In particular, salinosporamide A is a novel rare bicyclic beta-lactone gamma-lactam isolated from an obligate marine acti- nobacterium, S. tropica (Feling et al., ...
chapter twenty-four
Bioactive compounds from actinomycetes and their antiviral properties Present trends and future prospectives Avilala Janardhan, Arthala Praveen Kumar, and Golla Narasimha Contents 24.1 Introduction......................................................................................................................... 479 24.2 Antiviral activity.................................................................................................................480 24.3 Screening methods............................................................................................................. 481 24.4 Viral entry inhibition assay............................................................................................... 482 24.5 Viral replication inhibition assay..................................................................................... 482 24.6 Anticancer activity..............................................................................................................483 References...................................................................................................................................... 483
24.1 Introduction The search for bioactive compounds in nature is a multistep procedure that begins with the selection of suitable sources and then the biological, chemical, or physical interactions of metabolites with test systems that are then qualitatively or quantitatively evaluated (Omura, 1992). This is called screening. Natural products are the organic molecules derived from primary or secondary metabolism of living organisms such as microorganisms. Among them, 50%–60% are produced by plants (alkaloids, flavonoids, terpenoids, steroids, and carbohydrates), and 5% have a microbial origin (Berdy, 2005). The natural products from plants (Han et al., 2007; Huang et al., 2008; Huo et al., 2008), fungi (Krohn et al., 2001; Lin et al., 2002; Wu et al., 2004; Gao et al., 2007), bacteria (Lin et al., 2008), and actinomycetes (Xie et al., 2006; Tang et al., 2007) are the most anti-infectious, anticancer, antibacterial, antiviral, anti-inflammatory, antimalarial, and antidiabetic drugs on the market today. The increasing role in the production of natural products such as antibiotics and other drugs for treatment of serious diseases has been dramatic, but the development of resistance in pathogens and tumor cells has become a major problem and requires much research effort to screen it. The basic premises of a screening program are as follows: (1) drugs operate in a dose–response manner and produce toxicity in higher doses; (2) each class of drug has a characteristic dose–response profile; (3) for the majority of drugs, route of administration produces only a quantitative change in action; (4) absolute potency is not of major importance in therapeutics; and (5) it is possible to predict usefulness and toxicity of a new compound by utilizing a dose–response spectra library of various prototype drugs. The criteria of a good screening program are that it should be simple, economical, reliable, able to pick up new unexpected or unique activity, unbiased, 479
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and comprehensive (Irwin, 1962; Lucas and Lewis, 1944; Taylor et al., 1952; Laurence and Bacharach, 1964; Turner, 1965; Mantegazza and Piccinni, 1966; Turner and Hoborn, 1971; Dhawan and Srimal, 1984, 1992; Kamboj and Dhawan, 1989). Viruses cause many important diseases in humans, with viral-induced emerging and reemerging infectious diseases representing a major health threat to the public. In addition, viruses can also infect livestock and marine species, causing huge losses of many vertebrate food species. Effective control of viral infection and disease has remained an unachieved goal, due to the virus’ intracellular replicative nature and readily mutating genome, as well as the limited availability of antiviral drugs and measures. In relation to infectious diseases, the exploration of the marine environment represents a promising strategy in the search for active compounds, whereas there is a need for new medicines, due to the appearance of resistance to available treatments in many microorganisms, specifically concerning antiviral activities. Among the microorganisms, the actinomycetes are the gram-positive bacteria belonging to the order Actinomycetales, which play a significant role in the production of new metabolites (Goodfellow et al., 1988; Demain, 1995). Especially, the Streptomyces and Micromonospora strains have proven to produce novel antibiotics (Omura et al., 2001; Watve et al., 2001; Bentlley et al., 2002). The screening of microbial natural products leads to the discovery of novel chemicals for the development of new therapeutic agents (Bull et al., 2000). So, it is necessary to continue the screening for new metabolites and evaluate the potential of less-known and new bacterial strains so that the new and improved compounds for future use against drug-resistant bacteria or for chemical modification purposes may be developed (Kurtboke, 2005).
24.2 Antiviral activity Some compounds have been used for testing antiviral activity in our laboratory. Marine antiviral agents (MAVAs) (Fujioka and Loh, 1996) can be used for the biological c ontrol of human enteropathogenic virus contamination and disease transmission in sewagepolluted waters, as chemotherapy for viral diseases of humans and lower animals, as well as the biological control of viral diseases of marine animals. The seeding of MAVAs under natural conditions, or when marine mammals are kept in captivity for various uses, could control viral disease transmission within these select populations. It is clear that the marine environment will play a vital role in the future development and trials of antiinfective drugs. The purpose of this study was to establish an in vitro model to screen marine extracts for antiviral activity and to evaluate some marine extracts for their antiviral potential, with a long-term goal of discovering new marine compounds to be used as potential antiviral drug candidates. Viruses cause many diseases in animals; effective control of viral diseases and infections has remained an unachieved goal due to virus intracellular replicative nature and readily mutating genome. Due to the limited availability of antiviral drugs, the use of natural products as drugs was well established. Different studies were conducted to determine the effectiveness of the natural products. Ager (1984) had done experiments on 25 isolates of actinomycetes, which were given as feed for cultured shrimp and tested for their ability to reduce the white spot syndrome virus (WSSV) infection in shrimp. Among these 25 isolates, 6 isolates have shown to be most potential. The pentalactones are extracted from the fermentation broth of Streptomyces sp. M-2719 has been reported to be active against several DNA viruses (Kumar et al., 2006). Researchers have reported that guanine-7-Noxide produced by Streptococcus sp. was found to inhibit in vitro replication of fish herpes virus, rhabdovirus, and infectious pancreatic necrovirus (Nakagawa et al., 1985).
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Chapter twenty-four: Bioactive compounds from actinomycetes and their antiviral properties 481 The antibiotic SF 2487 from Actinomadura sp. was found to exhibit antiviral activity against influenza virus in vitro (Hasobe et al., 1985). A Streptomyces sp. isolated from Brazilian tropical forest soil possessed antiviral activity against herpes simplex virus type 1 (HSV-1) on HEP-2 cells (Hatsu et al., 1990). An antibiotic enriching from Streptomyces lavendulae showed inhibition of influenza A and influenza B virus in vitro (Sacrament et al., 2004). Current antiviral drugs comprise of over 40 compounds that have been officially approved for clinical use. Among these drugs, half of them were used to treat HIV infections (Bhakuni et al., 1990; Schaeffer and Krylov, 2000; Tziveleka et al., 2003; Mayer and Hamann, 2005). MAVAs were used for biological control of human enteropathogenic virus contamination and disease transmission in sewage-polluted water (Fujioka and Loh, 1996). MAVAs represent a significantly unique natural marine resource whose multipotential uses include the following applications: (1) One is the biological control of human enteropathogenic virus contamination and disease transmission in sewage-polluted waters. This application would be particularly important to communities that utilize the coastal waters for recreational activities and for food industries (e.g., fish, shellfish), as well as to those regions of the country, such as Hawaii, where the loss of these marine resources would have a devastating effect on the lifestyle and economy of the people. (2) The other one is the chemotherapy of viral diseases of humans and lower animals. To be of practical use, it is imperative that MAVAs are isolated from pure cultures, identified, and characterized. Their spectrum and mechanism of antiviral activity should also be clearly established. Their active principle and moieties should be identified and chemically characterized in order to facilitate application of biotechnological methods for increased yields and costeffective production. Currently, it appears that there have been only a few compounds derived from marine actinobacteria with antiviral activity. Benzastatin C (56), a 3-chloro-tetrahydroquinolone alkaloid obtained from Streptomyces nitrosporeus, showed antiviral activity in a dosedependent manner with EC50 values of 1.92, 0.53, and 1.99 g/mL against HSV-1, HSV-2, and vesicular stomatitis virus, respectively (Lee et al., 2007). Kumar et al. (2006) reported the antiviral property of a marine Streptomyces against WSSV in penaeid shrimp. WSSV infection can cause cumulative mortality up to 100% within 3–10 days, thereby causing considerable economic loss to the shrimp farmers.
24.3 Screening methods It is suggested that the potential antiviral agents must be screened in a living cell or animal host. Testing for antiviral activity is usually performed in cell culture or embryonated chicken eggs and animal models. In vitro antiviral testing using cell cultures involves the virus of interest and a primary or permanent cell line that can support its multiplication. The cells are infected with the virus, or already viral-infected cell lines are exposed to the extracted compound. If the compound has antiviral activity, the multiplication of the virus will be inhibited, which will be evident from the morphology of the cell monolayer. It is important to assess the toxic effect of the test substance on cells at each dilution. This can be done by examining the uninfected cell monolayers exposed to the extracted compound only. From the observed ED50 and LD50 of the compound, its therapeutic index is calculated. Several viral targets are studied to estimate the antiviral effect of compound in a cell culture system. Some of these are viral DNA polymerase activity, ribonucleotide diphosphate reductase, mRNA polyadenylation and RNA-dependent RNA polymerase, terminal deoxynucleotidyl transferase, thymidine kinase, uracil-DNA glycolase, d-UTPase, and reverse transcriptase. Testing for antiviral activity in chicken eggs is very simple.
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Here, prophylactic and therapeutic assays may be carried with different test substances since a wide choice of routes and timing of application of both virus and antiviral agents is possible. There are three main routes by which the bioactive compound could be administered into embryonated eggs: allantoic cavity inoculation, amniotic cavity inoculation, and chorioallantoic membrane inoculation. The virus and the compound may be given through different routes; it depends on the type of virus and the compound. The test substance can be given before, along with, or after virus infection. Testing in animal models has relatively the maximum predictive value among the various methods employed for detecting antiviral activity. Testing in these model systems can identify both antiviral activity and antiviral agents. The ideal animal model should have three features: (1) use of a human virus with minimal alteration by adaptation; (2) use of the natural route of infection and size of inoculum as in humans; and (3) similarity of infection, pathogenesis, host response, drug metabolism, and drug toxicity. Animal models exist for both local and systemic virus infections. Antiviral activity of a test substance can also be assessed by titrating the virus in blood and other target organs. The details of these models are described (Bhakuni et al., 1990).
24.4 Viral entry inhibition assay Cells at exponential growth phase were harvested and seeded into multiwell plates at densities that would allow the formation of an approximately 90% cell monolayer overnight. Marine extracts were diluted with serum-free medium to twice the effective safe concentrations, as determined by the cytotoxicity tests. A 250 μL solution of each extract at twice the maximum nontoxic concentration (e.g., 200 μg/mL for those found to be nontoxic at 100 μg/mL) was mixed with an equal volume of the virus dilution. Positive controls were made by mixing 250 μL of virus dilution with 250 μL of serum-free medium with 0.2% DMSO, in order to yield a final DMSO concentration of 0.1%. The 500 μL virus/ extract mixtures were preincubated for 1 h, along with controls, and then assayed for viral infectivity using the optimized plaque assay protocols. Antiviral effect of each extract was categorized as having no meaningful inhibition (
