Yuichiro Hirayama

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.

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).

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.

Design, synthesis, and biological evaluations of aplyronine A-mycalolide B hybrid compound

A hybrid compound consisting of aplyronine A and mycalolide B was synthesized, and its biological activities were evaluated. The hybrid compound was found to have somewhat more potent actin-depolymerizing activity than aplyronine A. In contrast, the hybrid compound possessed about 1000-fold less cytotoxicity than aplyronine A. These results indicated that there is no direct correlation between actin-depolymerizing activity and cytotoxicity.

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.

New Cytotoxic Norditerpenes from the Australian Nudibranchs Goniobranchus Splendidus and Goniobranchus Daphne

This study reports the isolation and characterisation of six new metabolites with ‘gracilin’-type carbon skeletons and of aplytandiene-3 from the Australian nudibranch Goniobranchus splendidus. The structure of gracilin G is revised, and the C-6 configuration deduced by comparison of calculated 3JC/H values with values measured using the EXSIDE pulse sequence. A lactone isolated from Goniobranchus daphne contains a rearranged spongionellin-type skeleton. Screening of selected metabolites revealed significant cytotoxicity against a HeLa S3 cell line by five of the new terpenes.

Enzyme-catalyzed cationic epoxide rearrangements in quinolone alkaloid biosynthesis

Epoxides are highly useful synthons and biosynthons in the construction of complex natural products during total synthesis and biosynthesis, respectively. Among enzyme-catalyzed epoxide transformations, a notably missing reaction, compared to the synthetic toolbox, is cationic rearrangement that takes place under strong acids. This is a challenging transformation for enzyme catalysis, as stabilization of the carbocation intermediate upon epoxide cleavage is required. Here, we discovered two Brønsted acid enzymes that can catalyze two unprecedented epoxide transformations in biology. PenF from the penigequinolone pathway catalyzes a cationic epoxide rearrangement under physiological conditions to generate a quaternary carbon center, while AsqO from the aspoquinolone pathway catalyzes a 3-exo-tet cyclization to forge a cyclopropane-tetrahydrofuran ring system. The discovery of these new epoxide-modifying enzymes further highlights the versatility of epoxides in complexity generation during natural product biosynthesis.

Integration of Chemical, Genetic, and Bioinformatic Approaches Delineates Fungal Polyketide–Peptide Hybrid Biosynthesis

To identify natural products and their associated biosynthetic genes from underutilized, difficult-to-manipulate microbes, chemical screening and bioinformatic analysis were employed to identify secondary metabolites and a potentially associated biosynthetic gene cluster. Subsequently, a heterologous expression system was used to confirm the identity of the gene cluster and the proposed biosynthetic mechanism. This approach successfully identified the curvupallide and spirostaphylotrichin biosynthetic pathways in endophytic fungus Curvularia pallescens and the short-chain pyranonigrin biosynthetic pathway in Aspergillus niger.

Analysis of the aplyronine A-induced protein-protein interaction between actin and tubulin by surface plasmon resonance.

The antitumor macrolide aplyronine A induces protein-protein interaction (PPI) between actin and tubulin to exert highly potent biological activities. The interactions and binding kinetics of these molecules were analyzed by the surface plasmon resonance with biotinylated aplyronines or tubulin as ligands. Strong binding was observed for tubulin and actin with immobilized aplyronine A. These PPIs were almost completely inhibited by one equivalent of either aplyronine A or C, or mycalolide B. In contrast, a non-competitive actin-depolymerizing agent, latrunculin A, highly accelerated their association. Significant binding was also observed for immobilized tubulin with an actin-aplyronine A complex, and the dissociation constant KD was 1.84μM. Our method could be used for the quantitative analysis of the PPIs between two polymerizing proteins stabilized with small agents.

Polyketide Synthase–Nonribosomal Peptide Synthetase Hybrid Enzymes of Fungi

Fungi are known to produce various nonribosomal peptide-derived compounds that exhibit useful bioactivities. However, understanding of how those fungal secondary metabolites are biosynthesized still remains limited. In this review, we focus on recent efforts in engineering select fungal species to make them amenable to efficient genetic modifications for identifying genes responsible for the biosynthesis of secondary metabolites. The fungi discussed in this review are Chaetomium globosum, Aspergillus fumigatus, A. niger, A. nidulans, and A. oryzae. This review also discusses how the engineered fungi are used in deciphering the mechanism of natural product biosynthesis, primarily through heterologous reconstitution of biosynthetic pathways of interest. In particular, potential involvement of enzymatic Diels–Alder reactions in the secondary metabolite biosynthesis is discussed in details. Compounds discussed here are cytochalasans, such as chaetoglobosins and cytochalasins, Sch 210972, equisetin, and pyrrolocins.