Computer-aided identification, synthesis and evaluation of substituted thienopyrimidines as novel inhibitors of HCV replication

Article abstract of DOI:10.1016/j.ejmech.2016.07.035

A structure-based virtual screening technique was applied to the study of the HCV NS3 helicase, with the aim to find novel inhibitors of the HCV replication. A library of ～450000 commercially available compounds was analysed in silico and 21 structures were selected for biological evaluation in the HCV replicon assay. One hit characterized by a substituted thieno-pyrimidine scaffold was found to inhibit the viral replication with an EC₅₀value in the sub-micromolar range and a good selectivity index. Different series of novel thieno-pyrimidine derivatives were designed and synthesised; several new structures showed antiviral activity in the low or sub-micromolar range.

Full text of DOI:10.1016/j.ejmech.2016.07.035

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Accepted Manuscript
Computer-aided identification, synthesis and evaluation of substituted
thienopyrimidines as novel inhibitors of HCV replication
Marcella Bassetto, Pieter Leyssen, Johan Neyts, Mark M. Yerukhimovich, David N.
Frick, Andrea Brancale
PII:
S0223-5234(16)30583-9
10.1016/j.ejmech.2016.07.035
EJMECH 8753
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 June 2016
Revised Date: 24 June 2016
Accepted Date: 18 July 2016
Please cite this article as: M. Bassetto, P. Leyssen, J. Neyts, M.M. Yerukhimovich, D.N. Frick, A.
Brancale, Computer-aided identification, synthesis and evaluation of substituted thienopyrimidines as
novel inhibitors of HCV replication, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.07.035.
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ACCEPTED MANUSCRIPT
Computer-aided identification, synthesis and evaluation of
substituted thienopyrimidines as novel inhibitors of HCV
replication.
c
Marcella Bassettoa 1, Pieter Leyssenb, Johan Neytsb, Mark M. Yerukhimovichc, David N. Frick ,
Andrea Brancalea
aCardiff School of Pharmacy and Pharmaceutical Sciences, Cardiff, King Edward VII Avenue, Cardiff CF103NB, UK
bRega Institute for Medical Research, University of Leuven, Belgium
c
Department of Chemistry & Biochemistry, University of Wisconsin- Milwaukee, Milwaukee, Wisconsin 53211, United States
Abstract
A structure-based virtual screening technique was applied to the study of the HCV NS3 helicase, with
the aim to find novel inhibitors of the HCV replication. A library of ~450000 commercially available
compounds was analysed in silico and 21 structures were selected for biological evaluation in the HCV
replicon assay. One hit characterised by a substituted thieno-pyrimidine scaffold was found to inhibit
the viral replication with an EC50 value in the sub-micromolar range and a good selectivity index.
Different series of novel thieno-pyrimidine derivatives were designed and synthesised; several new
structures showed antiviral activity in the low or sub-micromolar range.
Key words
Structure-based virtual screening; substituted thieno-pyrimidines; anti-HCV activity.
1. Introduction
Hepatitis C virus (HCV) is one of the first causes of chronic liver disease and it currently affects 130-
150 million people worldwide [1]. The infection becomes chronic in 60-85% of patients and leads to
the development of hepatic steatosis, fibrosis, cirrhosis and hepatocellular carcinoma [2, 3]. A vaccine
against this virus is still not available, while the standard of care was for long a combination of
pegylated interferon (pegIFN) and ribavirin, a therapy not specific for HCV and efficient in 50% of
treated patients, with many associated side effects [4]. HCV single-stranded, positive-sense, 9.6 kb
RNA genome encodes six non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A and NS5B), all
essential for the viral replication [5]. In the last decade great progress has been made in the
development of safe and highly potent inhibitors of HCV replication targeting the NS3 protease [6, 7],
the NS5B polymerase [8], the NS4B protein [9] and the NS5A protein [10]. Even if all-oral treatment
regimens of chronically infected patients with a combination therapy of either viral NS5B polymerase,
NS3 protease or NS5A inhibitors are showing promising results, these treatments are associated with
high costs [11, 12], while resistant viral strains have been reported for each clinically approved direct-
1 Corresponding author
E-mail address: bassettom@cardiff.ac.uk (Dr. M. Bassetto)
1

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acting antiviral [13-17]. These limitations highlight the need for new therapeutics that will reduce the
costs of treatment and avoid the development of resistance.
Despite the abundance of structural and mechanistic information on the HCV NS3 helicase [18], this
enzyme is still underexploited, with very few inhibitors reported so far, none of which taken into
clinical development [19]. The protein main function, essential for the viral replication [20, 21], is the
unwinding of double-stranded RNA sequences, formed as an intermediate of the viral nucleic acid
synthesis; this process is permitted by ATP hydrolysis [22, 23].
Due to the essential role of a functional NS3 helicase for HCV replication, this enzyme was chosen as
target for the computer-aided identification and synthesis of viral replication inhibitors. The SPECS
library of commercially available small molecules [24] was screened in silico on the enzyme known
RNA binding cleft, leading to the selection of 21 compounds that were analysed in the HCV replicon
assay. A first hit was found to inhibit the viral replication with an EC50 in the sub-micromolar range
and a good selectivity index. Its thieno-pyrimidine scaffold was the starting point for the design and
synthesis of different series of new analogues, with which the main chemical features were explored.
Several new derivatives were found to inhibit the HCV replication with EC50 values in the low or sub-
micromolar range.
2 Results and discussion
2.1 Structure-based virtual screening
The HCV NS3 helicase is formed by three domains and occupies the C-terminal portion of the NS3
protein. It presents multiple ligand-binding regions, the main ones being an ATP binding site in the
cleft separating domain 1 from domain 2 and a single-stranded nucleic acid binding site at the interface
of the three domains [25]. The potential interference with the known nucleic acid binding cleft was
evaluated to screen the SPECS library of commercially available compounds [24]. The 3KQH crystal
structure, which corresponds to the enzyme high-affinity open conformation bound to a single-stranded
DNA substrate [26], was used for all the analyses performed. The area defined by Glu493, Asn556 and
Phe557 was chosen as centre for the creation of a pharmacophoric filter, and the residues essential for
the helicase unwinding activity (Thr269, Arg393, Thr411, Glu493 and Trp501) [27] were considered to
build the pharmacophore using MOE 2014.10 (Figure 1) [28].
2