Virtual screening, synthesis and biological evaluation of DNA intercalating antiviral agents

Article abstract of DOI:10.1016/j.bmcl.2017.06.035

This paper describes computer-aided design of new anti-viral agents against Vaccinia virus (VACV) potentially acting as nucleic acid intercalators. Earlier obtained experimental data for DNA intercalation affinities and activities against Vesicular stomatitis virus (VSV) have been used to build, respectively, pharmacophore and QSAR models. These models were used for virtual screening of a database of 245 molecules generated around typical scaffolds of known DNA intercalators. This resulted in 12 hits which then were synthesized and tested for antiviral activity against VaV together with 43 compounds earlier studied against VSV. Two compounds displaying high antiviral activity against VaV and low cytotoxicity were selected for further antiviral activity investigations.

Full text of DOI:10.1016/j.bmcl.2017.06.035

Accepted Manuscript
Virtual screening, synthesis and biological evaluation of DNA intercalating an-
tiviral agents
Kyrylo Klimenko, Sergey Lyakhov, Marina Shibinskaya, Alexander Karpenko,
Gilles Marcou, Dragos Horvath, Marina Zenkova, Elena Goncharova, Rinat
Amirkhanov, Andrei Krysko, Sergei Andronati, Igor Levandovskiy, Pavel
Polishchuk, Victor Kuz'min, Alexandre Varnek
PII:
S0960-894X(17)30634-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.06.035
BMCL 25069
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 March 2017
9 June 2017
11 June 2017
Please cite this article as: Klimenko, K., Lyakhov, S., Shibinskaya, M., Karpenko, A., Marcou, G., Horvath, D.,
Zenkova, M., Goncharova, E., Amirkhanov, R., Krysko, A., Andronati, S., Levandovskiy, I., Polishchuk, P.,
Kuz'min, V., Varnek, A., Virtual screening, synthesis and biological evaluation of DNA intercalating antiviral
agents, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.06.035
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Virtual screening, synthesis and biological evaluation of DNA
intercalating antiviral agents
Kyrylo Klimenko,a,b Sergey Lyakhov,b Marina Shibinskaya,b Alexander Karpenko,b Gilles
Marcou,a Dragos Horvath,a Marina Zenkova,с Elena Goncharova,c Rinat Amirkhanov,c Andrei
Krysko,b Sergei Andronati,b Igor Levandovskiy,f Pavel Polishchuk,b,d Victor Kuz’min,b and
Alexandre Varnek*a,e
a Laboratoire de Chemoinformatique, (UMR 7140 CNRS/UniStra)
Université de Strasbourg,,
1, rue B. Pascal, Strasbourg 67000, France,
e-mail : varnek@unistra.fr
b A.V. Bogatsky Physico-Chemical Institute of NAS of Ukraine,
Lyustdorfskaya doroga, 86, Odessa 65080, Ukraine
c Institute of Chemical Biology and Fundamental Medicine,
Siberian Branch of Russian Academy of Sciences,
8 Lavrentiev Avenue, Novosibirsk 630090, Russia
d Institute of Molecular and Translational Medicine,
Palacky University Olomouc, Hněvotínská 1333/5,
Olomouc 779 00, Czech Republic
e Federal University of Kazan, Kremlevskaya str., 18, Kazan, Russia
f
Department of Organic Chemistry, Kiev Polytechnic Institute, Pr. Pobedy 37, 03056 Kiev, Ukraine.
Abstract: This paper describes computer-aided design of new anti-viral agents against Vaccinia virus (VaV)
potentially acting as nucleic acid intercalators. Earlier obtained experimental data for DNA intercalation affinities and activities
against Vesicular stomatitis virus (VSV) have been used to build, respectively, pharmacophore and QSAR models. These
models were used for virtual screening of a database of 245 molecules generated around typical scaffolds of known DNA
intercalators. This resulted in 12 hits which then were synthesized and tested for antiviral activity against VaV together with
43 compounds earlier studied against VSV. Two compounds displaying high antiviral activity against VaV and low cytotoxicity
were selected for further antiviral activity investigations.
Keywords: antiviral activity, vaccinia virus, structure-activity modelling, virtual screening, DNA
affinity
Highlights:
virtual screening to discover potential nucleic acids intercalators
two hits active against Vaccinia virus were found
intercalating mechanism was proposed for hits
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Viral diseases have a severe negative impact on human life worldwide1,2 which motivates researchers to develop new
antiviral drugs. Most of known target-specific antiviral compounds inhibit certain viral proteins, e.g. protease or polymerase 3.
Such compounds are rather selective, have low toxicity and the reduced risk of adverse effects. Corresponding drug
discovery projects are frequently supported by different chemionformatics tools. Thus, a combination of QSAR and docking
methods were used to identify a novel influenza virus neuraminidase inhibitor which is more potent than the commercialized
drug Oseltamivir4. The virtual screening procedure involving similarity search, shape-based and pharmacophore models was
used to discover HIV-1 reverse transcriptase dual inhibitors5.
Broad spectrum antiviral agents may, however, be more advantageous than target-specific compounds in controlling
multiple emerging pathogens 6. There exist several major groups of broad-spectrum antivirals. One of them includes
interferon and interferon inducers. Interferon is a protein produced as an immune response, inducing synthesis of protein
kinase which phosphorylates initiation factor of translation and, therefore, prevents synthesis of viral proteins. The second
group includes nucleotide analogs, i.e., substances which resemble DNA or RNA nucleotide but have an inappropriate
nitrogenous base. Being captured by proteins or tRNA involved in the virus reproduction processes; they may lead to the
synthesis of a non-coding sequences in viral nucleic acids.7 The third group includes nucleic acid intercalators which may
8
entry between the parallel pairs of bases in double helix of DNA or RNA. To our knowledge, in silico approaches are rarely
used in the design of broad spectrum antivirals and no computer-aided design of intercalators was reported so far.
In this study, we performed ligand-based virtual screening of new promising nucleic acid intercalators using Quantitative
Structure-Activity Relationships (QSAR) and pharmacophore models. Selected hits were synthesized and tested
experimentally against Vaccinia virus (VaV), which is a double-stranded DNA virus of the Poxviridae family similar to
potential biothreat variola virus. It is widely used in the laboratory as a model system to study various aspects of viral biology
and virus-host interactions. 23 Vaccinia viruses’ reproduction takes place entirety in the cytoplasm that might facilitate the
interaction of organic molecules with viral DNA leading to higher compounds’ bio-performance.
In order to build the models we collected a data set of 167 intercalators previously synthesized and tested at A.V.
Bogatsky Physico-Chemical Institute 9 (BPCI), see Table A1 in Supplementary Material. All compounds have similar motifs: a
polycyclic planar fragment (scaffold) linked to a basic amino group (Figure 1). According to the type of the scaffold, the data
set was divided on seven classes of compounds, as it is shown in Figure 1. The binding constants (Ki) values measured by
10
substitution of ethidium bromide in DNA
were reported for all compounds of this data set. For 117 out of 167 compounds
another kind of a biological response - maximum antiviral effect, Emax (%), against Vesicular stomatitis virus (VSV) within 0.2
9
- 620 µM concentration range was measured as described in . We believe that in the context of development of broad-
spectrum antiviral agents, the models obtained for anti-VSV activity can be used to discover molecules active against VaV.
This hypothesis looks reasonable assuming that both antiviral activities are related to intercalation of small molecules into
viral nucleic acids.
The pharmacophore model has been built with the LigandScout11 software on a subset of 161 compounds with lg(Ki) ≥ 4
recognized as reasonable DNA intercalators. ZINC database12 was used as a source of decoys for validation of
pharmacophore models. Three best pharmacophore models (Figure 2 and Table A3) having the highest recall and precision
values (0.66-1.00) were selected for virtual screening workflow. The classification model able to distinguish compounds with
higher (Emax ≥ 50%) and lower (Emax < 50%) maximum antiviral effect against VSV has been built using the Random Forest
method13 and simplex descriptors14 Earlier, similar technique was successfully used in QSAR modeling of various antiviral
15-17
18
activities.
The model’s applicability domain was assessed with Euclidean distance-based method.
The model was
trained on 94 compounds randomly selected from the PCI dataset. It performed well on the test set containing remaining 23
compounds (balanced accuracy BA = 0.78). More details about model development are given in the Section 2 of the
Supplementary Materials.
Developed models have been applied to screen a library of 245 virtual compounds generated as a combination of
scaffolds and some typical fragments available as building blocks at BPCI. This library was, at first, screened with the
pharmacophore models retaining 42 compounds. Screening of remaining compounds with the classification model resulted in
12 hits which then were synthesized in BPCI, see synthesis description in Section 3 of Supplementary Material.
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In view of development of broad-spectrum antiviral agents, the molecules discovered in virtual screening were
completed by 43 active compounds earlier studied in BPCI against VSV. Resulting set of 55 compounds was screened with
previously developed QSPR model 19 assessing aqueous solubility and with the PASS software 20 assessing affinities to the
wide spectra of biological targets. According to these calculations, all 55 compounds didn’t display any predicted side effects,
toxicity and mutagenicity and had predicted solubility in water was larger than 10-5 mol/l.
Figure 1. Seven classes of compounds according to core structures (CS) present in the modeling dataset.
Figure 2. Examples of retrieved hits by pharmacophore models
At the next step, all these compounds have been tested for their cytotoxicity via MTT assay and Real-time cell
analysis21. Antiviral activity against Vaccinia virus was determined using GFP expression quantitation and plaque forming
units’ assay23. Interferon inducing capacity was assessed by decrease of a cytopathic effect caused by virus. The detailed
description of experiments and experimental values for all compounds are provided in the Section 4 of the Supplementary
materials.
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15 out of 55 compounds were not soluble enough in 20% aqueous DMSO in order to complete sample preparation. The
cytotoxicity of 40 compounds with respect to CV-1 cells was measured at samples concentration 0.1, 10, 100 µM. Cell
viabilities observed for the most active compounds at concentration 10 µM are listed in Table 1. The values of CC50 (half-
maximal cytotoxicity concentration) were evaluated for each sample at time point 24 h. Based on these data concentration 10
µM was chosen for screening of antiviral activity of the compounds because at this concentration we observed no or slight
cytotoxicity for most of them.
Insertion of the DNA sequence encoding GFP into the thymidine kinase (TK) gene of Vaccinia virus significantly
improves tracking of the virus without interfering with its ability to replicate. Vaccinia virus strain LIVP-GFP expressed GFP
under the control of the early-late VACV VV7.5 promoter which resulted in efficient GFP expression during all stages of viral
infection so that one can easily monitor the development of viral infection by measuring the level of GFP. Compounds
antiviral activity at 10 µM was evaluated in experiments with CV-1 cells infected with LIVP-GFP similarly to 24. CV-1 cells
were treated with the compounds (10 µM) in duplicate. After 4 h of incubation the medium was removed and cells were
infected by LIVP-GFP at a multiplicity of infection (MOI) of 0.01. LIVP-GFP-infected cells were incubated for additional 24 h
prior to being processed for flow cytometry. The Relative expression of GFP is used for the primary assessment of the
antiviral activity since it shows a decline in viral proteins formation (Table 1). The screening performed showed that five
compounds reduced GFP expression more than twice (Table 1) whereas other compounds lack GFP expression inhibition
potency (see Table A5 of Supplementary Material). These five compounds were retained for further testing. Notice that one
of them (171) was designed in this work whereas four others (115, 114, 111 and 67) were previously studied against VSV.
Incubation of the cells with compounds 171, 67, 115, 111 (Figure 3) at concentration 30 - 50 µM prior to infection results
in 8 - 10 fold inhibition of GFP expression which reflects the strong antiviral activity of these compounds. Due to relatively
high cytotoxicity of the compound 114 (CC50 ≈ 50 µM) its antiviral activity was evaluated at concentrations not exceeding 10
µM: even at this relatively low concentration 114 twice reduce GFP expression level, thus showing rather pronounced
antiviral activity.
171
115
114
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111
67
Figure 3. Structures of the most active compounds identified in this study.
Table 1. CC50 and the antiviral activity measured by Flow cytometry analysis of GFP expression in the infected cells (Cells
viability and Relative expression of GFP are given at concentration of 10 µM). K- (negative control) – untreated, uninfected
cells (cells autoflurescence). K+ (positive control) – untreated, infected cells. Information on all 40 compounds GFP
expression test results is given in Table A5.
Compound
Cells viability, %
CC50, µM
Relative expression of GFP, %
ID
K+
100
8
K-
171
114
67
79 10
74 8
≈100
≈50
100
≈50
≈50
23
28
42
45
48
84 16
88 9
115
111
79 6
We applied the plaque forming assay to analyze the effect of compounds 171, 67, 115, 111, 114 on viral infection
development and infectious viral particles production in CV-1 cells. The virus titer was measured in the medium of infected
cells pre-incubated for 4 h prior to infection with or without (control) above-mentioned compounds taken at different
concentration (Table 2). In parallel real-time monitoring of cell viability using xCelligence Real-Time Cell Analyzer was
ꢁ

performed (see Figure A1 in Supplementary materials). Data obtained using xCelligence system are in a good agreement
with the results of MTT test.
Analysis of the antiviral activity showed that pre-incubation of CV-1 cells with the compounds resulted in the decrease of
virus titer in cell medium by 0.5 – 1 lg(PFU/ml) and these results are consistent with the data obtained by flow cytometry.
There are two lead compounds which exhibit antiviral activity in a concentration dependent manner, namely 171 (∆titre = 0.9 lg
PFU/ml) and 67 (∆titre = 0.8 lg PFU/ml); for other tested compounds the differences in the viral titter were less pronounced
(∆titre = 0.5 – 0.7 lg PFU/ml). As for 114 (∆titre = 0.5 – 0.7 lg PFU/ml) no dependence of the antiviral activity on the compound
concentration was observed together with stimulation of cell proliferation. Compounds 115 and 111 inhibited viral infection
only at the highest concentration used (30 µM) by 0.5 – 0.6 lg PFU/ml and at this concentration 25% of CV-1 cells died.
Based on these experiments, SI values for these compounds have been calculated (Table 3).
2 out of 40 compounds tested in the antiviral screening, namely 171 and 67, display a prominent activity by
approximately tenfold decrease in infectious viral particles production. Compound 67 is characterized additionally by
somewhat lower cytotoxicity in comparison with 171 (under similar conditions 90 and 75% of cells remained viable for 67 and
171, respectively).
Antiviral activity of the studied compounds could be a result of either direct inhibition of viral infection by virus life cycle
disruption or by inducing interferon (IFN) production by the cells. In order to analyze whether compounds work as IFN-α/β
inducers we estimated the level of IFN in murine fibroblasts, infected with murine encephalomyocarditis virus (EMCV) after
treatment with the selected compounds. Studied compounds did not induce IFN-α/β production on a detectible level. Taking
into account that efficacy of induction of IFN-α/β expression varied significantly in different cell lines we additionally tested
induction of IFN-α in mouse spleen cells treated (stimulated) with the compounds 171, 67, 115, and 111. In these
experiments no induction of IFN-α after the treatment of mouse spleen cells with compounds was observed. Noteworthy,
Cycloferon used as a positive control, stimulated IFN expression both in murine fibroblasts and in the mouse spleen cells.
Thus, two most promising compounds 171 and 67 inhibit virus reproduction by at least 8 and 6 folds, respectively in
considerably lower concentrations than their CC50, which makes them eligible candidates for further antiviral research. The
discovered hits were tested for DNA affinity (Ki) according to the procedure reported previously 10. They display reasonable
intercalating activity: lg(Ki) = 6.03 and 5.20 for compounds 171 and 67, respectively.
Table 2. Antiviral activity of potent compounds measured by classical plaque forming assay. n.d. – activity not determined. 1)
Virus titer in the infected cell incubated in the presence of 0.002, 0.02 or 0.1% of DMSO in the cell medium was 3.1 0.3
PFU/ml, similar to K+.
Compound
Virus titer,
C, µM
Viable cells, %
ID
lg(PFU/ml)
K+1)
171
100 0.1
107.8 0.1
96.9 0.2
75.6 0.1
3.3 0.1
1
3.1 0.1
10
50
2.8 0.1
2.4 0.1
ꢅꢁ

67
1
103.6 0.2
131.4 0.1
90.0 0.1
113.7 0.1
110.3 0.1
75 0.0
3.4 0.1
2.8 0.1
2.5 0.0
3.5 0.1
3.3 0.0
2.7 0.1
3.6 0.2
3.3 0.1
2.6 0.1
2.8 0.0
2.8 0.1
2.6 0.1
n.d.
10
50
1
115
111
114
10
30
1
107.2 0.1
110.1 0.1
75 0.0
10
30
1
97.1 0.1
125.3 0.1
125 0.0
5
10
50
17.2 0.0
Table 3. Antiviral activity and cytotoxicity of the compounds tested
Compound
aCC50, µM
ID
Selectivity
Index(SI)c
bIC50, µM
171
67
170
500
92
5
34
50
4
10
22
19
<1
115
111
114
81
4
30
<30
a
cytotoxic concentration 50%, compound concentration required to reduce cell viability by 50% assessed by the
MTT assay.
b inhibitory concentration 50%, compound concentration producing 50% inhibition of virus replication assessed by
the PFU assay.
c selectivity index: ratio between cytotoxic concentration (CC50) and inhibitory concentration (IC50).
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In this work, several broad spectrum antiviral agents acting as nucleic acids intercalators were discovered. First, 12
compounds were selected in virtual screening with the help of developed pharmacophore and QSAR models and then
synthesized. Together with previously studied 43 compounds, they were tested experimentally against Vaccinia virus. Out of
these, 2 molecules displayed high antiviral activity, reasonable DNA affinity and low toxicity. The interferon induction capacity
has not been detected for these compounds which supports our hypothesis about intercalation mechanism of their antiviral
activity.
Supplementary Material
It contains information about the development and performance of the virtual screening tools, synthesis of hit compounds
and the detailed description of the biological assays
Acknowledgements
Kyrylo Klimenko thanks the French Embassy in Ukraine for the PhD fellowship. Elena Goncharova and Rinat Amirkhanov
received financial support from ICBFM SB RAS.
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ꢀꢉꢁ

Highlights:
virtual screening to discover potential nucleic acids intercalators
two hits active against Vaccinia virus were found
intercalating mechanism was proposed for the hits
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