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Transcript of SIV 2012 Anticoli S. def
A new allosteric inhibitor of the hepatitis C virus NS5B polymerase is effective on HCV replication in infected cell culture system
Simona Anticoli, PhD
11th National Congress of the Italian Society for VirologyOrvieto, September 18, 2012
A chronic state is established in as many as 80% of infected individuals; about 10-20% of chronically infected patients develop liver cirrhosis and hepatocellular carcinoma over about 20 years
Hepatis C virus infection is a rapidly increasing global health problem, with about 3% of the world’s population infected
HCV infection
Nucleocapsid Protein,
Assembly
C21 30-35
TRANSLATION PROCESSING
E1 E2 p7 NS2 NS3 4A 4B 5A 5B
Envelope Glicoproteins,Assembly and Entry
Calcium Channel
Serine Protease/Helicase
Zn2+ protease NS3 cofactor
Phosphoprotein
Phosphoprotein,Replication,
IFN-resistance
RNA-dependent RNA polymerase
70 7 23 70 4-10 30 56-58 68Kd
Hepatitis C virus (HCV) genome
structural5’UTR
IRES,Translation Translation,
Replication
nonstructural3’UTR
RibavirinPegylated
IFN-+
• Efficacy: sustained virological response (SVR) in approximately 80% and 40-50% of patients infected with HCV genotypes 2-3 and 1 respectively• Side effects: flu-like symptoms, anorexia, insomnia, depression, skin rash, hair loss, neutropenia
• Efficacy: The SVR rate for patients with the most difficult to treat genotype-1 HCV is about 70%. Limited efficacy in partial non-responders and null responders to a prior course of Peg-IFN-/ ribavirin. • Side effects: Skin disorders, including rash and pruritus, anemia, nausea and diarrhea
RibavirinPegylated
IFN-
+
Protease Inhibitor
New HCV Standard of Care for patients infected with HCV genotype 1
Current anti-HCV drugs
Telapreviror
Boceprevir
Need of new and more effective therapeutic strategies…
Direct acting antivirals targets
NS5B polymerase inhibitors
NS3/4 protease inhibitors
NS5A inhibitors
NS5A inhibitors
HCV NS5B RNA polymerase inhibitors
BI207127
FilibuvirVX-222
SetrobuvirABT-333ABT-072 Tegobuvir
Welsch et al., Gut 2012
NI-site
Our study is aimed to:
identify new effective non nucleoside inhibitors of HCV NS5B
pre-clinically test their effectiveness to inhibit HCV replication, using an infectious cell culture system
Structure-based virtual screening studies into the palm domain of HCV NS5B polymerase
Definition of a library of commercial compounds (14.400 compounds) Library filtering by Lipinski rules of five Definition of a database of active compounds taking into account different chemotype Evaluation of active database by fingerprint analysis (MolPrint2D) Screening of commercially available compounds by fingerprint analysis (500 selected
compounds) Tanimoto analysis to remove similar compounds Biological evaluation of 10 higher scoring compounds Docking analysis of selected compounds by MolPrint2D Biological evaluation of docking selected compound (Visual Inspection)
N-Phenyl 1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamide
RS4398
NS5B polymerase residual enzymatic activity is 7% upon treatment with 10 mM
RS4398 reduces NS5B polimerase activity
J6 (2a genotype)
JFH1 (2a genotype)
Tscherne et al., J. Virol. 2006
Jc1Luc(FL-J6/JFH1-5’C19RLuc 2Ubi)
RNA transcript
Tranfection
Huh7.5 cells
production
filtered supernatant
Infection
Detection(IF, RTQ-PCR,
Luciferase activity assay)
HCV infectious cell culture (HCVcc) system
Wakita et al., Nat. Med. 2005
RNA transcript Huh7.5 cells
2 cellular passagestranfection
Supernatant 10 days p.t.
naive Huh7.5 cells
Huh7.5 cells 10 days p.t.
Infection
Cell culture adaptedJc1Luc stock virus
2 cellular passages
Generation of cell culture adapted Jc1Luc virus(Jc1LUCcc)
105 TCID50/ml
RS4398 reduces intracellular HCV replication in transfected Huh7.5 cell line
CTR 5 mM 10 mM
CTR 5 mM 10 mM
Transfection
RS4398
6 days1 cell
passage
Hour post-treatment
48 h 72 h
A
B
* ** *
** *
* ** p ˂0,05 vs control
p ˂0,01 vs control
Luc assayRTQ-PCR
CTR 10 mM
C
* *
74% reduction
RS4398 reduces the release of HCV virions from transfected Huh7.5 cell line
Transfection
RS4398
6 days1 cell passage
Hour post-treatment
72 h
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
HCV
RNA
copi
es/m
l (x
105)
0
20
40
60
80
100
120
100 37
HCV
RNA
copi
es/m
l (%
)
CTR 10 mMCTR 10 mM
* * * *
* * p ˂ 0,01 vs control
A B
RTQ-PCR assay of viral RNA in culture supernatants
63% reduction
RS4398 toxicity in Huh7.5 cell line
CTR5 mM
10 mM
48 h post-treatment 72 h post-treatment
RS4398 reduces intracellular HCV replication in infected Huh7.5 cell line
0
0.5
1
1.5
2
2.5
3
3.5
Intr
acel
lula
r HCV
RN
A
(c
opie
s/m
g ce
llula
r RN
A)
(
x 10
4)
0
20
40
60
80
100
120
100 30
Intr
acel
lula
r HCV
RN
A
(
% co
pies
)
RTQ-PCR
Viral adsorption period 72h
Virus removal
3 h Hour post infection
Infection
RS4398
* *
CTR 10 mM CTR 10 mM* p ˂0,05 vs control
RTQ-PCR assay of intracellular viral RNA
A B
70% reduction
Conclusions
RS4398 effectively decreased HCV replication in our in vitro system, especially when used at 10 mM for 72 h
RS4398 treatment of transfected Huh7.5 cells decreased intracellular HCV level as well as virus release in cell culture supernatants
RS4398 can be the basis of additional chemical refinements that focus on improving antiviral potency
Chemical modifications to obtain analogs with major antiviral efficacy are currently in progress
Thanks to…
Department of Public Health and Infectious Diseases
Sapienza University of Rome
Lucia NencioniAnna Teresa Palamara
Department of Infectious, Parasitic And Immune-Mediated Disease
Istituto Superiore di Sanità
Anna RuggieriGiovanni Rezza
Department of Drug Chemistryand Technologies
Sapienza University of Rome
Francesco PiscitelliRomano Silvestri
Huh7.5 cell line
Huh-7 cell lines harboring SG-Neo subgenomic HCV replicons
were cured of HCV RNA by prolonged treatment with IFN
Mutation in the first CAARD domain
Unable to stimulate IFN induction
Adapted from Arnaud et al., Plos One 2010
HCV life cycle