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