Antiviral and Antiparasitic secondary metabolites from Actinobacteria

1. Antiviral and Antiparasitic secondary metabolites from Actinobacteria Under the guidance of Dr D.Dhanasekaran Submitted by Rubini .V Reg.No.10MIC1521 II M.Sc.,Microbiology

2. Introduction Actinobacteria play a significant role in biotechnology, because of the important natural producers. The diversity of actinobacteria secondary metabolites is unrivaled and unmatched medical significance(50%of the antibiotics). Marine actino bacteria to synthesize diverse compounds It will inevitably focus on the intensive research. Thispresent review describe the antiviral and antiparasitic compounds from actinnobacteria.

3. Antiviral compounds

4. Screening of antiviral compounds- Antiviral assay • Vero cells were grown in minimum essential medium (MEM) supplemented with 5% bovine serum. • HSV-1 strain F and HSV-2 Strain G. Vero cell viability was measured by the MTT method. • The CC50 (cytotoxic concentration 50%) was calculated as the compound concentration required to reduce cell viability by 50%. • Antiviral activity was evaluated by reduction of virus plaque formation.

5. Plaque assay • The cell lines were cultured and then seeded into multiwell plates. • The serial 10-fold dilutions of stock virus were made and 100 μl/well of each viral dilution were added to the plates. • Plates were incubated for 1 hour, then inoculum’s from each well was completely removed . • Plates were then incubated for3-4 days to allow viral plaque development. • Viral plaques were visualized by the addition of 2 ml/well of crystal violet staining solution for at least 2 hours.

6. Cytotoxicity assay • Viral attachment/entry inhibition assay • Viral replication inhibition assay • Plaque assay

7. Cell lines are used for screening of antiviral activity

8. Macrolide FK506 The antiviral potential of the macrolide FK506, produced by Streptomyces tsukubaensis (Reis et al 2006). Cell lines were infected with different Orthopoxviruses and treated with FK506(Castro et al. 2003). The macrolide inhibited the replication of the prototypic Orthopoxvirus, vaccinia virus strain WR, with an IC50 of 12.05µmol/1 ). Progeny production of other Orthopoxviruses was also inhibited by FK506 at noncytotoxic concentrations, as evaluated by the neutral-red uptake assay and metabolic labelling of cellular proteins by Western blot assay,

9. Poly peptide NP-06 • Polypeptide NP-06, produced by Streptomyces sp, which is active against human immunodeficiency virus (HIV) in vitro. NP-06 is a hydrophobic 21-mer oligopeptide, NP-06 appears to block the early stage of HIV-1 infection. • It most likely at the stage of virus-cell fusion. • NP-06 blocked the replication of a primary HIV-1 isolate, HIV- 1ERS104pre (11), with an IC50 and an IC90 of 1.and3.9 mM, respectively.

10. Bripiodionen • Bripiodionen, a new natural product, was isolated from Streptomyces sp. WC76599, (Shu et al.,1997). • During the screening of microbial fermentation extracts for their ability to inhibit human cytomegalovirus protease. • The structure of Bripiodionen was elucidated by spectroscopic methods. • Compound Bripiodionen displayed inhibitory activity against human cytomegalovirus protease with an IC50 value of 30 µM.

11. Streptomyces Derived substance EM2487 • EM2487, a Streptomyces-derived substance, selective inhibitor of human T lymphotropic virus type I replication in persistently infected cells (Wang et al.,2002 ). • Its 50% effective concentrations for HTLV-I p19 antigen production were 3.6 and 1.2 μM in MT-2and MT-4 cells, respectively. • The 50% cytotoxic concentrations of EM2487 were 30.6 and 5.7 μM in MT-2 and MT-4 cells, respectively. • The compound also displayed selective inhibition of HTLV-I production in peripheral blood mononuclear cells obtained from patients.

12. Geldanamycin • Geldanamycin (GA) is an antibiotic produced by Streptomyces hygroscopicus. • It targeting the ADP/ATP binding site of heat shock protein 90 (Hsp90). GA active against HSV-1 (Huan Li et al.,2004). • HSV-1 replication in vitro was significantly inhibited by GA with an 50% inhibitory concentration of 0.093µ M . • It inhibited cellular growth 50% in comparison with untreated controls of 350 µM. • GA exhibits broad-spectrum antiviral activity. GA new antiviral agent for HSV-1

13. Antiviral compounds from actinobacteria

14. Antiparsasitic compounds • Diseases caused by tropical parasites affect hundreds of millions of people worldwide. • It have been largely neglected for drug development because they affect poor people in poor regions of the world. • Most of the current drugs used to treat these diseases are decades old and have many limitations, including the emergence of drug resistance. • These neglected diseases present unique challenges to drug development.

15. Screening of antiparasitic compounds • Antiplasmodial Activity Assay • Nematicidal activity • Hatching inhibition analysis • Toxicity assay • parasite proliferation assays in P. falciparum

16. Ivermectin • Avermectins produced by Streptomvces avermitilis. Ivermectin (dihydroavermectin BI) is used in practice. • It is a potent antiparasitic compound active against a broad spectrum of nematode and arthropod parasites (Ikeda and Omura1995;MacNeil 1995). • It shows potent microfilaricidal activity against the major filarial parasites of humans, Wuchereria bancroftii, Brugia mahoi, Loa loa and Mansonella ozzardi. • it is as effective as currently available drugs against the intestinal nematodes Ascaris lumbricoides, Trichuris trichiura and Enterobius vermicularis. • It against the human hookworms, it shows only partial efficacy.

17. Antimalarial β-Carboline and Indolactam Alkaloids • Four new β-carboline alkaloids, designated marinacarbolines A−D , were obtained from the fermentation broth of Marinactinospora thermotolerans SCSIO 00652 • It is a deep Sea Isolate, a new actinomycete belonging to the family Nocardiopsaceae (Hongbo Huang et al., 2011). • It exhibited antiplasmodial activities against Plasmodium falciparum lines 3D7 and Dd2, with IC50 values ranging from 1.92 to 36.03 μM. M. thermotolerans. • The new compounds were inactive against a panel of eight tumor cell lines (IC50 > 50 μM)

18. Antimycin A9 • A new antimycin group antibiotic, antimycin A9, was isolated from a cultured broth of Streptomyces sp. K01-0031 • It have aromatic 8-acyl residue. • It showed potent nematocidal and insecticidal activities against Caenorhabditis elegans and Artemia salina, respectively (K. Shiomi et al., 2005). • Compound 1 showed slightly more potent activity than other known antimycins. Compound 8 was only weakly active against A. salina. • Minimum inhibitory concentraction 0.05 0.2 A. salina C. elegans

19. Fervenulin • Fervenulin produced byStreptomyces sp. CMU-MH021 • It inhibited egg hatch and increased juvenile mortality of the root-knot nematode Meloidogyne incognita in vitro (Lumyong et al., 2011). • Nematicidal activity of fervenulin was assessed using the broth microdilution technique. • The lowest minimum inhibitory concentrations (MICs) of the compound against egg hatch of M. incognita was 30 lg/ml and juvenile mortality of M. incognita increasing was observed at 120 lg/ml. • The concentration of 250 lg/ml fervenulin showed killing effect on second-stage nematode juveniles of M. incognita after 96 h incubation.

20. valinomycin ,staurosporine and butenolide • Streptomyces sp. strains from Mediterranean sponges, screening for antiinfective activities. • Three known compounds namely, cyclic depsipeptide valinomycin, indolocarbazole alkaloid staurosporine and butenolide (Sheila et al.,2010). • These compounds exhibited novel anti-parasitic activities

21. Antiparasitic compounds from actinobacteria isolates

22. Conclusion • Substantially increased interest in natural products, especially those originating from marine organisms,. • It potential of marine bacteria to synthesize diverse compounds will inevitably bring marine actinbacteria into the focus of intensive research. • The fact that actinobacteria are very diverse, and each species might have special requirements not only for growth, but also for the production of secondary metabolites. • Its major advantage is that, if successful, it will result in the production of a compound which structure, novelty and biological activity can be assessed straight away.

23. However, this cultivation-dependent technology is unlikely to reveal the full biosynthetic potential. The second approach, genome-based bioprospecting, Its success will depend on such factors as development of efficient tools for bioinformatic analysis of the genomes allowing identification of unique biosynthetic gene clusters. It seems likely that combination of these approaches may result in establishment of robust pipelines for drug discovery from marine actinobacteria in the near future.

24. Thank u for ur kind attention