Streptomyces are a major group of soil bacteria that produce wide range of bioactive compounds including antibiotics. Daunorubicin is a chemotherapeutic agent for treatment of certain types of cancer, which is produced as a secondary metabolite by S. peucetius. Owing to the significance of this drug in treating cancer, understanding the molecular mechanism of its biosynthesis will assist in the genetic manipulation of this strain for better drug yields. Additionally, the knowledge can also be applied to design hybrid antibiotics that can be made in vivo by transferring genes from one Streptomyces species to another. Biosynthesis of daunorubicin in S. peucetius is accomplished by the function of 30 enzyme-coding genes in a sequential and coordinated fashion. In addition to these enzymes, three transcriptional regulators DnrO, DnrN and DnrI regulate this multi-step process by forming a coherent feed forward loop regulatory circuit, consequently controlling the entire enzyme coding genes. Since daunorubicin is a DNA intercalating drug, maintaining an optimal intracellular drug concentration is pivotal to prevent self-toxicity. Commencement of daunorubicin biosynthesis also activates the feedback mechanisms mediated by the metabolite. At exceeding intracellular concentrations, daunorubicin intercalates into DNA sequences and impedes the binding of these transcription factors. This feedback repression is relieved by a group of self-resistance genes, which concurrently efflux the excess intracellular daunorubicin. This review will discuss the mechanistic role of each transcription factor and their interplay in initiating and maintaining the biosynthesis of daunorubicin in S. peucetius.

Streptomyces peucetius self-resistance genes drrA and drrB encode membrane-associated proteins that function like an ABC transporter for the efflux of daunorubicin and to maintain a constant subinhibitory physiological concentration of the drug within the cell. In this study, the drrA and drrB operons were disrupted for investigating drug production, self-resistance and regulation. The drrA-drrB null mutant was highly sensitive to daunorubicin. A 10-fold decrease in drug production was observed in the null mutant compared with the wild-type strain. We propose that the absence of a drug-specific efflux pump increases the intracellular concentration of daunorubicin, which is sensed by the organism to turn down drug production. Quantitative real-time PCR analysis of the mutant showed a drastic reduction in the expression of the key regulator dnrI and polyketide synthase gene dpsA. However, the expression of regulatory genes dnrO and dnrN was increased. Feedback regulation based on the intracellular daunorubicin concentration is discussed.

DrrC is a DNA-binding protein of Streptomyces peucetius that provides self-resistance against daunorubicin, the antibiotic produced by the organism. DrrC was expressed in E.coli and purified by using N-terminal MBP-tag which retained DNA-binding property in spite of the tag. Mobility shift assay confirmed the interaction of 313bp DNA that has the dnrI promoter, daunorubicin and MBP-DrrC in the presence of ATP. Biotinylated and immobilized 313bp DNA was intercalated with daunorubicin to observe the release of the drug when MBP-DrrC is allowed to act on the DNA. The release of daunorubicin was recorded by absorption and fluorescence spectroscopy. The experiments proved that daunorubicin was released from DNA in the presence of MBP-DrrC. Fluorescence emission of daunorubicin had a maximum peak at 591nm. However, emission spectrum of released daunorubicin showed hypochromism with a maximum peak at 584nm that is possibly because it is in complex with MBP-DrrC. We propose that DrrC naturally binds at intercalated sites to eject daunorubicin; in the process both drug and protein are dislodged from DNA. Like UvrA, DrrC possibly scans the DNA for intercalated daunorubicin. When it encounters daunorubicin, DrrC dislodges it, thereby allowing DNA replication and transcription to go on unhindered. Thus a novel self resistance mechanism by DNA repair is mediated by DrrC.

DnrO is a transcription factor that regulates biosynthesis of secondary metabolite daunorubicin (DNR) in Streptomyces peucetius. DNR is a DNA-intercalating drug widely used in cancer chemotherapy. Binding of DnrO close to its promoter fulfils dual functions, namely activation of dnrN and repression of dnrO. DnrN protein binds to a sequence close to the dnrI promoter to activate it, which is essential for turning on biosynthetic genes. In this study, we analyzed the inhibition of DNA-DnrO complex formation by DNR and its effect on dnrO and dnrN expression. The intracellular concentration of drug required to alter the expression of these two genes was determined in vitro. Based on the results, a model is proposed which describes the modulation of dnrN and dnrO expression by intracellular stoichiometric concentration of the drug DNR and protein DnrO. This regulatory mechanism would maintain optimal intracellular drug concentrations in S. peucetius. This would imply that the organism has an adaptive mechanism to escape the cytotoxicity of DNR in addition to its self-resistance.

BackgroundNeuraminidase (NA) is a prominent surface antigen of Influenza viruses, which helps in release of viruses from the host cells after replication. Anti influenza drugs such as Oseltamivir target a highly conserved active site of NA, which comprises of 8 functional residues (R118, D151, R152, R224, E276, R292, R371 and Y406) to restrict viral release from host cells, thus inhibiting its ability to cleave sialic acid residues on the cell membrane. Reports on the emergence of Oseltamivir resistant strains of H1N1 Influenza virus necessitated a search for alternative drug candidates. Pleconaril is a novel antiviral drug being developed by Schering-Plough to treat Picornaviridae infections, and is in its late clinical trials stage. Since, Pleconaril was designed to bind the highly conserved hydrophobic binding site on VP1 protein of Picorna viruses, the ability of Pleconaril and its novel substituted derivatives to bind highly conserved hydrophobic active site of H1N1 Neuraminidase, targeting which oseltamivir has been designed was investigated.Result310 novel substituted variants of Pleconaril were designed using Chemsketch software and docked into the highly conserved active site of NA using arguslab software. 198 out of 310 Pleconaril variants analyzed for docking with NA active site were proven effective, based on their free binding energy.ConclusionPleconaril variants with F, Cl, Br, CH3, OH and aromatic ring substitutions were shown to be effective alternatives to Oseltamivir as anti influenza drugs.

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Daunorubicin forms specific complex with an extracellular protease in the Streptomyces peucetius culture. The drug-protein complex co-migrates in non-denaturing PAGE as a red band. De novo peptide sequencing by nano-LC-ESI-MS/MS and MASCOT analysis identified the daunorubicin binding protein as serine protease precursor. The same protease precursor was purified sans the daunorubicin, from the mutant named ΔDPSAmut, which is deficient in daunorubicin production. Daunorubicin was added to ΔDPSAmut culture and the protease readily formed the daunorubicin-protease complex. Ability of serine protease precursor to form a selective complex with daunorubicin was confirmed by this study. Selective binding of protease to daunorubicin was seen as self-resistance determinant for the organism to survive toxic levels of the drug outside the cell. Daunorubicin-protease complex placed on S. peucetius lawn did not produce clearing zone around it, whereas daunorubicin purified from the complex did produce the clearing zone. Thereby it is concluded that the protease sequesters daunorubicin to prevent its entry into cells. Sequestration of daunorubicin by extracellular protease helps the organism to maintain a steady state sub-inhibitory level of drug around the cells. A new self-resistance determinant is reported here.