Phytochem Rev (2013) 12:459465 DOI 10.

1007/s11101-012-9237-5

Interkingdom signaling by structurally related cyanobacterial and algal secondary metabolites

Received: 31 October 2011 / Accepted: 12 May 2012 / Published online: 30 May 2012 Springer Science+Business Media B.V. 2012

Abstract Several groups of structurally-related compounds, comprised of either ve or six-membered ring structures with attached lipophilic carbon chains and in some cases possessing halogen atoms, have been isolated from various marine algae and lamentous cyanobacteria. The related compounds considered in the present work include the coibacins, laurenciones, honaucins, malyngamides and the tumonoic acids. Members of all of these compound families were assayed and found to inhibit the production of
L. Gerwick (&) P. Boudreau H. Choi S. Mascuch F. A. Villa K. L. Malloy W. H. Gerwick Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, 9500 Gilman Dr MC 0212, La Jolla, CA 92093, USA e-mail: lgerwick@ucsd.edu F. A. Villa Department of Chemistry, Western Arizona College, Yuma, AZ, USA M. J. Balunas Division of Medicinal Chemistry, Department of Pharmaceutical Sciences, University of Connecticut, Storrs, Manseld, CT, USA M. E. Teasdale D. C. Rowley Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, RI, USA W. H. Gerwick Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA

nitric oxide in lipopolysaccharides-stimulated macrophages, indicating their anti-inammatory potential. In addition, several of these same marine natural products were found to inhibit quorum sensing mediated phenotypes in Vibrio harveyi BB120 and/ or Escherichia coli JB525. The mechanism and evolutionary signicance for inhibition of these cellular processes in prokaryotic and eukaryotic systems are speculated on and discussed. Keywords Quorum sensing Anti-inammatory Lactone Acyl chain Marine natural products

Introduction Hundreds of marine natural products (secondary metabolites) have been isolated from cyanobacteria and macroalgae, making these phyla rich sources for new compound discovery. In particular, many of these natural products have been submitted for bioactivity screening and potential development as anti-cancer agents (Tidgewell et al. 2010). This emphasis on cancer could partially result from priorities set by the funding agencies and partially from the fact that many natural products are found to be cytotoxic (Nagle and Paul 1999; Nagarajan et al. 2012). However, many of the natural products isolated display other types of bioactivity, for example, quorum sensing inhibition, anti-microbial activity, anti-inammatory activity or inhibition or activation of neuronal receptors or ion

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channels (Choi et al. 2012; Villa et al. 2010; Mo et al. 2009; Li et al. 2001). Chronic inammation has been implicated as one of the causes underlying such common diseases such as cancer, arthritis, heart diseases, skin diseases, asthma and inammatory bowel disease (Adcock et al. 2008; Grivennikov et al. 2010). However, our supply of antiinammatory treatments is quite limited. At this point, the corticosteroids and the non-steroidal anti-inammatory drugs (NSAIDs) are the most commonly used; however, these options can have severe side effects. More small molecule anti-inammatory agents, which have different modes of action, are needed to treat chronic inammation, both as a disease prevention and a cost containment measure. Quorum sensing (QS) in bacteria is a concentration dependent process that involves intercellular signaling. Both Gram-negative and Gram-positive bacteria emit chemical signals that promote or disrupt their own cellular responses such as sporulation, swarming, bioluminescence, DNA transfer, biolm formation, production or repression of virulence factors and other secondary metabolites (Pappas and Winans 2003; Zhang et al. 2002; Ni et al. 2009; Ng and Bassler 2009). Molecules known as autoinducers regulate gene expression during these QS events. The activation of the QS physiological response is dependent upon the concentration of the autoinducer reaching a certain threshold, below which activity is not induced (Teng et al. 2011). Biolm formation and other QS related responses can increase the pathogenicity of certain bacteria, and thus, there is a strong interest in nding inhibitors of the QS response. For example, in cystic brosis (CF), colonization and QS-induced biolm formation of the lung by Pseudomonas aeruginosa leads to chronic pneumonia, a condition that has few effective treatments at present (Drenkard and Ausubel 2002). Biolm formation is also a persistent problem on the surfaces of indwelling catheters and QS inhibitors have been used to prevent these from developing (Thomsen et al. 2011). As exemplied above, QS inhibitors can be useful therapeutic agents that inhibit biolm formation without inhibiting growth, thus circumnavigating the pitfalls of developing antimicrobial resistance (Ni et al. 2009; Galloway et al. 2011). Several cyanobacterial and algal compounds have been identied in recent years that possess activity in both inhibition of QS as well as the production of nitric

oxide (NO) in macrophages. Some of the compounds that inhibited the NO production were also tested for potential anti-oxidant activity and none so far has exhibited this property, hence indicating an intracellular mechanism of action for these compounds. In comparing this set of anti-inammatory natural products, it is striking that they possess similar structural features comprised of either ve- or six-membered rings which have an attached hydrophobic carbon chain. Intriguingly, the Gram-negative QS modulating molecules, characterized as acyl homoserine lactones, are structurally similar to the cyanobacterial and algal compounds described in this review, and have also been shown to possess anti-inammatory properties (Telford et al. 1998; Kravchenko et al. 2008). As a result, this structure type has been identied as having inter-kingdom signaling activity. Below, we describe several cyanobacterial and algal metabolites which have similar structural features and biological properties as modulators of both eukaryotic inammation and bacterial quorum sensing.

Laurenciones Laurencione was rst isolated and characterized from the Oregon red alga Laurencia spectabilis (Bernart et al. 1991). Subsequently, Lowery et al. (2005) determined that laurencione could induce bioluminescence in the Vibrio harveyi MM30 mutant system [this mutant is unable to produce autoinducer-2 (AI-2)] indicating that laurencione can act as a ligand for LuxP. To further investigate the biological activity of laurencione, we synthesized the natural product as well as the mono and diacetate analogs (Fig. 1). The natural product and diacetate derivatives were prepared according to literature procedures, and matched 1H NMR, 13C NMR, and HR-MS data previously recorded (Bernart et al. 1991; Aelterman et al. 1997). The monoacetate was obtained as a minor byproduct of the laurencione one-step synthesis reported by Aelterman et al. in which glacial acetic acid replaced dioxane/water as the reaction solvent [yellow-green oil; IR (neat) vmax 2,970, 1,739, 1,718, 1,421, 1,366, 1,242, 1,090, 1,042, 909, 818, 580 cm-1; 1H NMR (500 MHz, CDCl3) 4.37 (t, J = 5.0), 3.06 (t, J = 5.0), 2.37 (s), 2.03 (s); 13C NMR (125 MHz, CDCl3) 196.8, 196.1, 171.1, 59.1, 35.5, 23.5, 20.9; HR-ESI-TOFMS [M ? MeOH ? Na]? m/z 213.0734 (calcd for C8H14NaO4 213.0733)].

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Using an assay that measures acyl-homoserine lactone (AHL) induced green uorescent protein (GFP) production by Escherichia coli JB525 (Anderson et al. 2001; Teasdale et al. 2009), a decrease in the uorescence signal by 50% was observed when *600 lM laurencione was added. In addition, the two acetate analogs, laurencione monoacetate and diacetate were found to be slightly more potent since they reduced uorescence in the JB525 strain (IC50 * 150 lM and *55 lM, respectively) (Table 1). Laurencione and the mono and diacetate analogs were also assayed for their potential anti-inammatory activity by measuring their ability to inhibit NO production in lipopolysaccharides (LPS)-stimulated macrophages following the methods described in Villa et al. (2010). All three of these compounds inhibited NO production with IC50 values ranging from 15 to 25 lM (Table 1).

Honaucins Honaucins AC were isolated from a bloom of the cyanobacterium Leptolyngbya crossbyana that overgrows corals off the coast of the Big Island in Hawaii. Honaucin A (Fig. 1) was found to inhibit the production of NO in the macrophage assay with an IC50 of 4.0 lM, making it one of the most potent natural product inhibitors that we have identied to date (Choi et al. in press). In addition, honaucin A potently inhibits the production of bioluminescence (IC50 5.6 lM) in the Vibrio harveyi BB120 system (Table 1). We speculate that honaucin B and C may be artifacts of the isolation process; however, both of these molecules inhibit quorum sensing at IC50 values of 17.6 and 14.6 lM as well as the production of NO with IC50 levels of 4.5 and 7.8 lM, respectively (Choi et al. in press).

Coibacins The coibacin series of natural products was isolated from a marine lamentous cyanobacterium (Oscillatoria sp.) collected near the island of Coiba on the Pacic coast of Panama. In total, four compounds were isolated, coibacin AD (Fig. 1). The coibacins consist of a six-membered lactone ring that is substituted at C5 with an unsaturated acyl chain that varies in length (Fig. 1). Coibacins A, B and D were not assayed using the Vibrio harveyi BB120 quorum inhibition assay (Teasdale et al. 2009) due to limited supply of these compounds; however, to our surprise coibacin C did not show any inhibition of bioluminescence in this assay system. The possibility exists that the coibacins could be activators of bioluminescence; however, the above mentioned assay utilizing the Vibrio harveyi MM30 assay is not currently available to us. Nevertheless, coibacins AD were found to inhibit NO production of LPSstimulated macrophages with IC50s of 20, 5, 11 and 21 lM, respectively (Balunas et al. in progress). The length of the acyl chain with its imbedded cyclopropyl ring appears to be important for modulating the level of activity in this series (Fig. 1). The coibacins are biosynthetically interesting compounds due to their variations in acyl chain length, number and location of double bonds, and terminal cyclopropyl rings versus vinyl chloride functionalities (Gu et al. 2009a, b).

Malyngamides The malyngamide series of natural products have been isolated from various marine lamentous cyanobacteria, mainly from the genus Moorea (formerly Lyngbya) (Engene et al. 2012). Most of these are composed of a fatty acyl chain attached to an oxidized cyclohexyl ring, and these appear to biosynthetically derive from a mixed Non-Ribosomal Peptide Synthetase (NRPS) and Polyketide Synthase (PKS) pathway. The rst malyngamide structure was isolated from Lyngbya majuscula in 1979, and since then, at least 28 malyngamide analogs have been isolated from locations as diverse as Hawaii, Curac ao in the Caribbean, Papua New Guinea, Florida, Puerto Rico and Madagascar (Cardellina et al. 1979; Kwan et al. 2010; Malloy et al. 2011; Nagarajan et al. 2012). Malyngamide C acetate, F, F acetate, H, I, J, K, L and T were each evaluated in the LPS-stimulated macrophage assay, but only malyngamide F (5.4 lM) and F acetate (7.1 lM) inhibited NO production with IC50 values in the low lM range (Fig. 1, Table 1)(Villa et al. 2010). The selective inhibition of NO production by just these two malyngamides revealed the importance of the hydroxy or acetoxy substituents at C-6 of the cyclohexenone ring [e.g. malyngamide K, which lacks a substituent at that position, showed no inhibition of NO (Villa et al. 2010)]. Changes in transcription of Interleukin 1 (IL1), Interleukin 6 (IL6), tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide

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462 Fig. 1 Structures of the natural products discussed

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synthase (iNOS) were determined in the RAW 264.7 macrophage cell line with and without exposure to malyngamide F acetate. All of these cytokine and inammatory protein transcripts were down-regulated except for TNF-alpha which was modestly upregulated. Further experiments determined that malyngamide F acetate inhibits the MyD88 dependent pathway (Villa et al. 2010). Of this series, only 8-epi malyngamide C has been assessed for quorum sensing inhibition; it was very modestly active in the E. coli JM109 pSB1075 assay system (IC50 * 1,000 lM)

(Kwan et al. 2010). However, further testing of malyngamides F and F acetate in various QS inhibition assays is needed to determine if these also act as interkingdom signaling molecules.

Tumonoic acid The rst tumonoic acids were isolated from a mixed assemblage of Lyngbya majuscula and Schizothrix calcicola collected at Tumon Bay, Guam, and through

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Phytochem Rev (2013) 12:459465 Table 1 Bioactivity levels of marine algal and cyanobacterial natural products in the antiinammatory and quorum sensing inhibition assays

463

Compound

MW

Inhibition of NO (IC50, lM)#

Inhibition of quorum sensing (lM) Vibrio harveyi BB120 5.6 17.6 14.6 * * * * * * *100 * 62 * * * Escherichia coli JHB525 38.5 908 576 * * Inactive * *612 *150 *55 * Inactive

Honaucin A Honaucin B Honaucin C Coibacin A Coibacin B Coibacin C Coibacin D Laurencione Laurencione monoacetate Laurencione diacetate Tumonoic acid A

204.6 250.1 236.0 284.4 258.4 266.8 268.8 102.1 158.2 200.2 339.2 525.3 439.0 481.0

4 4.5 7.8 20 5 11 21 25 15 18 9.8 * 5.4 7.1

* Yet to be assayed
# Inhibition of nitric oxide production in RAW 264.7 macrophages

Tumonoic acid F Malyngamide F Malyngamide F acetate

biological screening, found not to be cytotoxic. Five tumonoic acids were isolated in total, tumonoic acid A, B, C, methyl tumonoate A and methyl tumonoate B (Harrigan et al. 1999). The tumonoic acids, classied as acyl proline derivatives, structurally resemble the AHL found in many Gram-negative bacteria. Subsequently, Clark et al. (2008) isolated tumonoic acids D through I from a collection of Blennothrix cantharidosmum from Papua New Guinea. Because of their structural similarity to AHL, these natural products were evaluated for their ability to inhibit QS regulated bioluminescence using the Vibrio harveyi BB120 system. Modest inhibition was determined for all six of the compounds with tumonoic acid F being the most potent inhibitor (IC50 = 62 lM; Table 1). More recently, from a survey of the lamentous marine cyanobacteria found growing at different locations around the island of Curac ao, the tumonoic acid derivative ethyl tumonoate A was isolated. This derivative was found to inhibit NO production in the LPS-stimulated macrophages with an IC50 of 9.8 lM (Engene et al. 2011).

Conclusion Several different classes of marine compounds exhibiting structural similarities were isolated from cyanobacteria and a red alga. These compounds were assessed for their anti-inammatory activity and QS

antagonism. The laurenciones, the malyngamides, the honaucins, the coibacins and the tumonoic acids all consist of a ve or six membered ring that is highly oxygenated and possesses an acyl chain of varying length and possessing different substituents such as halogen atoms. These various natural product classes show structural resemblance to the AHLs, known QS signaling molecules that have been isolated from bacteria such as Vibrio and Pseudomonas sp. Some AHLs have also been shown to decrease cytokine production in mice (Kravchenko et al. 2008), hence indicating their anti-inammatory properties. However, it remains uncertain if the compounds isolated from cyanobacteria function as quorum sensors or inhibitors in their natural environment. Moreover, it is unknown if they naturally function as inhibitors of the innate immune system found in marine invertebrates. We speculate that for lamentous marine cyanobacteria and algae it might be of evolutionary advantage to produce a single molecule that can interact with both prokaryotic and eukaryotic life forms, thus being able to prevent biolm formation by competing microorganisms and at the same time down-regulate the innate immune system of marine invertebrates with which they may associate. For example on corals, these compounds might give settling and growth advantages to the cyanobacteria or algae. This hypothesis needs to be tested more rigorously by using puried compounds, bacteria common in the

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coral environment, coral larvae and perhaps coral coelomocytes. However, several of the described natural products show potent inhibition of QS mediated phenotypes and inhibition of an inammatory response. Some of these compounds are being further evaluated for their mechanism of action and in vivo efcacy because there exists a great need for developing biolm inhibitors as well as new classes of antiinammatory therapeutics.
Acknowledgments This research was partially funded by the International Cooperative Biodiversity grant (U01 TW006634), the Ledger Benbough Foundation to L.G, NIH/FIC International Research Scientist Development Award (IRSDA) to M.J.B., NIGMS Training grant in marine biotechnology to S.M., NIH/ NIGMS Institutional Research and Academic Career Development Award (IRACDA) fellowship to F.V. and an E.W Scripps Fellowship to P.B.

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