This document discusses peptide-based inhibitors of the hepatitis C virus (HCV). It provides background on HCV and its structure, focusing on the non-structural proteins involved in replication. It then introduces a hexapeptide inhibitor that targets the NS3 protease and inhibits its activity. An assay was performed showing the hexapeptide is a competitive inhibitor. Further derivatives were developed incorporating additional inhibitors to enhance potency against the NS3 protease. Future work could develop highly effective oral HCV therapies with minimal side effects.

1. 1

2. Peptide Based Inhibitors of HCV
Made by
M.AQIB HANIF

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3. Contents
 Introduction of Hepatitis c
 Introduction of HCV
 Structure of HCV
 Mechanism of Action of HCV to Host Cell
 Replication
 Introduction of Inhibitor
 Performed assay
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4.  Observation
 Result
 Future Aspects
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5. Hepatitis C
 Global epidemic Disease
 Caused by HCV
 Millions of people suffering from it
 Not Only effect the function of Liver
 Causes Liver fibrosis and cirrhosis
 Leads to liver Cancer
(Webster et al,2009 ; Pinzani et al,2010).
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6.  Majority of Liver transplantation is due to Hepatitis C
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7. Signs and Symptoms
 Feeling very tired
 Sore muscles
 Joint pain
 Nausea or poor appetite
 Stomach pain
 Dark urine
 A yellow discoloration of the skin and whites of the
eyes, called jaundice.
 Fever
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8. Introduction of HCV
 Small enveloped Virus
 Positive sense single stranded RNA genome
 3010 Amino acids
Mode of Transmission
 Blood Transfusion
 Surgical equipments
 Breakdown in skin or
lining of mouth
• Incubation period is 40-120 days
(De Francesco R et al,2003 ; Beaulieu PL&Tsantrizos YS,2004).
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9. Diagnosis
 HCV Antibody-Elisa used to diagnose hepatitis c
infection. Not useful in acute phase as it takes 4 weeks
after infection
 HCV RNA – Various techniques are available e.g PCR
Treatment
 Interferon
 Ribavirin
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10. Structure Of HCV
Structural Proteins
• Form the viral particle including core protein
Non structural Proteins
• Viral proteins required for RNA replication
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11. Types of Non structural Proteins
 NS2
 NS3
 NS4a
 NS4b
 NS5a
 NS5b
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12. NS2
 21-23 kDa trans-membrane protein
 Essential for completion of the viral replication cycle
in vitro and in vivo
 contains highly hydrophobic N-terminal residues
 N-terminal is hydrophobic while c terminal plays
important role in NS2/3 protease activity.
(Khromykh AA& Westaway EG,1997 ;
Pietschmann T et al,2006).
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13. NS3
 67 kda protein
 N terminal has serine protease activity while C
terminal has helicase activity
• NS3 N terminus is involved in cleavage between
NS3,4a, 4b,5a and 5b.
• The proposed catalytic activity of HCV NS3 is due to
three amino acid residues His- 1083, Asp-1107 and Ser-
1165.
(Bartenschlager R et al,1993)
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14. NS4a
 54 amino acids protein
 acts as a cofactor for NS3 protein
 N-terminus is highly hydrophobic
 involved in targeting NS3 to the ER membrane
(Wolk B et al,2000).
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15. NS4b
 Small hydrophobic 27 kDa protein
 Play role in targeting
NS5a
• hydrophilic phospho-protein
• Role in viral replication, modulation of cell signaling
pathways and interferon response .
(Macdonald A et el,2004 ; Reed KE et al,1997).
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16. NS5b
 65 kDa in size
 Important role in synthesis of new RNA genome
(Behrens SE et al,1996).
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17. Mode of Entry
 Interaction between virus and target cell
 Lipoproteins are the interaction mediators
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18. Replication of Viral RNA
 After interaction and entry nucleo-capsid is released
into the cytoplasm
 Virus is decapsidated
 Translation of HCV RNA
is initiated by binding the
5/-IRES to ribosome.
 Translation of HCV RNA
occurs at rough endoplasmic
reticulum.
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19.  NS5B RNA-dependent RNA polymerase replicates the
genome by the synthesis of negative strand RNA
 Negative strand RNA serves as a template for the
synthesis of positive strand RNA.
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20. Inhibitors
 An inhibitor is a molecule that binds to an enzyme
and decreases its activity.
 Blocking an enzyme's activity can kill a pathogen.
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21. Hexa peptide inhibitor
 DDIVPC-OH (Hexa peptide) is a competitive
inhibitor of the hepatitis C virus NS3 protease
complaxed with NS4A cofactor peptide.
 The amino terminus of NS3, is responsible for an auto-
catalytic cleavage at the NS2/3 junction
 NS3 protease is responsible for the proteolysis of
NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B .
(Grakoui et al,1993 ; Grakoui et al,1993)
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22.  NS3 protease is a chymotrypsinkrypsin-like serine
protease
 developed an enzymatic assay using the NS3 protease
complaxed with the NS4A peptide cofactor (NS3-4&
protease)
 N-terminal hexapeptide product DDIVPC-OH of a
substrate derived from the NSSA/5B cleavage site
inhibited the enzyme
(Mori et al,1997)
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23. Protease Assay
 An enzymatic assay
 Designed for the quantitative determination
of proteases present in a protein sample, using a dye-
labeled protein substrate
 The proteases present in the sample of interest will
digest the protein substrate and release dye labeled
peptides.
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24. Performance
 enzymatic assay was performed in 50 mM Tris-HCl,
pH 7.5, 30% glycerol, 1 mg/mL BSA, 1 mM TCEP. 25 pM
of the substrate
 various concentrations of inhibitor were incubated
with 25 nM of protease and 2.5 uM of the NS4A-
derived peptide
 The separation of substrate horn products was
performed by adding avidincoated agarose beads to
the assay mixture followed by filtration.
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25.  A non-linear curve tit using the Hill model was then
applied to the % inhibition-concentration data and
50% effective concentration (IC,) was calculated
through the use of SAS
 (Statistical Software System, SAS Institute Inc., Gary,
N.C.).
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26. Result
 Found that DDIVPC-OH , corresponding to the N-
terminal cleavage product of a NS5a/5B derived
peptide substrate, inhibits the NS3-4,,, protease.
 Hexapeptide is a competitive inhibitor of the enzyme
with a Ki app of 14 pM.
 Structure-activity studies on led to inhibitors with I&
values in the low micromolar range .
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27. a-Ketoamides, a-Ketoesters and a-
Diketones as HCV NS3 Protease
Inhibitors
 In an attempt to enhance the inhibitory
 Potency a-keto-amides, a-keto-esters, and a-di ketones,
used as inhibitors of related serine
Proteases
 Designed target 2 by incorporation of these three
inhibitors into the backbone of 1.
 Protease assay showed positive effects. Activity of
Hexapeptide is increased by the addition of inhibitors.
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28. Future aspects
 Currently available therapies for the treatment of
chronic hepatitis C are effective in half of patients
 Expensive, often poorly tolerated,
 Unsuitable for certain patient populations.
 The ideal therapy would be highly effective, orally
bioavailable, have minimal side effects, be cost
effective, and suitable for the majority of patients with
hepatitis C.
 These inhibitors will show ideal properties.
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29. References
 Webster, D. P.; Klenerman, P.; Collier, J.; Jeffery, K. J.
Lancet Infect. Dis. 2009, 9,108.
 De Francesco R, Tomei L, Altamura S, Summa V,
Migliaccio G: Approaching a new era for hepatitis C
virus therapy: inhibitors of the NS3-4A serine protease
and the NS5B RNA dependent RNA polymerase.
Antiviral Res 2003, 58:1-16.
 Khromykh AA, Westaway EG: Subgenomic replicons of
the flavivirus Kunjin: construction and applications. J
Virol 1997, 71:1497-1505.
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30.  Bartenschlager R, Ahlborn-Laake L, Mous J, Jacobsen H:
Nonstructural protein 3 of the hepatitis C virus encodes a
serine-type proteinase required for cleavage at the NS3/4
and NS4/5 junctions. J Virol 1993, 67:3835-3844
 Wolk B, Sansonno D, Krausslich HG, Dammacco F, Rice
CM, Blum HE, Moradpour D: Subcellular localization,
stability, and trans-cleavage competence of the hepatitis C
virus NS3-NS4A complex expressed in tetracycline-
regulated cell lines. J Virol 2000, 74:2293-2304.
 Macdonald A, Crowder K, Street A, McCormick C, Harris
M: The hepatitis C virus NS5A protein binds to members of
the Src family of tyrosine kinases and regulates kinase
activity. J Gen Virol 2004, 85:721-729.
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31.  Behrens SE, Tomei L, De Francesco R: Identification
and properties of the RNA-dependent RNA
polymerase of hepatitis C virus. Embo J 1996, 15:12-22.
 Grakoui, A.; Wychowski, C.; Lin, C.; Feinstone, S.M.;
Rice, C.M. J. Viral. 1993,67, 1385
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