coral communities, and symbiotic relationships

The discovery of new ecologically active compounds often triggers the development of basic scientific concepts in the field of biological sciences, since such compounds have direct physiological and behavioral effects on other living organisms. We have focused on the identification of natural key compounds that control biologically and physiologically intriguing phenomena. We describe three recent aspects of chemical ecology that we have investigated: natural toxins, coral communities, and symbiotic relationships. Blarina toxin (BLTX) is a lethal mammalian venom that was isolated from the short-tailed shrew. Duck-billed platypus venom shows potent Ca2+ influx in neuroblastoma cells. The venom of the solitary wasp contains arginine kinase-like protein and is used to paralyze its prey to feed its larva. The ecological behaviors of corals are controlled by combinations of small molecules. The polyol compound symbiodinolide may serve as a defense substance for symbiotic dinoflagellates to prevent digestion of their host animals. These compounds reveal the wonder of nature, in both terrestrial and marine ecological systems.

Aplysiasecosterol A: A 9,11‐Secosteroid with an Unprecedented Tricyclic γ‐Diketone Structure from the Sea Hare Aplysia kurodai

A new 9,11-secosteroid having an unprecedented tricyclic γ-diketone structure, aplysiasecosterol A (1), was isolated from the sea hare Aplysia kurodai. The structure was determined by one- and two-dimensional NMR spectroscopic analysis, molecular modeling studies, a comparison of experimental and calculated ECD spectra, and a modified Moshers method. Aplysiasecosterol A (1) exhibited cytotoxicity against human myelocytic leukemia HL-60 cells. A biosynthetic pathway for 1 from a known cholesterol was proposed and includes twice α-ketol rearrangements and an intramolecular acetalization.