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Cyanopeptolins, depsipeptides from cyanobacteria
System. Appl. Microbiol. 19, 133-138 (1996) © Gustav Fischer Verlag· Stuttgart· lena . New York
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Cyanopeptolins, Depsipeptides from Cyanobacteria JDRGEN WECKESSER1:f, CORNEL MARTIN 1
2
2
,
and CLEMENS JAKOBI!
Institut fiir Biologie II, Mikrobiologie, Albert-Ludwigs-Universitat, 5chanziestraBe 1, D-79104 Freiburg, Germany Dr. Willmar 5chwabe Arzneimittel, Willmar-5chwabe-5traBe 4, D-76227 Karlsruhe, Germany
Received December 19, 1995
Summary The wide range of secondary metabolites from cyanobacteria includes cyclic depsipeptides. They can be filed into several classes. Members of class V, the cyanopeptolins, are proposed in this review to be defined as (a) cyanobacterial 19-membered cyclic depsipeptides cyclized by an ester-linkage of the hydroxyl-group of threonine with the carboxy terminus of the C-terminal amino acid of a proposed linear precursor, (b) an unusual 3-amino-6-hydroxy-2-piperidone unit, and (c) a cis-configurated amide linkage between the amino acids in position 3 and 4. The cyanobacterial depsipeptides known so far, matching with this definition, are the cyanopeptolins A, B, C, and D, the aeruginopeptins 228-A, 228-B, 95-A, and 95-B, microcystilide A, the micropeptins A, B, and 90, compound A90nOA, and the sulfate-containing cyanopeptolins 5 and 55. The cyanopeptolins and related depsipeptides from cyanobacteria may serve a biochemical and pharmacological tools, because most of them exhibit protease-inhibitory and/or cytotoxic activities. The structure-function relationships of the serine protease inhibitor A90nOA and trypsin is known by a cocrystallization study. There are indications for a possible cyanobacterial origin of cyanopeptolin-like compounds found in marine eukaryotes.
Key words: Cyanobacteria - Microcystis sp. - Depsipeptides - 3-Amino-6-hydroxy-2-piperidone - Cyanopeptolins - Protease-inhibitors - Cytotoxicity
Introduction Cyanobacteria are a rich source of biologically active peptides, but presently attention is paid mostly to hepatotoxic, cyclic peptides of the microcystin/nodularin class [for recent reviews see Carmichael (1994) and Rinehart et a1. (1994)] and their multiple effects as inhibitors of protein phosphatases 1 and 2A [(for recent reports see Claeyssens et a1. (1995), Rabouille et a1. (1995), Hayakawa and Kohama (1995), and Murphy et a1. (1995)]. Other cyclic peptides from cyanobacteria, like the bistratamide-related westiellamide from Westiellopsis prolifica (Prinsep et aI., 1992), laxaphycins from Anabaena laxa (Frankmolle et aI., 1992a,b), and the hormothamnins from Hormothamnion enteromorphoides (Gerwick et aI., 1992) * Corresponding author 10 System. Appl. Microbiol. Vol. 19/2
have cytotoxic and/or antimicrobial including antifungal activity. The linear peptide microginin from Microcystic aeruginosa is an angiotensin-converting enzyme inhibitor (Okino et aI., 1993a). The likewise linear aeruginosin 298A from the freshwater strain Microcystis aeruginosa NIES298 (Murakami et aI., 1994) as well as the monosulfated aeruginosins 98-A and B from Microcystis aeruginosa NIES-98 (Murakami et aI., 1995) inhibit the serine protease trypsin potently, and less potently plasmin and thrombin (Murakami et aI., 1995). This review is focusing on cyanopeptolins and related cyclic depsipeptides in cyanobacteria (for a definition of depsipeptides see Schroder and Lubke, 1965). Like other cyclic peptides, the cyclodepsipeptides are supposed to be non-ribosomally synthesized. The termination of biosyn-
134
J. Weckesser, C. Martin, and C.]akobi
thesis occurs by lactonization with a terminal or side chain hydroxyl group, giving a lactone or branched lactone (Kleinkauf and von Dohren, 1995). Overview on cyanobacterial depsipeptides The cyclic depsipeptides isolated so far from cyanobacteria may be filed into five classes: (I) Cryptophycin A and a number of respective structural variants (the monochlorinated L-O-methyltyrosine and the non-chlorinated D-O-methyltyrosine unit series) from Nostoc sp. GSV-224 and ATCC 53789 (Trimurtulu et al., 1994). (II) Majusculamide C, a lipophilic heptacyclodepsipeptide found in Lyngbya majuscula (Carter et al., 1984). (III) Hapalosin from the cyanobacterium Hapalosiphon welwitschii W. & G. S. West (Stratmann et al., 1994). (IV) Microviridin, a tricyclic tetradecadepsipeptide containing two ester-linkages, from Microcystis viridis NIES-102 (Ishitsuka et al., 1990) and microviridins A, Band C from Microcystis aeruginosa (Okino et al. (1995). (V) The cyanopeptolins, as discussed in the following.
a
c
General structure and variations
Among the cyanobacterial peptides described so far, cyclic depsipeptides containing an unusual 3-amino-6-hydroxy-2-piperidone (Ahp) unit (Martin et al., 1993; Lee et al., 1994), have been found frequently. This class of molecules, found by Martin et al. (1993) in axenically grown Microcystis aeruginosa PCC 7806, were termed by these authors "cyanopeptolins". They may be defined as cyanobacterial products which contain the following structural characteristics: (a) a 19-membered depsipeptide ring cyclisized by an ester-linkage of the hydroxyl-group of threonine with the carboxy terminus of the C-terminal amino acid of a proposed linear precursor, (b) the Ahp unit, and (c) cis-configuration of the amide linkage between amino acids in positions 3 and 4 (Fig. 1). The cyanobacterial depsipeptides known so far matching with the definition of cyanopeptolins differ from the cyanopeptolins A-D from Microcystis aeruginosa PCC 7806 in the composition of distinct positions within the ring as well as in the amide-linked side chain (Fig. 1, Tab. 1). They com-
Ho,soT OH(OSO,H)
OH
b
Cyanopeptolins and related compounds
H~~~ ~'('Cr0
d
e
Rl ....
T
('-/)~HN\ HO
"'~HN o
Ji. .2.JiL 3 I
HO
0
H)O
I
R2
I
NH~~-\-- 0
--6-0~~f'o~:J~o3 ___);"CH,: g: ---Y-H(OH) R3
Side chain
0
5
4
7
Fig. 1. Right: Overall structure of depsipeptides of the cyanopeptolin type described so far for Microcystis sp. and Microchaete loktakensis strains (for references see Table 1). From the amino acids (Table 1), only the part involved in the ring formation is drawn in the figure, their residual part is indicated by Rl, R2, and R3. The 3-amino-6-hydroxy-2-piperidone (Ahp) unit can be thought of as a glutamate in which the side chain carboxyl has been reduced to an aldehyde and the aldehyde cyclized with the n+ 1 amide to form a cyclic hemiaminol (Lee et al., 1994). The numbers 1-7 account for the positions of the structural units. Left, a-f: Structures of the side chains (see also Table 1).
Cyanopeptolins
135
Table 1. Structural variations of cyanopeptolins and related compounds from cyanobacteria. For the position of Rl, R2, R3, H (OH), and the side chains see Fig. 1. For references: Cyanopeptolins A-D (Martin et al., 1993); aeruginopeptins 228-A, 228-B, 95-A 95-B (Harada et al., 1993); microcystilide A, Tsukamoto et al. (1993); micropeptins A und B (Okino et al., 1993b); micropeptin 90 (Ishida et al., 1995); A90nOA (Lee et al., 1994); cyanopeptolin 5 and cyanopeptolin 55 (jakobi et al., 1995a, b). All compounds listed match with the definition of cyanopeptolins (see text) Cyanopeptolin-type compounds (class V of cyanobacterial depsipeptides)
Rl
R2
R3
H(OH)
Side chain
Cyanopeptolin A Cyanopeptolin B Cyanopeptolin C Cyanopeptolin D Aeruginopeptin 228-A Aeruginopeptin 228-B Aeruginopeptin 95-A Aeruginopeptin 95-B Microcystilide A Micropeptin A Micropeptin B Micropeptin 90 Compound A90nOA Cyanopeptolin S Cyanopeptolin SS
Arg Lys N-Methyl-Lys N,N-Dimethyl-Lys Tyr ThTyr h Tyr ThTyr Tyr Lys Lys Arg Arg Arg Arg
Leu Leu Leu Leu Thr Thr Thr Thr Leu Leu Leu Phe Leu Ile Ile
Val Val Val Val Ile Ile Ile Ile Ile Val Val Val Val Ile Ile
H H H H H H H H OH OH OH OH OH H H
Asp-hexanoic acid c Asp-hexanoic acid c Asp-hexanoic acid c Asp-hexanoic acidc Gln-Hplae,g Gln-Hpla e Gln-Thr-Hpla f Gln-Thr-Hpla f Gln-D-Hpla e Glu-octanoic acid d Glu-hexanoic acid d Monosulfated glyceric acid b Leu-monosulfated glyceric acid b Monosulfated glyceric acid' Disulfated glyceric acid'
,-f,
Indices account for the side chains in Fig. 1; g, Hpla, 4-hydroxyphenyllactic acid; h, ThTyr, tetrahydrotyrosine
prise the aeruginopeptins from Microcystis aeruginosa T AC 95 and M228 (Harada et al., 1993), microcystilide A from Microcystis aeruginosa NO-15-1840 (Tsukamoto et al., 1993), the micropeptins A and B from Microcystis aeruginosa NIES-100 (Okino. et aL, 1993b), micropeptin 90 from Microcystis aeruginosa NIES-90 (Ishida et aL, 1995), compound A90nOA from Microchaete 10ktakensis, and the cyanopeptolins Sand SS from a Microcystis sp. bloom in a german lake (jakobi et al., 1995a,b). As far as known, the variations of amino acids in the hexapeptide ring of cyanopeptolins and related compounds seem to vary significantly as follows (Fig. 1, Table 1): At the R1 position occur mainly basic amino acids (Arg, Lys, N-Me-Lys, N,N-Di-Me-Lys) or Tyr and tetrahydrotyrosine (ThTyr). The R2 position is occupied by strongly varying amino acids (Thr, Leu, lie, Phe). In contrast, the R3 position with lie or Val seems to be rather conserved. High variability is found in the side chain, where aromatic (4-hydroxyphenyllatic acid) and hydrophilic (Gin, Thr, Asp) amino acids, or non amino acid constituents (glyceric acid, aliphatic fatty acids) occur (Table1).
Biological activities The most conspicuous properties of cyclic depsipeptides of cyanobacterial origin published so far are protease inhibitory, fungicidal, cytotoxic, and antitumor activities (Table2). Among the cryptophycin A group, the major tumor-selective cytotoxin in Nostoc sp. GSV 224 showed in vivo excellent activity against solid tumors implanted in mice revealing very low ICso-values against KB (a human nasopharyngeal carcinoma cell line), and LoVo (a human
colorectal adenocarcinoma cell line), respectively (Trimurtulu et al., 1994). It appeared to have the same mode of action as vinblastine but differed from the latter in irreversibly inhibiting tubulin polymerization. Majusculamide C inhibits growth of fungal plant pathogens (Carter et aL, 1994) and is structurally similar to dolastatin 11, a major antineoplastic compound in the sea hare Dolabella auricularia (Pettit et aL, 1993; see also below). Hapalosin has multidrug-resistance (MDR)-reversing activity in a vinblastine-resistant subline of a human ovarian adenocarcinoma line (Stratmann et al., 1994). Microviridin strongly inhibits tyrosinase activity (Ishitsuka et aL, 1990), and microviridins Band C are elastase-inhibitors (Okino et aL, 1995). Among the cyanopeptolins and related compounds, microcystilide A revealed cell-differentiation-promoting activity in a cell differentiation assay using HL-60 human leucocytes (Tsukamoto et aL, 1993). Micropeptin A and B inhibit the serine proteases plasmin and trypsin but not thrombin, chymotrypsin and elastase (Table 2). Similarly, micropeptin 90 inhibits only plasmin and trypsin among the serine proteases, and also the cysteine protease papain (Ishida et aL, 1995). The mechanism of inhibition was suggested to be similar to that of compound A90nOA (Lee et aL, 1994). Cyanopeptolin Sand SS inhibit trypsin and plasmin more potently than thrombin and both revealed to be nontoxic to isolated rat hepatocytes (jakobi et al., 1995b). Cyanopeptolin SS was toxic to Daphnia magna STRAUS, while Cyanopeptolin S was not toxic in the concentrations tested (Table 2). The elegant study of Lee et aL (1994) pointed on structure-function relationships of the cyanopeptolin-like potent serine protease inhibitor compound A90720A by
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J. Weckesser, C. Martin, and C. Jakobi
Table 2. Biological activities of cyanobacterial depsipeptides Inhibition (IC 50 given in !-tg/ml)
Depsipeptide
Serine protease Trypsin
Plasmin
Thrombin
Other biological activities Cysteine protease
Chymotrypsin
Elastase
Papain
Cryptophycin A group
Tumor-selective cytotoxicity' (at 3 and 6 pg/ml) Fungicide MDR-reversing activityb Tyrosinase-inhibitory activity (at 3.3 x 10-4 M).
Majusculamide C Hapalosin Microviridin 0.044 0.084
Microviridin B Microviridin C Microcystilide A Micropeptin A Micropeptin B Micropeptin 90
0.071 0.025 2.0
0.026 0.035 0.1
None None
Compound A90nOA Cyanopeptolin A Cyanopeptolin S
0.01 <0.2 <0.2
< 1.0
<5.0
Cyanopeptolin SS
<0.2
< 1.0
<5.0
0.03
0.275
None None None (at 10)
None None None (at 10)
Cell-differentiation-promoting activityc (at 0.5 mg/ml) Weak cytotoxicity (IC50 0.5 mg/ml) None (at 10) Daphnia toxicity: None « 11.5 !-tg/ml) Daphnia toxicity: 60% at 7.5 !-tg/ml
• Against the human carcinoma cell lines KB and LoVo, respectively (Trimurtulu et aI., 1994) b Multidrug-resistance (MDR)-reversing activity (Stratmann et aI., 1994) C In a human leucocyte (HL-60) and against HCT116 and HCTVP35 cell lines (Tsukamoto et aI., 1993) For references: Cryptophycin A group (Trimurtulu et aI., 1994); majusculamide C (Carter et aI., 1984); hapalosin (Stratmann et aI., 199.4); microviridin (Ishitsuka et aI., 1990); microviridin Band C (Okino et aI., 1995); microcystilide A (Tsukamoto et aI., 1993); micropeptins A und B (Okino et aI., 1993b); micropeptin 90 (Ishida et aI., 1993); compound A90nOA (Lee et aI., 1994); cyanopeptolin A Uakobi et aI., 1995a); cyanopeptolin Sand cyanopeptolin SS Uakobi et aI., 1995b)
crystallization and structural elucidation of a bovine trypsin-compound A90nOA complex at 1.9 A resolution by single crystal X-ray diffraction. The depsipeptide interacts tightly with trypsin in a non-covalent, substrate-like manner through hydrogen bonds, hydrophobic interactions and steric complementarity. It imitates the canonical conformation of the exposed binding loop of the so-called 'small' protease inhibitors using peptidal an nonpeptidal elements, whereby the Ahp unit (see Fig. 1) plays an essential role by determining the binding conformation of the inhibitor and preventing its dissociation by its transannular hydrogen bonds.
Are the cyclodepsipeptides obtained from marine eukaryotes of cyanobacterial origin? An Ahp unit-containing cyclic depsipeptide, dolastatin
13, has also been found in the shell-less marine mollusc
Dolabella auricularia (Pettit et aI., 1989). However, the finding of such depsipeptides in axenically grown cyanobacteria led later to the suggestion that the true producers
of dolastatin 13 were cyanobacteria (Ishida et aI., 1995), and Tsukamoto et al. (1993) stated: "There has been a persisting speculation that a number of interesting compounds - many of them are peptides - found in Dolabella are derived from dietary alga." Furthermore, dolastatin 11 from the same origin like dolastatin 13 (Pettit et aI., 1993) and arenastatin A from the sponge Dysidea aranaria (Kobayashi et aI., 1994) were similar to majusculamide C and to cryptophycins, respectively. It is of interest in this context, that motuporin, structurally very similar to the cyanobacterial hepatotoxin nodularin, has been found in a marine sponge and was strongly suggested to be a cyanobacterial product (de Silva et aI., 1992). Similar observations were made for scytophycin-type compounds found in both, marine sponges and freshwater cyanobacteria [(cited in Tsukamoto et al. (1993)]. Recently, the structurally similar oscillamide A and anabaenopeptins were isolated from Oscillatoria agardhii NIVA CYA 18 (Sano and Kaya, 1995) and Anabaena (los-aquae NRC 525-17 (Harada et aI., 1995), respectively. They had a high resemblance with keramamide A from Theonella sp. (Kobayashi et aI., 1991).
Cyanopeptolins
Concluding remarks An attempt was made herein to point out that cyanopeptolins and related depsipeptides from cyanobacteria may serve as biochemical and/or pharmacological tools. Knowledge of their mode of action is feasable now, since e.g. cocrystallization of serine protease inhibitor compound A90nOA with trypsin (Lee et al., 1994) and microcystin-LR with the catalytic subunit of the protein phosphatase 1 (Goldberg et al., 1995) have already been performed successfully. Cyanopeptolins and related depsipeptides may be more widely distributed in cyanobacteria than notized so far. It is notable that cyanopeptolins can be simultaneously produced together with microcystins (Martin et al., 1993; Harada et al., 1993; Jakobi et al., 1995a,b). These peptides may contribute to chemotaxonomy of cyanobacteria, whereby the possible origin of some depsipeptides found in marine eukaryotes has to be considered.
References Carmichael, W. W.: The toxins of cyanobacteria. Scient. Amer. 270, 64-72 (1994) Carter, D. c., Moore, R. E., Mynderse, j. S., Niemczura, W. P., Todd, j. S.: Structure of majusculamide C, a cyclic depsipeptide from Lyngbya majuscula. J. Org. Chern. 49, 236-241 (1984) Claeyssens, S., Francois, A., Chedeville, A., Lavoinne, A.: Microcystin-LR induced an inhibition of protein synthesis in isolated rat hepatocytes. Biochem. J. 306, 693-696 (1995) De Silva, E. D., Williams, D. E., Andersen, R. J., Klix, H., Holmes, C. F. B., Allen, T. M.: Motuporin, a potent phosphatase inhibitor isolated from the Papua New Guinea sponge Theonella swinhoei Gray. Tetrahedron Lett. 33, 1561-1564 (1992) Frankmolle, W. P., Larsen, 1. K., Caplan, F. R., Patterson, G. M. 1., Kniibel, G., Levine, I. A., Moore, R. E.: Antifungal cyclic peptides from the terrestrial blue-green alga Anabaena laxa. I. Isolation and biological properties. J. Antibiot. 45, 1451-1457 (1992a) Frankmolle, W. P., Kniibel, G., Moore, R. E., Patterson, G. M. 1.: Antifungal cyclic peptides from the terrestrial blue-green alga Anabaena laxa. II. Structures of laxaphycins A, B, D and E. ]. Antibiot. 45, 1458-1466 (1992b) Gerwick, W. H., Jiang, Z. D., Agarwal, S. K., Farmer, B. T.: Total structure of hormothamnin A, a toxic cyclic undecapeptide from the tropical marine cyanobacterium Hormothamnion enteromorphoides. Tetrahedron 48, 2313-2324 (1992) Goldberg, j., Huang, H.-B., Kwon, Y.-G., Greengard, P., Nairn, A. c., Kurlyan, j.: Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase 1. Nature 376, 745-753 (1995) Harada, K., Mayumi, T., Shimada, T., Suzuki, M., Kondo, F., Watanabe, M. F.: Occurrence of four depsipeptides, aeruginopeptins, together with microcystins from toxic cyanobacteria. Tetrahedron Lett. 34, 6091-6094 (1993) Harada, K.-I., Fujii, K., Shimada, T., Suzuki, M., Sano, H., Adachi, K., Carmichael, W. W.: Two cyclic peptides, anabaenopeptins, a third group of bioactive compounds from the cyanobacterium Anabaena flos-aquae NRC 525-17. Tetrahedron Lett. 36, 1511-1514 (1995)
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Hayakawa, K., Kohama, K.: Reversible effects of okadaic acid and microcystin-LR on the ATP-dependent interaction between actin and myosin. J. Biochem. 117,509-514 (1995) Ishida, K., Murakami, M., Matsuda, H., Yamaguchi, K.: Micropeptin 90, a plasmin and trypsin inhibitor from the blue-green alga Microcystis aeruginosa (NIES-90). Tetrahedron Lett. 36, 3535-3538 (1995) Ishitsuka, M. 0., Kusumi, T., Kakisawa, H., Kaya, K., Watanabe, M. M.: Microviridin: A novel tricyclic depsipeptide from the toxic cyanobacterium Microcystis viridis. J. Am. Chern. Soc. 112,8180-8182 (1990) Jakobi, c., Oberer, 1., Quiquerez, c., Konig, W. A., Weckesser, j.: Cyanopeptolin 5, a sulfate-containing depsipeptide from a water bloom of Microcystis sp. FEMS Microbiol. Lett. 129, 129-134 (1995a) Jakobi, c., Rinehart, K. 1., Neuber, R., Mez, K., Weckesser, J.: Cyanopeptolin 55, a disulfated depsipeptide from a water bloom in Leipzig (Germany): structural elucidation and biological activities. Phycologia, submitted for publication (1995b) Kleinkauf, H., von Dohren, H.: The nonribosomal peptide biosynthetic system - on the origins of structural diversity of peptides, cyclopeptides and related compounds. Antonie van Leeuwenhoek 67, 229-242 (1995) Kobayashi, j., Sato, M., Ishibashi, M., Shigemori, H., Nakamura, T., Ohizumi, Y.: Keramamide A, a novel peptide from the okinawan marine sponge Theonella sp. J. Chern. Soc. Perkin Trans. I, 2609-2611 (1991) Kobayashi, M., Aoki, S., Ohyabu, N., Kurosu, K., Wang, W., Kitagawa, I.: Arenastatin A, a potent cytotoxic depsipeptide from the okinawa marine sponge Dysidea arenaria. Tetrahedron Lett. 35, 7969-7972 (1994) Lee, A. Y., Smitka, T. A., Bonjouklian, R., Clardy, j.: Atomic structure of the trypsin-A90720A complex: a unified approach to structure an function. Chern. BioI. 1, 113-117 (1994) Martin, c., Oberer, 1., Ino, T., Konig, W. A., Busch, M., Weckesser, j.: Cyanopeptolins, new depsipeptides from the cyanobacterium Microcystis sp. PCC 7806. J. Antibiot. 46, 15501556 (1993) Murakami, M., Okita, Y., Matsuda, H., Okino, T., Yamaguchi, K.: Aeruginosin 298-A, a thrombin and trypsin inhibitor from the blue-green alga Microcystis aeruginosa (NIES-298). Tetrahedron Lett. 35, 3129-3132 (1994) Murakami, M., Ishida, K., Okino, T., Okita, Y., Matsuda, H., Yamaguchi, K.: Aeruginosins 98-A and B, trypsin inhibitors from the blue-green alga Microcystis aeruginosa (NIES-98). Tetrahedron Lett. 36, 2785-2788 (1995) Murphy, j., Crompton, C. M., Hainey, S., Codd, G. A., Hutchison, C. j.: The role of protein phosphorylation in the assembly of a replication competent nucleus: investigation in Xenopus egg extracts using the cyanobacterial toxin microcystin-LR J. Cell Sci. 108,235-244 (1995) Okino, T., Matsuda, H., Murakami, M., Yamaguchi, K.: Microginin, an angiotensin-converting enzyme inhibitor from the blue-green alga Microcystis aeruginosa. Tetrahedron Lett. 34: 501-504 (1993a) Okino, T., Murakami, M., Haraguchi, R., Munekata, H., Matsuda, H., Yamaguchi, K.: Micropeptins A and B, plasmin and trypsin inhibitors from the blue-green alga Microcystis aeruginosa. Tetrahedron Lett. 34, 8131-8134 (1993b) Okino, T., Matsuda, H., Murakami, M., Yamaguchi, K.: New microviridins, elastase inhibitors from the blue-green alga Microcystis aeruginosa. Tetrahedron Lett. 39, 10679-10686 (1995) Pettit, G. R., Kamano, Y., Herald, C. 1., Dufresne, c., Cerny, R. 1., Herald, D. 1., Schmidt, j. M., Kizu, H.: Isolation and
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structure of the cytostatic depsipeptide dolastatin 13 from the sea hare Dolabella auricularia. J. Am. Chern. Soc. 111, 5015-5017 (1989) Pettit, G. R., Kamano, Y., Herald, C. 1., Fujii, Y., Kizu, H., Boyd, M. R., Boettner, F. E., Doubek, D. 1., Schmidt, J. 1., Chapuis, J. c., Michel, c.: Isolation of dolastatins 10-15 from the marine mollusc Dolabella auricularia. Tetrahedron 49, 9151-9170 (1993) Prinsep, M. R., Moore, R. E., Levine, I. A., Patterson, G. M. 1.: Westiellamide, a bistratamide-related cyclic peptide from the blue-green alga Westiellopsis prolifica. J. Natural Prod. 55, 140-142 (1992) Rabouille, c., Misteli, T., Watson, R., Warren, G.: Reassembly of golgi stacks from mitotic golgi fragments in a cell-free system. J. Cell BioI. 129, 605-618 (1995) Rinehart, K. 1., Namikoshi, M., Choi, B. W.: Structure and biosynthesis of toxins from blue-green algae (cyanobacteria). J. Appl. Phycol. 6, 159-176 (1994)
Sano, T., Kaya, K.: Oscillamide Y, a chymotrypsin inhibitor from toxic Oscillatoria agardhii. Tetrahedron Lett. 36, 5933-5936 (1995) Schroder, E., Lubke, K.: The Peptides. Academic Press, New York and London (1965) Stratmann, K., Burgoyne, D. 1., Moore, R. E., Patterson, G. M. 1., Smith C. D.: Hapalosin, a cyanobacterial cyclic depsipeptide with multidrug-resistance reversing activity. J. Org. Chern. 59, 7219-7226 (1994) Trimurtulu, G., Ohtani, I., Patterson, G. M. 1., Moore, R. E., Corbett, T. H., Valeriote, F. A., Demchik, 1.: Total structures of cryptophycins, potent antitumor depsipeptides from the blue-green alga Nostoc sp. strain GSV 224. J. Am. Chern. Soc. 116,4729-4737 (1994) Tsukamoto, S., Painuly, P., Young, K. A., Yang, X., Shimizu, Y., Cornell, 1.: Microcystilide A: a novel cell-differentiationpromoting depsipeptide from Microcystis aeruginosa NO-151840. J. Am. Chern. Soc. 115, 11046-11047 (1993)
Jurgen Weckesser, Institut fUr Biologie II, Mikrobiologie, Albert-Ludwigs-Universitat Freiburg, SchanzlestraBe 1, D-79104 Freiburg i.Br., Germany; Tel. +49-761-203-2638; Fax +49-761-203-2647; email: [email protected]
Minireview
Cyanopeptolins, Depsipeptides from Cyanobacteria JDRGEN WECKESSER1:f, CORNEL MARTIN 1
2
2
,
and CLEMENS JAKOBI!
Institut fiir Biologie II, Mikrobiologie, Albert-Ludwigs-Universitat, 5chanziestraBe 1, D-79104 Freiburg, Germany Dr. Willmar 5chwabe Arzneimittel, Willmar-5chwabe-5traBe 4, D-76227 Karlsruhe, Germany
Received December 19, 1995
Summary The wide range of secondary metabolites from cyanobacteria includes cyclic depsipeptides. They can be filed into several classes. Members of class V, the cyanopeptolins, are proposed in this review to be defined as (a) cyanobacterial 19-membered cyclic depsipeptides cyclized by an ester-linkage of the hydroxyl-group of threonine with the carboxy terminus of the C-terminal amino acid of a proposed linear precursor, (b) an unusual 3-amino-6-hydroxy-2-piperidone unit, and (c) a cis-configurated amide linkage between the amino acids in position 3 and 4. The cyanobacterial depsipeptides known so far, matching with this definition, are the cyanopeptolins A, B, C, and D, the aeruginopeptins 228-A, 228-B, 95-A, and 95-B, microcystilide A, the micropeptins A, B, and 90, compound A90nOA, and the sulfate-containing cyanopeptolins 5 and 55. The cyanopeptolins and related depsipeptides from cyanobacteria may serve a biochemical and pharmacological tools, because most of them exhibit protease-inhibitory and/or cytotoxic activities. The structure-function relationships of the serine protease inhibitor A90nOA and trypsin is known by a cocrystallization study. There are indications for a possible cyanobacterial origin of cyanopeptolin-like compounds found in marine eukaryotes.
Key words: Cyanobacteria - Microcystis sp. - Depsipeptides - 3-Amino-6-hydroxy-2-piperidone - Cyanopeptolins - Protease-inhibitors - Cytotoxicity
Introduction Cyanobacteria are a rich source of biologically active peptides, but presently attention is paid mostly to hepatotoxic, cyclic peptides of the microcystin/nodularin class [for recent reviews see Carmichael (1994) and Rinehart et a1. (1994)] and their multiple effects as inhibitors of protein phosphatases 1 and 2A [(for recent reports see Claeyssens et a1. (1995), Rabouille et a1. (1995), Hayakawa and Kohama (1995), and Murphy et a1. (1995)]. Other cyclic peptides from cyanobacteria, like the bistratamide-related westiellamide from Westiellopsis prolifica (Prinsep et aI., 1992), laxaphycins from Anabaena laxa (Frankmolle et aI., 1992a,b), and the hormothamnins from Hormothamnion enteromorphoides (Gerwick et aI., 1992) * Corresponding author 10 System. Appl. Microbiol. Vol. 19/2
have cytotoxic and/or antimicrobial including antifungal activity. The linear peptide microginin from Microcystic aeruginosa is an angiotensin-converting enzyme inhibitor (Okino et aI., 1993a). The likewise linear aeruginosin 298A from the freshwater strain Microcystis aeruginosa NIES298 (Murakami et aI., 1994) as well as the monosulfated aeruginosins 98-A and B from Microcystis aeruginosa NIES-98 (Murakami et aI., 1995) inhibit the serine protease trypsin potently, and less potently plasmin and thrombin (Murakami et aI., 1995). This review is focusing on cyanopeptolins and related cyclic depsipeptides in cyanobacteria (for a definition of depsipeptides see Schroder and Lubke, 1965). Like other cyclic peptides, the cyclodepsipeptides are supposed to be non-ribosomally synthesized. The termination of biosyn-
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J. Weckesser, C. Martin, and C.]akobi
thesis occurs by lactonization with a terminal or side chain hydroxyl group, giving a lactone or branched lactone (Kleinkauf and von Dohren, 1995). Overview on cyanobacterial depsipeptides The cyclic depsipeptides isolated so far from cyanobacteria may be filed into five classes: (I) Cryptophycin A and a number of respective structural variants (the monochlorinated L-O-methyltyrosine and the non-chlorinated D-O-methyltyrosine unit series) from Nostoc sp. GSV-224 and ATCC 53789 (Trimurtulu et al., 1994). (II) Majusculamide C, a lipophilic heptacyclodepsipeptide found in Lyngbya majuscula (Carter et al., 1984). (III) Hapalosin from the cyanobacterium Hapalosiphon welwitschii W. & G. S. West (Stratmann et al., 1994). (IV) Microviridin, a tricyclic tetradecadepsipeptide containing two ester-linkages, from Microcystis viridis NIES-102 (Ishitsuka et al., 1990) and microviridins A, Band C from Microcystis aeruginosa (Okino et al. (1995). (V) The cyanopeptolins, as discussed in the following.
a
c
General structure and variations
Among the cyanobacterial peptides described so far, cyclic depsipeptides containing an unusual 3-amino-6-hydroxy-2-piperidone (Ahp) unit (Martin et al., 1993; Lee et al., 1994), have been found frequently. This class of molecules, found by Martin et al. (1993) in axenically grown Microcystis aeruginosa PCC 7806, were termed by these authors "cyanopeptolins". They may be defined as cyanobacterial products which contain the following structural characteristics: (a) a 19-membered depsipeptide ring cyclisized by an ester-linkage of the hydroxyl-group of threonine with the carboxy terminus of the C-terminal amino acid of a proposed linear precursor, (b) the Ahp unit, and (c) cis-configuration of the amide linkage between amino acids in positions 3 and 4 (Fig. 1). The cyanobacterial depsipeptides known so far matching with the definition of cyanopeptolins differ from the cyanopeptolins A-D from Microcystis aeruginosa PCC 7806 in the composition of distinct positions within the ring as well as in the amide-linked side chain (Fig. 1, Tab. 1). They com-
Ho,soT OH(OSO,H)
OH
b
Cyanopeptolins and related compounds
H~~~ ~'('Cr0
d
e
Rl ....
T
('-/)~HN\ HO
"'~HN o
Ji. .2.JiL 3 I
HO
0
H)O
I
R2
I
NH~~-\-- 0
--6-0~~f'o~:J~o3 ___);"CH,: g: ---Y-H(OH) R3
Side chain
0
5
4
7
Fig. 1. Right: Overall structure of depsipeptides of the cyanopeptolin type described so far for Microcystis sp. and Microchaete loktakensis strains (for references see Table 1). From the amino acids (Table 1), only the part involved in the ring formation is drawn in the figure, their residual part is indicated by Rl, R2, and R3. The 3-amino-6-hydroxy-2-piperidone (Ahp) unit can be thought of as a glutamate in which the side chain carboxyl has been reduced to an aldehyde and the aldehyde cyclized with the n+ 1 amide to form a cyclic hemiaminol (Lee et al., 1994). The numbers 1-7 account for the positions of the structural units. Left, a-f: Structures of the side chains (see also Table 1).
Cyanopeptolins
135
Table 1. Structural variations of cyanopeptolins and related compounds from cyanobacteria. For the position of Rl, R2, R3, H (OH), and the side chains see Fig. 1. For references: Cyanopeptolins A-D (Martin et al., 1993); aeruginopeptins 228-A, 228-B, 95-A 95-B (Harada et al., 1993); microcystilide A, Tsukamoto et al. (1993); micropeptins A und B (Okino et al., 1993b); micropeptin 90 (Ishida et al., 1995); A90nOA (Lee et al., 1994); cyanopeptolin 5 and cyanopeptolin 55 (jakobi et al., 1995a, b). All compounds listed match with the definition of cyanopeptolins (see text) Cyanopeptolin-type compounds (class V of cyanobacterial depsipeptides)
Rl
R2
R3
H(OH)
Side chain
Cyanopeptolin A Cyanopeptolin B Cyanopeptolin C Cyanopeptolin D Aeruginopeptin 228-A Aeruginopeptin 228-B Aeruginopeptin 95-A Aeruginopeptin 95-B Microcystilide A Micropeptin A Micropeptin B Micropeptin 90 Compound A90nOA Cyanopeptolin S Cyanopeptolin SS
Arg Lys N-Methyl-Lys N,N-Dimethyl-Lys Tyr ThTyr h Tyr ThTyr Tyr Lys Lys Arg Arg Arg Arg
Leu Leu Leu Leu Thr Thr Thr Thr Leu Leu Leu Phe Leu Ile Ile
Val Val Val Val Ile Ile Ile Ile Ile Val Val Val Val Ile Ile
H H H H H H H H OH OH OH OH OH H H
Asp-hexanoic acid c Asp-hexanoic acid c Asp-hexanoic acid c Asp-hexanoic acidc Gln-Hplae,g Gln-Hpla e Gln-Thr-Hpla f Gln-Thr-Hpla f Gln-D-Hpla e Glu-octanoic acid d Glu-hexanoic acid d Monosulfated glyceric acid b Leu-monosulfated glyceric acid b Monosulfated glyceric acid' Disulfated glyceric acid'
,-f,
Indices account for the side chains in Fig. 1; g, Hpla, 4-hydroxyphenyllactic acid; h, ThTyr, tetrahydrotyrosine
prise the aeruginopeptins from Microcystis aeruginosa T AC 95 and M228 (Harada et al., 1993), microcystilide A from Microcystis aeruginosa NO-15-1840 (Tsukamoto et al., 1993), the micropeptins A and B from Microcystis aeruginosa NIES-100 (Okino. et aL, 1993b), micropeptin 90 from Microcystis aeruginosa NIES-90 (Ishida et aL, 1995), compound A90nOA from Microchaete 10ktakensis, and the cyanopeptolins Sand SS from a Microcystis sp. bloom in a german lake (jakobi et al., 1995a,b). As far as known, the variations of amino acids in the hexapeptide ring of cyanopeptolins and related compounds seem to vary significantly as follows (Fig. 1, Table 1): At the R1 position occur mainly basic amino acids (Arg, Lys, N-Me-Lys, N,N-Di-Me-Lys) or Tyr and tetrahydrotyrosine (ThTyr). The R2 position is occupied by strongly varying amino acids (Thr, Leu, lie, Phe). In contrast, the R3 position with lie or Val seems to be rather conserved. High variability is found in the side chain, where aromatic (4-hydroxyphenyllatic acid) and hydrophilic (Gin, Thr, Asp) amino acids, or non amino acid constituents (glyceric acid, aliphatic fatty acids) occur (Table1).
Biological activities The most conspicuous properties of cyclic depsipeptides of cyanobacterial origin published so far are protease inhibitory, fungicidal, cytotoxic, and antitumor activities (Table2). Among the cryptophycin A group, the major tumor-selective cytotoxin in Nostoc sp. GSV 224 showed in vivo excellent activity against solid tumors implanted in mice revealing very low ICso-values against KB (a human nasopharyngeal carcinoma cell line), and LoVo (a human
colorectal adenocarcinoma cell line), respectively (Trimurtulu et al., 1994). It appeared to have the same mode of action as vinblastine but differed from the latter in irreversibly inhibiting tubulin polymerization. Majusculamide C inhibits growth of fungal plant pathogens (Carter et aL, 1994) and is structurally similar to dolastatin 11, a major antineoplastic compound in the sea hare Dolabella auricularia (Pettit et aL, 1993; see also below). Hapalosin has multidrug-resistance (MDR)-reversing activity in a vinblastine-resistant subline of a human ovarian adenocarcinoma line (Stratmann et al., 1994). Microviridin strongly inhibits tyrosinase activity (Ishitsuka et aL, 1990), and microviridins Band C are elastase-inhibitors (Okino et aL, 1995). Among the cyanopeptolins and related compounds, microcystilide A revealed cell-differentiation-promoting activity in a cell differentiation assay using HL-60 human leucocytes (Tsukamoto et aL, 1993). Micropeptin A and B inhibit the serine proteases plasmin and trypsin but not thrombin, chymotrypsin and elastase (Table 2). Similarly, micropeptin 90 inhibits only plasmin and trypsin among the serine proteases, and also the cysteine protease papain (Ishida et aL, 1995). The mechanism of inhibition was suggested to be similar to that of compound A90nOA (Lee et aL, 1994). Cyanopeptolin Sand SS inhibit trypsin and plasmin more potently than thrombin and both revealed to be nontoxic to isolated rat hepatocytes (jakobi et al., 1995b). Cyanopeptolin SS was toxic to Daphnia magna STRAUS, while Cyanopeptolin S was not toxic in the concentrations tested (Table 2). The elegant study of Lee et aL (1994) pointed on structure-function relationships of the cyanopeptolin-like potent serine protease inhibitor compound A90720A by
136
J. Weckesser, C. Martin, and C. Jakobi
Table 2. Biological activities of cyanobacterial depsipeptides Inhibition (IC 50 given in !-tg/ml)
Depsipeptide
Serine protease Trypsin
Plasmin
Thrombin
Other biological activities Cysteine protease
Chymotrypsin
Elastase
Papain
Cryptophycin A group
Tumor-selective cytotoxicity' (at 3 and 6 pg/ml) Fungicide MDR-reversing activityb Tyrosinase-inhibitory activity (at 3.3 x 10-4 M).
Majusculamide C Hapalosin Microviridin 0.044 0.084
Microviridin B Microviridin C Microcystilide A Micropeptin A Micropeptin B Micropeptin 90
0.071 0.025 2.0
0.026 0.035 0.1
None None
Compound A90nOA Cyanopeptolin A Cyanopeptolin S
0.01 <0.2 <0.2
< 1.0
<5.0
Cyanopeptolin SS
<0.2
< 1.0
<5.0
0.03
0.275
None None None (at 10)
None None None (at 10)
Cell-differentiation-promoting activityc (at 0.5 mg/ml) Weak cytotoxicity (IC50 0.5 mg/ml) None (at 10) Daphnia toxicity: None « 11.5 !-tg/ml) Daphnia toxicity: 60% at 7.5 !-tg/ml
• Against the human carcinoma cell lines KB and LoVo, respectively (Trimurtulu et aI., 1994) b Multidrug-resistance (MDR)-reversing activity (Stratmann et aI., 1994) C In a human leucocyte (HL-60) and against HCT116 and HCTVP35 cell lines (Tsukamoto et aI., 1993) For references: Cryptophycin A group (Trimurtulu et aI., 1994); majusculamide C (Carter et aI., 1984); hapalosin (Stratmann et aI., 199.4); microviridin (Ishitsuka et aI., 1990); microviridin Band C (Okino et aI., 1995); microcystilide A (Tsukamoto et aI., 1993); micropeptins A und B (Okino et aI., 1993b); micropeptin 90 (Ishida et aI., 1993); compound A90nOA (Lee et aI., 1994); cyanopeptolin A Uakobi et aI., 1995a); cyanopeptolin Sand cyanopeptolin SS Uakobi et aI., 1995b)
crystallization and structural elucidation of a bovine trypsin-compound A90nOA complex at 1.9 A resolution by single crystal X-ray diffraction. The depsipeptide interacts tightly with trypsin in a non-covalent, substrate-like manner through hydrogen bonds, hydrophobic interactions and steric complementarity. It imitates the canonical conformation of the exposed binding loop of the so-called 'small' protease inhibitors using peptidal an nonpeptidal elements, whereby the Ahp unit (see Fig. 1) plays an essential role by determining the binding conformation of the inhibitor and preventing its dissociation by its transannular hydrogen bonds.
Are the cyclodepsipeptides obtained from marine eukaryotes of cyanobacterial origin? An Ahp unit-containing cyclic depsipeptide, dolastatin
13, has also been found in the shell-less marine mollusc
Dolabella auricularia (Pettit et aI., 1989). However, the finding of such depsipeptides in axenically grown cyanobacteria led later to the suggestion that the true producers
of dolastatin 13 were cyanobacteria (Ishida et aI., 1995), and Tsukamoto et al. (1993) stated: "There has been a persisting speculation that a number of interesting compounds - many of them are peptides - found in Dolabella are derived from dietary alga." Furthermore, dolastatin 11 from the same origin like dolastatin 13 (Pettit et aI., 1993) and arenastatin A from the sponge Dysidea aranaria (Kobayashi et aI., 1994) were similar to majusculamide C and to cryptophycins, respectively. It is of interest in this context, that motuporin, structurally very similar to the cyanobacterial hepatotoxin nodularin, has been found in a marine sponge and was strongly suggested to be a cyanobacterial product (de Silva et aI., 1992). Similar observations were made for scytophycin-type compounds found in both, marine sponges and freshwater cyanobacteria [(cited in Tsukamoto et al. (1993)]. Recently, the structurally similar oscillamide A and anabaenopeptins were isolated from Oscillatoria agardhii NIVA CYA 18 (Sano and Kaya, 1995) and Anabaena (los-aquae NRC 525-17 (Harada et aI., 1995), respectively. They had a high resemblance with keramamide A from Theonella sp. (Kobayashi et aI., 1991).
Cyanopeptolins
Concluding remarks An attempt was made herein to point out that cyanopeptolins and related depsipeptides from cyanobacteria may serve as biochemical and/or pharmacological tools. Knowledge of their mode of action is feasable now, since e.g. cocrystallization of serine protease inhibitor compound A90nOA with trypsin (Lee et al., 1994) and microcystin-LR with the catalytic subunit of the protein phosphatase 1 (Goldberg et al., 1995) have already been performed successfully. Cyanopeptolins and related depsipeptides may be more widely distributed in cyanobacteria than notized so far. It is notable that cyanopeptolins can be simultaneously produced together with microcystins (Martin et al., 1993; Harada et al., 1993; Jakobi et al., 1995a,b). These peptides may contribute to chemotaxonomy of cyanobacteria, whereby the possible origin of some depsipeptides found in marine eukaryotes has to be considered.
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Jurgen Weckesser, Institut fUr Biologie II, Mikrobiologie, Albert-Ludwigs-Universitat Freiburg, SchanzlestraBe 1, D-79104 Freiburg i.Br., Germany; Tel. +49-761-203-2638; Fax +49-761-203-2647; email: [email protected]