Hepatitis C Virus (HCV):
Serine Protease Inhibitors
Sarah Burke, Andrew Krasley, Maria Winters
Some Statistics
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3% of the world
population and 1.3%
of the United States
are infected with HCV.
HCV is the leading
cause of liver
transplantation.
HCV is 5x more
widespread than HIV.
http://www.hivandhepatitis.com/2008icr/aasld/docs/111808_c.html
HCV Basics
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HCV has surface proteins
which recognize the liver
cells it ultimately infects.
HCV has an icosohedral
core like HIV.
HCV contains singlestranded positive sense
RNA.

http://people.rit.edu/japfaa/infectious.html
This means that it is similar
to mRNA and can therefore
be immediately translated
by the host cell.
How To Contract HCV
http://www.youtube.com/watch?v=tQIUV_cSll0
HCV in the Body
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HCV targets hepatocytes (liver cells) because the
liver is its main replication site.
HCV is very similar to HIV in that it attaches itself to
the hepatocyte and releases its genetic material
into the cell. It then “hijacks” the cell and replicates.
Ultimately the liver becomes inflamed and cirrhosis
can occur and lead to death.
Effect of HCV on Liver
Uniform and smooth healthy liver.
Liver from individual who
died from cirrhosis.
Current Treatments: Interferons
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Interferons (IFN) are glycoproteins which stimulate the
immune system. IFN-α can be injected into the
bloodstream as a treatment of HCV.
More recently PEGylated-IFN-α’s have been used.
PEG gives the IFN- α better PK properties such as
solubility and half-life.
Current Treatments: Ribavirin
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Ribavirin is a prodrug
which resembles RNA
nucleotides.
The mechanism of
action is unknown, but
it somehow interferes
with production of
viral RNA and
prevents HCV from
replicating.
Downfalls of Current Therapies
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Only effective on 40-50% of patients
with the most common form of HCV.
IFNs not directly act on the virus and the
virus can quickly replicate to overcome
ribavirin.
They cause side effects such as:
 Fatigue,
fever, and headaches
 Depression and insomnia
 Cough, skin rash, and chest pain
New Target: NS3 Protease
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Hepatitis C RNA encodes for a polyprotein that is 3000
amino acids long.
This is cleaved by viral and host proteases to form
structural and nonstructural proteins.
The nonstructural (NS) proteins are further processed by
two viral proteases, NS2/3 and NS3.
More About the NS3 Protease
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180 amino acid serine
protease with a His-57,
Asp-81, and Ser-139
catalytic triad.
In order to be actived it
must be bound to an
NS4a cofactor.
Located at the Nterminal end of the NS3
protein.
Contains a shallow
active site on the surface
of the enzyme.
http://www.biomedcentral.com/1472-6807/5/1
Two types of NS3 Protease Inhibitors
Competive, noncovalent
inhibitors
α-ketoamides
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Form a reversible
covalent bond with
serine in the active
site.
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Aim to bind more
strongly to active site
than substrate
through noncovalent
interactions.
Mechanism of Action of α-ketoamides
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These are typically peptides containing
an electrophile that form reversible
covalent bonds with serine139 in the
active site.
Serine Protease Mechanism
Alpha-Ketoamide Inhibitors
http://www.rcsb.org/pdb/explore/jmol.do?structureId=2OC0&bionumber=1
General PK Information
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Challenges in Modern Drug Discovery: A Case Study of Boceprevir, an
HCV Protease Inhibitor for the Treatment of Hepatitis C Virus Infection
F. George Njoroge et al.
Selecting a Target
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NS3-NS4A protease
Screened 4 million compounds
 No
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leads generated
Structure based design
 Look
at substrate-enzyme active site interactions
 Account for oral bioavailability/PK properties
 Exploit H-bonding and hydrophobic interaction
Testing Inhibitors
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Cell-based replicon system of HuH-7 cells
HCV NS3 protease continuous assay
Lack of small animal model
http://huh7.com/
 Severe
immunodeficiency disease (SICD) mice
 Chronically infected chimpanzees
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Many are compared against human neutrophil
elastase (HNE)
Many are compared against CYPs
Creating a Drug
cyclopropylalanine
phenylglycine
isobutyl carbamate cap
carboxylic acid
cyclopropylalanine
cyclohexylglycine
gem-dimethylcyclopropylproline
tert-butyl carbamate
dimethylamide
tert-butylglycine
P2’
substituted hydrazine urea
macrocyclization
tert-alkyl ether linkage
dimethylcyclopropyl-proline
macrocyclization
Boceprevir
α-center
urea
cyclobutylalanine
Performance
X-ray of Bioceprevir Bound in Pocket
dimethylcyclopropylproline
H-bond
urea capping group
tert-butylglycine
cyclobutylalanine
Discovery of Narlaprevir (SCH 900518): A Potent, Second
Generation HCV NS3 Serine Protease Inhibitor
Ashok Arasappan et al.
Major Goals
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Design a second generation HCV NS3 Serine
Protease Inhibitor (α-ketoamide class of slowbinding reversible inhibitors)
Eliminate purification issues by developing a
molecule that exists as a single isomer
Focus on in vitro potency and PK profile
Boceprivir
Narlaprivir
Crystal Structure of Bound Boceprivir
SAR Studies Lead to t-butyl Sulfone
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Initial attempts showed improvement in replicon
potency and rat PK properties
Monkey exposure was more of a challenge
t-butyl sulfone moiety showed an EC90 = 100 nM
with good bioavailability in monkey plasma
Synthesis: Route 1
Curtis Rearrangement
Mixture of P1
Diastereomers
Synthesis: Route 2
Optimized Stereoselective Synthesis of 37
SAR Studies Investigating P1
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P’ 2o allyl amides showed desirable rat PK profile
Allyl (or small alkyl groups) were not tolerated well
with certain P1 groups
Identification of the best P’-P1 combination was
desirable.
P’ SAR of Inhibitors of
Type 15
Table 3 : Discovery of P1 Moiety
Synthesis of P1-P’ intermediate
Synthesis of Intermediate
Passerini Reaction Mechanism
Comparison of 37 & 70
Compound
AUC
(monkey/rat/dog)
µM
Bioavailability
(monkey/rat/dog)
Boceprivir
0.1/1.5/3.1
4-11%/26%/30%
37
1.1/6.5/0.9
46%/46%/29%
70
-/2.1/0.5
-/21%/16%
NS3 Protease Bound X-ray Structure
Containing 37
Discovery of a Potent and Selective Noncovalent Linear
Inhibitor of the Hepatitis C Virus NS3 Protease
Montse Llinas-Brunet* et al
Major Goals of this Research
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After the failure of their initial macrocyclic lead
compound, these authors set out to find a new series
of potent noncovalent inhibitors.
Goal was to develop a linear tripeptide with single
digit nM EC50 and IC50 values.
They also wanted to study the PK properties of their
compounds, specifically plasma concentrations and
oral bioavailability.
Previous Studies: Macrocyclic Inhibitors
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Vinyl-ACCA
Previously reported Cterminus carboxylic acid
containing noncovalent
inhibitor of NS3 protease.
P1 residue: 1-amino-2vinylcyclopropylcarboxylic
acid (vinyl-ACCA).
Advancement of this
compound was discontinued
because of cardiotoxicity in
monkeys.
Previous Studies: Linear Inhibitors
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The authors have previously published on linear
inhibitors like compound 2 which are easier and less
costly to synthesize.
This peptide backbone forms many favorable
interactions with the active site.
Synthesis
Synthesis
Synthesis
SAR Studies on Capping Group
SAR Studies on Aminothiazol and
Quinoline Moieties
PK Studies
More SAR Studies
PK Studies