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Molecular recognition studies of dasabuvir, tegobuvir, and
setrobuvir against natural Hepatitis C Virus NS5B
polymerase variants
Dr Nadia Marascio, PhD
Microbiology and Virology Specialist
Department of Health Sciences, Unit of Clinical Microbiology, School of Medicine, University of
"Magna Graecia", Viale Europa, Germaneto, 88100 Catanzaro, Italy.
19 Ottobre 2016
Background
•Management of Hepatitis C virus (HCV) infection changed after development of
direct-acting antiviral (DAAs) agents, including NS5B polymerase inhibitors.
• Several amino acid (AA) changes give rise to resistance associated
substitutions (RASs), which could lead to therapy failure in HCV1b positive
patients.
Aims
•To detect NS5B polymerase
RASs and polymorphisms in
baseline sequences from HCV1b
infected DAA-naïve patients.
•To predict stability and binding
affinity of drugs, carrying C316N
RAS and in combination with
specific polymorphisms.
Materials and Methods
NS5B gene
19 baseline serum
sample from
HCV1b positive
DAA-naïve patients
Direct sequenced
Sanger sequencing
ABIPRISM3500
Genetic Analyzer
Genetic variability analysis
- All sequences were aligned with the Clustal W algorithm and manually edited by MEGA v.5.5.2. Subtype was
determined by Bioafrica Oxford HCV and COMET HCV typing tool.
- Phylogenetic reconstruction was performed in PhyML v.3.0.
- The AA positions of RASs and polymorphisms were determined by Geno2pheno[hcv]0.92 tool and aligning
generated sequences to HCV1b AJ238799 reference by Clustal W.
In silico study on 6 isolates carring C316N
- X-Ray models of NS5B (Protein Data Bank 3H2L).
- Mutants were generated by single residue replacement and were performed 200 ns of a Molecular Dynamics
simulations.
- The obtained structures were submitted to molecular recognition studies by means of Glide docking program For
the most populated poses, the Emodel score.
Patients' characteristics: the 6 patients who carried C316N RAS
Patient ID
Gender
Age
Liver stiffness
(KPa)*
Risk factors#
DAAs
Therapy
Baseline viral load
(IU/ml)
SVR 12
HCV05
M
71
20.4
Surgery and Cohabitation
TVR
3250000
No
HCV06
M
69
6.5
Surgery and Cohabitation
TVR
1580000
Yes
HCV10
F
68
13.9
Cohabitation
SMV/DCV
3070000
No
HCV14
M
69
4.6
Surgery
TVR
3250000
Yes
HCV18
F
69
Not Available
Surgery
TVR
3610000
Yes
HCV19
M
46
6.9
Surgery and Tatoo
TVR
4290000
Yes
* According to Castéra L et al, 2005, KPa ≤ 7.1 = F0-F1 (minimal fibrosis), 7.1 < KPa ≤ 9.5 = F2 (moderate fibrosis), 9.5 < KPa ≤ 14.5 = F3 (severe fibrosis),
and KPa > 14.5 = F4 (cirrhosis)
# Surgery
and cohabitation with HCV-antibodies (Ab) positive individuals or blood transfusion, tattoo are transmission risks reported by the same patient
Phylogenetic analysis to confirm genotype/subtype determined by LiPA assay
92
Phylogenetic analysis of 43 HCV1b NS5B
sequences. The Maximum likelihood tree, inferred
by PhyML v.3.0, contains 19 newly HCV isolates
(red) and 24 HCV references strains (black),
downloaded from Los Alamos HCV Sequence
Database.
Baseline RAS and polymorphisms detected for NS5B target region
Patient ID
NS5B polymerase
RASs
Polymorphisms
HCV05
C316N
Q309R V338A
HCV06
C316N
R254K S300T V338A
HCV10
C316N
S300T S335N V338A
HCV14
C316N
R254K S335N V338A
HCV18
C316N
V322I S335N V338A
HCV19
C316N
Q309R S335N V338A
Apo-polymerase stability
The thermodynamic profile of WT and C316N mutant polymerase, calculated with respect to the
initial structure during the MD simulation, expressed in kcal/mol.
In presence of C316N RAS alone we observed a minor stability of NS5B polymerase
Molecular docking
3D representation of the best predicted pose of a,d) DSV, b,e) STV, c,f) TGV in the WT and C316N
complexes, respectively.
Minor binding affinity in presence of C316N between drugs and polymerase
Glide (kcal/mol) score and Emodel (kcal/mol) values for drugs bound to wild-type and mutated
polymerase complexes
Compensative polymorphisms (clusters) plus C316N restored binding affinity between
STV and polymerase, but not for DSV and TGV.
Conclusions
In presence of specific clusters there is a greater binding affinity between STV and NS5B
polymerase.
The 316N RAS and associated natural substitutions in HCV polymerase should be
evaluated in patients who had virological breakthrough to previous treatment.
The study may be useful to guide therapeutic approach with DAAs.
Thanks to co-authors
Grazia Pavia1, Isabella Romeo2, Carmine Talarico2, Giosuè Costa2, Emilia Zicca1, Giorgio
Settimo Barreca1, Vincenzo Pisani3, Chiara Costa3, Carlo Torti3, Anna Artese2, Stefano Alcaro2,
Maria Carla Liberto1, Alfredo Focà1.
1Department
of Health Sciences, Unit of Clinical Microbiology, School of Medicine, University of
"Magna Graecia", Viale Europa, Germaneto, 88100 Catanzaro, Italy.
2 Department
of Health Sciences, University of "Magna Graecia", Viale Europa, Germaneto, 88100
Catanzaro, Italy.
3 Department
of Medical and Surgical Sciences, Unit of Infectious and Tropical Diseases, School of
Medicine, University of "Magna Graecia", Viale Europa, Germaneto, 88100 Catanzaro, Italy
Thank you all
for attention !!!!
“Magna Graecia” University