Characterization of a  protease inhibitor PISC-2002 wit h anti-influenza virus activity  isolated from  Streptomyces chrom...
The Protease Inhibitors - what are they? <ul><li>Diverse group of proteins classified according their structure and protea...
Influenza Virus Morphology <ul><li>envelope glycoproteins: </li></ul><ul><li>hemagglutinin (HA) and neuraminidase (NA)  </...
Influenza A serotypes <ul><li>Influenza A viruses  infect a wide variety of hosts: humans, horses, pigs, ferrets and birds...
Influenza Virus Replication www-micro.msb.le.ac.uk/3035/Orthomyxoviruses.html
Genus  Streptomyces unique mycelial, sporulating life cycle   products with broad medical application Protease Inhibitors ...
Cell toxicity and virus-inhibitory effects of cell-free extracts from  Streptomyces  on human (H3N2) and bird (H7N1) virus...
Purification of PISC-2002 Ammonium Sulphate Precipitation (80%, 24  h , 4°С)  Anion  E xchange  S eparation Reversed Phase...
Reversed Phase Chromatography RP-FPLC
Analytical  RP-HPLC
Homology between PISC-2002 and SSI family inhibitors Amino acid sequence a  Main fraction. b  Minor fraction. Species   ( ...
PISC-2002 Anti-influenza virus targets (www-micro.msb.le.ac.uk/3035/Orthomyxoviruses.html)
Spectrum of inhibitory activity
Virus-inhibitory effect of PISC-2002 on the reproduction of A/Rostock (H7N1)   (one cycle of viral replication)
Effect of the protease inhibitor  on the survival rate of mice in the experimental influenza A/Aichi-a infection
Infectious titer in mice lungs  in the experimental influenza A/Aichi-a infection
PISC-2002 protects mice from infection (morphological changes in mice lungs at the 9-th day of influenza infection) a  –  ...
Conclusions <ul><li>PISC-2002 significantly suppres the reproduction of human and bird influenza strains. </li></ul><ul><l...
Acknowledgments <ul><li>Julia Serkedjieva, PhD </li></ul><ul><li>Iskra Ivanova, PhD </li></ul><ul><li>Svetla Danova, PhD <...
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Lidiya Angelova-Duleva PhD thesis

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Lidiya Angelova-Duleva PhD thesis

  1. 1. Characterization of a protease inhibitor PISC-2002 wit h anti-influenza virus activity isolated from Streptomyces chromofuscus     Lidiya Angelova-Duleva, PhD
  2. 2. The Protease Inhibitors - what are they? <ul><li>Diverse group of proteins classified according their structure and protease inhibitory specificities. </li></ul><ul><li>Widely used in medicine and agriculture: </li></ul><ul><li>- Anticancer (multiple myeloma) </li></ul><ul><li>- Antiviral (HIV, Hepatitis C) </li></ul><ul><li>- Antiprotozoal (malaria) </li></ul><ul><li>- Anti-pests </li></ul>
  3. 3. Influenza Virus Morphology <ul><li>envelope glycoproteins: </li></ul><ul><li>hemagglutinin (HA) and neuraminidase (NA) </li></ul><ul><li>matrix proteins M1 </li></ul><ul><li>Integral membrane protein M2 </li></ul><ul><li>RNA + nucleoprotein (RNPs) </li></ul><ul><li>polymerase polypeptides( PA, PB 1 and PB2) </li></ul><ul><li>non-structural peptides (NS1 and NS2) </li></ul>Paul Digard, Dept Pathology, University of Cambridge http://www.microbiologybytes.com/virology/Orthomyxoviruses.html
  4. 4. Influenza A serotypes <ul><li>Influenza A viruses infect a wide variety of hosts: humans, horses, pigs, ferrets and birds. </li></ul><ul><li>There are 16 different HA serotypes and 9 different NA subtypes. </li></ul><ul><li>Pigs and birds are believed to be particularly important reservoirs, generating pools of genetically/antigenically diverse viruses which get transferred back to the human population via close contact between humans and animals. </li></ul><ul><li>Most pathogenic human subtypes are: H3N2, H1N2 and H2N2. </li></ul><ul><li>Bird flu H5N1 </li></ul><ul><li>Swine flu H1N1 </li></ul>
  5. 5. Influenza Virus Replication www-micro.msb.le.ac.uk/3035/Orthomyxoviruses.html
  6. 6. Genus Streptomyces unique mycelial, sporulating life cycle products with broad medical application Protease Inhibitors FLU Proteolytic cleavage of HA from subtilizin-like host proteases (H5 and H7) or trypsin-like host proteases (other H types) INFECTION Project Overview
  7. 7. Cell toxicity and virus-inhibitory effects of cell-free extracts from Streptomyces on human (H3N2) and bird (H7N1) virus replication in MDCK cells * n. t. – not tested   Strain     Cell toxicity TC50 (µg ml-1)   A/Rostock (H7N1 )   A/Aichi (H3N2)   ED 50 (µg ml-1)   SI   ED 50 (µg ml-1)   SI Streptomyces sp. 3 В (30) >1000 32 > 32.0 32 32.0 Streptomyces sp.19-Б 67 17 4.0 5 13.0 Streptomyces sp. ДЕБ-3 25 8 3.2 8 3.2 Streptomyces chromofuscus 34-1 >1600 12.5 128 23 70 Streptomyces galbus var. achromogenes 695 0.3 > 0.3 - n. t. n. t. Streptomyces galbus var. achromogenes 695-2-206 0.3 >0.3 - n. t. n. t. Streptomyces sp. A 12 >1670 > 1670 - n. t. n. t. Streptomyces sp. 712 183 11.4 16.0 11.4 16.0 Rimantadin >32.0 0.2 2. 5 mg/ml >160 0.5 3.0 mg/ml >64.0 ε-AKK 50.0 mg/ml 25.0 16.6
  8. 8. Purification of PISC-2002 Ammonium Sulphate Precipitation (80%, 24 h , 4°С) Anion E xchange S eparation Reversed Phase Chromatography Analytical RP-HPLC
  9. 9. Reversed Phase Chromatography RP-FPLC
  10. 10. Analytical RP-HPLC
  11. 11. Homology between PISC-2002 and SSI family inhibitors Amino acid sequence a Main fraction. b Minor fraction. Species ( proteinase inhibitor ) S. chromofuscus (PISC-2002) a S. chromofuscus (PISC-2002) b AS L PAV SALVLTV S L PAV SALVLTV S. lividans (SLPI (STI1)) S L YAP SALVLTV GHG S. longisporus (STI 2) AS L YAP SALVLTV GHG S. coelicolor A3 (SIL 3) S L Y A P SALVLTV GHG
  12. 12. PISC-2002 Anti-influenza virus targets (www-micro.msb.le.ac.uk/3035/Orthomyxoviruses.html)
  13. 13. Spectrum of inhibitory activity
  14. 14. Virus-inhibitory effect of PISC-2002 on the reproduction of A/Rostock (H7N1) (one cycle of viral replication)
  15. 15. Effect of the protease inhibitor on the survival rate of mice in the experimental influenza A/Aichi-a infection
  16. 16. Infectious titer in mice lungs in the experimental influenza A/Aichi-a infection
  17. 17. PISC-2002 protects mice from infection (morphological changes in mice lungs at the 9-th day of influenza infection) a – non infected untreated mice b – infected untreated mice c – infected mice treated with P ISC-2002 a b c
  18. 18. Conclusions <ul><li>PISC-2002 significantly suppres the reproduction of human and bird influenza strains. </li></ul><ul><li>The penetration of viral particles and the late stages of viral replication are the most susceptible to inhibition. </li></ul><ul><li>PISC-2002 decrease the mortality of mice infected with influenza A/Aichi virus in a dose-dependent manner. </li></ul>
  19. 19. Acknowledgments <ul><li>Julia Serkedjieva, PhD </li></ul><ul><li>Iskra Ivanova, PhD </li></ul><ul><li>Svetla Danova, PhD </li></ul><ul><li>Michèle Dalgalarrondo, PhD </li></ul><ul><li>All colleagues from University of Sofia, Bulgarian Academy of Sciences, Sofia, Bulgaria; National Agronomic Institute (INRA), Nantes, France </li></ul>

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