Introduction

The main focus in recent decades for pharmaceutical discovery from natural products has been on microbial sources (bacterial and fungal), dating back to the discovery of penicillin from the mould fungus Penicillium notatum in the first half of the twentieth century.1 The emphasis on terrestrial microbes has more recently been augmented by the inclusion of microbes from marine sources. The majority of new chemical entities introduced as drugs (60 and 75% in areas of cancer and infectious disease, respectively) in market during the period of 1981–2002 are of natural origin.2, 3

In recent years, the pharmaceutical industry has invested in high-throughput screening systems, genomics and bioinformatic tools, rational design and combinatorial chemistry for the discovery of new bioactive compounds. Despite of these technological advances, the number of new entities reaching the market has declined from 53 to only 26 within a time span of 9 years (1996–2005). Currently, the pharmaceutical industry has to spend around 500 million to 2000 million dollars, depending on the therapy or the developing firm, to bring a new drug (a drug with a new chemical entity) to market.4 As a result, there is a scarcity of new therapeutic drugs reaching the market. On the other hand, the prevalence of diseases such as cancer, human immunodeficiency virus (HIV)–acquired immune deficiency syndrome, hematological and autoimmune disorders is increasing rapidly.

Currently available drugs are effective against only one-third of the diseases as a result of increased antibiotic resistance in pathogens. Thus, identification of new biologically active compounds is urgently required for development of new drugs. To fulfill the demand for new therapeutic drugs and to decrease the average costs involved in development, scientist should consider screening organisms from overlooked microbial sources, such as proteobacteria, bacterioidetes and cyanobacteria. The medicinal qualities of cyanobacteria were first recognized in 1500 BC when Nostoc sp. were used to treat gout, fistula and several forms of cancer5 but the most extensive modern work in this field was started in the 1990s by RE Moore and WH Gerwick.

Cyanobacteria

Cyanobacteria, a group of Gram-negative photoautotrophic prokaryotes, are capable of performing oxygenic photosynthesis. This unique class of microorganisms has many names, like cyanoprokaryotes, cyanophytes and blue–green bacteria. These names are derived because of the presence of a blue–green colored pigment, c-phycocyanin (C-PC), which is a pigment used for photosynthesis. These bacteria have a fossil record from 3.3 to 3.5 billion years,6 and are still among the most successful organisms on earth. They have a typical prokaryotic cell organization but like eukaryotes have an elaborate system of internal membranes responsible for both respiratory and photosynthetic electron transport. They have unique food storage compounds, myxophycean starch and cyanophycin. They accomplish water-oxidizing photosynthesis by using both Photosystem I and Photosystem II (Z scheme), but under anaerobic conditions they are capable of using only Photosystem I like purple bacteria. They use same electron transport machinery for both photosynthesis and respiration. Although photoautotrophy is the major mode of nutrition in cyanobacteria,7 some species exhibit a photoheterotrophic mode8 by utilizing glucose as a carbon and energy source in the dark. The morphology of cyanobacteria varies from unicellular to filamentous or colonial forms. The colonies are often surrounded by a mucilaginous or gelatinous sheath, depending on environmental conditions. Nostoc, a filamentous cyanobacterium may produce spherical colonies as much as 3 or 4 cm in diameter. Some of the filamentous cyanobacteria have three types of cells namely: vegetative cells, climate-resistant akinetes and thick-walled heterocysts. Cyanobacteria inhabit almost all the habitats on earth; from bare rock to soil and from water to air. Although they are in the main free living, symbioses of some species with plant and animal species are not uncommon.

Cyanobacteria have long been known for their ecological and agricultural impact; that is, as the primary colonizers of an ecosystem, their ability to fix atmospheric nitrogen and solubilize phosphates. In recent years, significant emphasis has been given to cyanobacterial biotechnology.9 Worldwide attention is drawn toward cyanobacteria for their possible role to help and harm humankind. Nowadays, these organisms are under consideration as an alternative source of energy because of their capabilities for generating hydrogen and ethanol. Products from some species (Aphanizomenon flos-aquae and Arthospira platensis) are available as supplements to provide a protein-rich diet. During recent decades, cyanobacteriologists have started to pay attention to the bioactivity of cyanobacteria.10, 11 A literature study of Journal of Natural Products (January 2007–October 2008), revealed the discovery of 38 new compounds from cyanobacteria among which 8 are antiprotozoal, 7 are antibacterial, 2 are antiviral, 6 are cytotoxic, 7 are protease inhibitors, 1 is a Ca2+ channel inhibitor and the remainder (7) show no bioactivity (Figure 1). The high degree of diversity in the bioactivities of cyanobacteria is due to the broad spectrum of its secondary metabolites. As cyanobacteria do not produce a particular class of chemicals, its secondary metabolite spectrum includes 40.2% lipopeptides, 5.6% amino acids, 4.2% fatty acids, 4.2% macrolides, 9.4% amides and others.12 The wide spectrum of cyanobacterial secondary metabolites can be explained on the basis of their ubiquitous occurrence and long evolutionary history. These metabolites exhibit a wide range of bioactivities that include some that may be relevant to the natural environment, such as antibacterial, antifungal, antiviral, cytotoxic, and others whose natural function may not be clear but may be important medicinally as anticancer agents, immunomodulators or protease inhibitors. Thus, these bioactive molecules simultaneously indicate the pharmaceutical potential of cyanobacteria.13, 14 Genome sequencing studies of cyanobacteria show significant diversity and novelty of genes responsible for bioactive proteins, ribosomal and non-ribosomal peptides, and peptide–polyketide hybrid molecules.15

Figure 1
figure 1

Bioactivity of cyanobacterial compounds. A full color version of this figure is available at the Journal of Antibiotics online

The presence of non-ribosomal peptide synthetase and polyketide synthetase genes reveal the potential for finding novel natural product drug leads from these organisms.16 It is well known that the non-ribosomal peptide synthetase–polyketide synthetase system produces a diverse family of compounds having biological and pharmacological properties; for example, the antibiotic—vancomycin, immunosuppressive agent—cyclosporine and anticancer agent—bleomycin.17 In this review, an attempt has been made to focus on the natural compounds from cyanobacteria, which have shown potent activity in vivo or in vitro, and have promise to be developed as therapeutic agents.

Anticancer activity

There is an urgent need of new anticancer drugs because tumor cells are developing resistance against currently available drugs, like vinca alkaloids and taxanes, which is thought to be a major cause of failure in the chemotherapeutic treatment of cancers. In addition, the incidence of new types of cancer such as gliobastoma is increasing rapidly. As a result, cancer is still among the leading cause of mortality worldwide. According to an estimate from the American Cancer Society,18 7.6 million people died from cancer in the world during 2007. The screening of cyanobacterial extracts for new anticancer compounds was initiated in the laboratory of Moore (Oregon State University) and Gerwick (University of Hawaii) in the 1990s. The following are few examples of promising anticancer cyanobacterial metabolites with established mechanisms of action (Table 1).

Table 1 Some anticancer compounds from cyanobacteria

Cryptophycins are potent anticancer agents produced by the cyanobacteria. Cryptophycin 1 was isolated from Nostoc sp. GSV224 in Moore's lab19 as anticancer agent. It has an IC50 of 5 pg ml–1 against KB human nasopharyngeal cancer cells, and 3 pg ml–1 against LoVo human colorectal cancer cells, and thus it was found to be 100–1000 times more potent than currently available anticancer drugs; for example, taxol or vinblastine. It also exhibits anticancer activity against adriamycin-resistant M 17 breast cancer and DMS 273 lung cancer cell lines. It is a highly potent suppressor of microtubule dynamics and blocks the cells in G2/M phase. There are several analogs of cryptophycin either naturally isolated or chemically synthesized. Cryptophycin-52 (LY 355073), a chemical analogue of cryptophycin 1, entered a clinical trial but produced only marginal activity.20 Currently two other analogues, cryptophicins 249 and 309, with improved stability and water solubility are being considered as second-generation clinical candidates.21 The enhanced efficacy of these compounds compared with that of Cr-52 (LY 355073) has empowered new efforts to move these compounds into clinical trials.22

Curacin A, first isolated from Lyngbya majuscula by Gerwick et al.,23 appeared to be a potent tubulin interactive compound but was so insoluble that its bioactivity could not be demonstrated in in vivo animal models. To improve on the solubility of natural curacin A, a number of soluble semi-synthetic derivatives have been generated using combinatorial chemical techniques. Currently, these compounds are undergoing preclinical evaluation as potential future anticancer drugs.

Dolastatin 10, which was originally isolated in very low quantity from the sea hare Dolabella auricularia, is actually a cyanobacterial metabolite, as confirmed by its direct isolation from a Symploca sp.24 It is a pentapeptide containing four unique amino acids, dolavaline, dolaisoleucine, dolaproline and dolaphenine. It is a potent antiproliferative agent with an ED50 of 4.6 × 10−5 μg ml–1. It binds to tubulin on the rhizoxin-binding site and affects microtubule assembly arresting the cell into G2/M phase. Unfortunately, in clinical tests, its phase II trial as a single agent was discontinued because of the development of peripheral neuropathy in 40% of patients and the lack of significant activity in patients with hormone refractory metastatic adenocarcinoma25 and recurrent platinum sensitive adenocarcinoma.26 An analogue of dolastatin 10, TZT-1027 (auristatin PE or soblidotin), which differs only in the absence of the thiazoline ring from the dolaphenine residue, was found to be effective in two human xenograft models, MX-1 breast carcinoma and LX-1 lung carcinoma in mice.27 It showed equivalent efficacy against both p53 normal and mutant cell lines.28 Bhaskar et al.29 demonstrated that a conjugate of auristatin with a monoclonal antibody directed to the adhesion molecule E-selectin inhibited the growth of prostate cancer cells by up to 85% in a mouse model.

Another member of the dolastatin family, dolastatin 15, is a linear peptide having an ED50 of 2.4 × 10−3 μg ml–1 against various cancer cell lines. It binds directly to the vinca alkaloid site on tubulin and blocks the transition into M phase. No clinical trials have been undertaken with this compound because of its structural complexity, low synthetic yield and poor water solubility. Cematodin (LU-103793), a water-soluble analogue of dolastatin 15, which has a terminal benzylamine moiety in place of dolapyrolidone, retains high cytotoxicity in vitro. It was found to be effective in a phase I trial for treatment of breast and other cancers by BASF Pharma (Varanasi, India), but a phase II trial was discontinued following unexpected results. Currently, ILX-651 (Synthadotin), a third-generation analogue with a terminal tert-butyl moiety in place of dolapyrolidone, has successfully completed a phase I clinical trial and a phase II trial has been recommended.30

Apratoxin A is a cyclodepsipeptide isolated from a Lyngbya sp. collected from Guam. This compound is cytotoxic to human tumor cell lines with IC50 values ranging from 0.36 to 0.52 nM in vitro,31 but showed only marginal activity against early stage adenocarcinoma in vivo. It induces G-1 phase cell cycle arrest and apoptosis.32

The photosynthetic pigment C-PC is reported to have various pharmacological characteristics including anti-inflammatory and anticancer activities because of its β-subunit. The recombinant β-subunit of C-PC has been demonstrated to have anticancer properties. Recombinant C-PC/β, tested on four different cell lines exhibited a high rate of proliferation inhibition and apoptotic induction. It has been shown that the recombinant protein interacts with membrane-associated β-tubulin and glyceraldehyde-3-phosphate dehydrogenase. In addition, the nuclear level of glyceraldehyde-3-phosphate dehydrogenase decreased significantly.33 These properties suggest that C-PC/β may have promise as a cancer prevention or therapy agent.

Tolyporphin from Tolypothrix nodosa exhibits a potent photosensitizing activity against tumor cells and is 5000 times more effective than the photodynamic treatment (photofrin II).34 Somocystinamide A (ScA) was isolated from the marine cyanobacterium Lyngbya majuscula. It is a pluripotent inhibitor of angiogenesis and tumor cell proliferation. It induces apoptosis in tumor and angiogenic endothelial cells. In vitro, picomolar concentrations of somocystinamide A are sufficient to disrupt proliferation and tubule formation in endothelial cells.35 In addition, there are numerous other anticancer cyanobacterial metabolites that have not been covered here as their mechanisms of action have yet to be determined.

Antiviral activity

The global spread of deadly viral diseases like HIV–acquired immune deficiency syndrome and dengue may have dramatic consequences. New potent and safe antiviral agents are urgently needed in this situation. Presently, the only approved anti-HIV treatment (HAART (highly active antiretroviral therapy) tri therapy), which is effective in controlling the progression of HIV infections has proved to be toxic, to induce strong viral resistance, and is unable to eradicate the causative viral agent.36 The following are three classes of cyanobacterial compounds with potent in vitro antiviral activity. Their structures are depicted in Table 2.

Table 2 Antiviral compounds isolated from cyanobacteria

Polysaccharides

The most significant antiviral cancer polysaccharides are spirulan and Ca-spirulan from Spirulina sp. These compounds from the extracts of cyanobacteria showed potent and broad-spectrum activity against HIV-1, HIV-2, H, influenza and a series of other enveloped viruses. They inhibit the reverse transcriptase activity of HIV-1 (like azidothymidine). These sulfated polysaccharides prevent virus-cell attachment and fusion with host cells. They also inhibit the fusion between HIV-infected and uninfected CD4+ lymphocytes, a mechanism that greatly enhances viral infectivity. These compounds have advantages as antiviral agents over other sulfated polysaccharides because of reduced anticoagulant properties.37 Also noteworthy is nostoflan, an acidic polysaccharide from Nostoc flagelliforme that exhibits potent virucidal activity against herpes simplex virus-1 (ref. 38).

Carbohydrate-binding proteins

Currently, two carbohydrate-binding proteins namely cyanovirin-N and scytovirin are being developed as potent virucidal drugs. These carbohydrate-binding proteins show antiviral activity by interfering with multiple steps in the viral fusion process.

Cyanovirin-N is an 101 amino acid long, 11 kDa polypeptide isolated from Nostoc ellipsosporum showing potent in vitro and in vivo activity against HIV and other lentiviruses in nanomolar concentrations.39 It interferes with the binding of HIV gp120 proteins with CD4+ receptors and the chemokine CCR5 or CXCR4 co-receptors of target cells, and thus, inhibits fusion of HIV virus with CD4 cell membrane. It is being developed as a vaginal gel for inhibiting the sexual transmission of HIV by Cellegy Pharmaceuticals, San Francisco, CA, USA. The development of a vaginal microbicide would be of major benefit for reducing the global spread of HIV-1 (ref. 40). It also inhibits herpes simplex virus-6 and measles virus in vitro.41

Scytovirin is a 95 amino acid long, 9.7 kDa polypeptide containing five intra chain disulphide bonds. It was first isolated from the aqueous extract of Scytonema varium.42 It binds to the envelope glycoprotein of HIV (gp120, gp160 and gp41) and inactivates the virus in low nanomolar concentrations. NMR analysis revealed that scytovirin has two domains and the first domain (a.a. 1–48) has similar anti-HIV activity to the full-length scytovirin.43

In addition, two cyclic depsipeptides, ichthyopeptins A and B, were also isolated from Microcystis ichthyoblabe. They show antiviral activity against influenza A virus with an IC50 value of 12.5 μg ml–1 (ref. 44).

Sulfoglycolipids

The natural sulfoglycolipids from cyanobacteria are also reported to inhibit HIV–reverse transcriptase and DNA polymerases.45

Antibacterial activity

If the incidence of bacterial resistance continues to rise, some diseases may become untreatable. Of late, the multi-drug-resistant bacteria causing nosocomial infections like methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococci and AmpC β-lactamase-producing Enterobacteriaceae have posed therapeutic challenges and are of great concern worldwide.46 Therefore, searches for new antibiotics to treat bacterial infections are urgently needed. In an effort to develop new antibiotics, scientists are screening cyanobacterial extracts for their antibacterial activity47, 48, 49 and found them potentially active against various bacteria. Unfortunately, very few antibacterial compounds from cyanobacteria have been structurally characterized to date (Table 3). Noscomin57 from Nostoc commune, showed antibacterial activity against Bacillus cereus (MIC 32 p.p.m.), Staphylococcus epidermidis (MIC 8 p.p.m.), Escherichia coli (MIC 128 p.p.m.) comparable to that of the standard drugs. Bhateja et al.57 reported the antibacterial activity of Anabaena extract against vancomycin-resistant S. aureus with an MIC of 32–64 μg ml−1. Carbamidocyclophanes are paracyclophanes isolated from Nostoc sp. CAVN 10. They show moderate antibacterial activity against Staphylococcus aureus.58 Raveh et al.50 isolated nine ambiguines from Fischerella sp., which showed antimicrobial activity. Ambiguine-I isonitrile showed more potent antibacterial activity than streptomycin against Bacillus subtilis and Staphylococcus albus. Recently, two new norbietane compounds, showing antibacterial activity against S. aureus, S. epidermidis, Salmonella Typhi, Vibrio cholarae, B. subtilis, B. cereus, E. coli and Klebsiela pneumoniae, were isolated from Micrococcus lacustris.51

Table 3 Some antibacterial compounds recently isolated from cyanobacteria

Antiprotozoal activity

According to an estimate of World Health Organization, more than one billion people throughout the world are suffering from tropical diseases caused by Plasmodium, Trypanosoma, Leishmania, Schistosoma and others.59 The failures in the treatment of these diseases, especially in cases of malaria60 and leishmaniasis,61 are due to development of resistance by these protozoa. On the other hand, progress in the advancement of drug discovery programs against these diseases is very slow.62 In an effort to encourage the development of effective and affordable treatment of these diseases, the Panamanian International Co-operative Biodiversity Group is screening extracts from terrestrial and marine sources. Recently, this project has reported the isolation of five classes of antiprotozoal compounds from cyanobacteria (Table 4). In addition, the protease inhibitor, nostocarboline,67 an alkaloid isolated from Nostoc sp. 78-12 A was also found to be active against Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani and Plasmodium falciparum with IC50 values ranging from 0.5 to 0.194 μM. Aerucyclamide C68 isolated from Microcystis aeruginosa PCC 7806 was also found to be active against T. brucei, and the already known aerucyclamide B against P. falciparum with submicromolar IC50 values. Clark et al.69 isolated six new acyl proline derivatives, tumonoic acids D-I, from the marine cyanobacterium Blennothrix cantharidosmum among which tumonoic acid I displayed moderate activity in an antimalarial assay (IC50 2 μM).

Table 4 Antiprotozoal compounds from cyanobacteria isolated in ICBG project

Protease inhibition activity

The discovery of new protease inhibitors may be of great pharmaceutical value. Jaspars and Lawton13 described some protease inhibitors of cyanobacterial origin, like microginins, aeruginosins and cyanopeptolins. Microginins are used in the treatment of high blood pressure. Serine protease inhibitors like cyanopeptolin are applied in the treatment of conditions such as asthma and viral infections. Some protease inhibitors isolated from cyanobacteria in the last 5 years are given in Table 5.

Table 5 Some protease inhibitors isolated from cyanobacteria

Recently, Taori et al.75 reported the isolation of two kempopeptins having protease inhibitory activity. Kempopeptin A, a cyclodepsipeptide from a marine Lyngbya sp. exhibited an IC50 of 0.32 μM against elastase and 2.6 μM against chymotrypsin, whereas kempopeptin B inhibited trypsin with an IC50 of 8.4 μM.

Immunomodulatory activity

Hayashi et al.76 investigated the effect of Spirulina in mice and reported increased phagocytic activity and increased antigen production in the test animals under study. Qureshi and Ali77 reported an increased phagocytic activity, increased antigen production and increased natural killer cell-mediated antitumor activity in chicken for the same cyanobacterium (Spirulina). In a preliminary small clinical study, an increase in 13.6-fold in interferon and 3-fold in interleukin (IL)-1β and -4 was observed in human blood cells incubated with Spirulina extracts.78 Khan et al.79 reported that different products prepared from Spirulina influence immune systems in various ways such as increasing the phagocytic activity of macrophages, stimulating antibody and cytokine production, increasing accumulation of natural killer cells into tissues and activating T and B cells. In a clinical study in Korea,80 a significant rise in plasma IL-2 concentration, and a significant reduction in IL-6 concentration in humans was observed after the consumption of Spirulina at home, 8 g day–1, for 16 consecutive weeks. Although Spirulina was found to be safe, other species of cyanobacteria contain metabolites, such as microcystin, that are cytotoxic to lymphocytes and inhibit membrane-bound leucine aminopeptidase.81 The latter appears to be linked to antigen-processing and antigen presentation response.82

The immunotoxicity of a cyanobacterial bloom extract containing microcystin was reported for the first time by Shen et al.83 Treatment with cyanobacterial bloom extract resulted in the inhibition of lipopolysaccharide-induced lymphoproliferation and the dose-dependent decrease in the numbers of antibody-forming cells in mice that were immunized by using T-dependent antigen sheep red blood cells. Thus, exposure to cyanobacterial bloom extract resulted in immunosuppression in mice. Shi et al.84 evaluated the effect of cyanobacterial bloom extract containing microcystins on the expression of multiple cytokines, including proinflammatory (IL-1β, tumor necrosis factor-α and IL-6) and Th1/Th2-related cytokines (IL-2, IL-4 and IL-10) in vivo. They observed the distinct patterns of expression of these cytokines suggesting a modulation of cytokine networks.

Conclusions

Cyanobacteria constitute a unique group of oxygenic photosynthetic bacteria and populate diverse habitats throughout the world. Their potential as a good source of new therapeutic lead compounds has been realized during the last two decades. We have discussed several bioactive molecules obtained from cyanobacteria showing a broad spectrum of activities, such as antitumor, antibacterial and antiviral effects and protease inhibition. Another advantage of cyanobacteria as a microbial source for drug discovery lies in the economy of their cultivation compared with other microorganisms, as they require only simple inorganic nutrients for growth. Thus, it seems that the cyanobacteria have the potential for expanded utilization in drug discovery.

Despite their potent biological activities, very few cyanobacterial compounds have entered clinical trials and no cyanobacterially derived compound has been approved by the Food and Drug Administration. In our opinion, the pharmaceutical potential of cyanobacteria deserves more scientific attention and interdisciplinary research. Further, to find novel compounds, cyanobacterial strains from still unexplored and extreme habitats should also be studied.