An integrated biological approach to guide the development of metal-chelating inhibitors of influenza virus PA endonuclease

Article abstract of DOI:10.1124/mol.114.095588

The influenza virus PA endonuclease, which cleaves capped cellular pre-mRNAs to prime viral mRNA synthesis, is a promising target for novel anti-influenza virus therapeutics. The catalytic center of this enzyme resides in the N-terminal part of PA (PA-Nte

Full text of DOI:10.1124/mol.114.095588

Supplemental material to this article can be found at:
http://molpharm.aspetjournals.org/content/suppl/2014/12/04/mol.114.095588.DC1.html
1521-0111/87/2/323–337$25.00
MOLECULAR PHARMACOLOGY
http://dx.doi.org/10.1124/mol.114.095588
Mol Pharmacol 87:323–337, February 2015
Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
An Integrated Biological Approach to Guide the Development of
Metal-Chelating Inhibitors of Influenza Virus PA Endonuclease s
Annelies Stevaert, Salvatore Nurra, Nicolino Pala, Mauro Carcelli, Dominga Rogolino,
Caitlin Shepard, Robert A. Domaoal, Baek Kim, Mercedes Alfonso-Prieto,
Salvatore A. E. Marras, Mario Sechi, and Lieve Naesens
Rega Institute for Medical Research, KU Leuven–University of Leuven, Leuven, Belgium (A.S., L.N.); Department of Chemistry
and Pharmacy, University of Sassari, Sassari, Italy (S.N., N.P., M.S.); Department of Chemistry, University of Parma, Parma, Italy
(M.C., D.R.); Center for Drug Discovery, Department of Pediatrics, School of Medicine, Emory University, Atlanta, Georgia (C.S.,
R.D., B.K.); Department of Pharmacy, Kyung-Hee University, Seoul, South Korea (B.K.); Institute for Computational Molecular
Science, Temple University, Philadelphia, Pennsylvania (M.A.P.); and Public Health Research Institute, Department of
Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, New Jersey (S.M.)
Received September 1, 2014; accepted December 4, 2014
ABSTRACT
The influenza virus PA endonuclease, which cleaves capped inhibition of Mn21 versus Mg21, the latter probably being the
cellular pre-mRNAs to prime viral mRNA synthesis, is a promising biologically relevant cofactor. This real-time MB assay allowed
target for novel anti–influenza virus therapeutics. The catalytic us to measure the enzyme kinetics of PA-Nter or perform high-
center of this enzyme resides in the N-terminal part of PA throughput screening. Several DKA derivatives were found to
(PA-Nter) and contains two (or possibly one or three) Mg21 or cause strong inhibition of PA-Nter, with IC50 values comparable to
Mn21 ions, which are critical for its catalytic function. There is that of the prototype L-742,001 (i.e., below 2 mM). Among the
great interest in PA inhibitors that are optimally designed to different compounds tested, L-742,001 appeared unique in
occupy the active site and chelate the metal ions. We focused having equal activity against either Mg21 or Mn21. Three
here on a series of b-diketo acid (DKA) and DKA-bioisosteric compounds (10, with a pyrrole scaffold, and 40 and 41, with an
compounds containing different scaffolds, and determined indole scaffold) exhibited moderate antiviral activity in cell
their structure-activity relationship in an enzymatic assay with culture (EC99 values 64–95 mM) and were proven to affect viral
PA-Nter, in order to build a three-dimensional pharmaco- RNA synthesis. Our approach of integrating complementary
phore model. In addition, we developed a molecular beacon enzymatic, cellular, and mechanistic assays should guide ongoing
(MB)–based PA-Nter assay that enabled us to compare the development of improved influenza virus PA inhibitors.
transcription process, referred to as “cap-snatching.” This
Introduction
reaction appears unique for Orthomyxo-, Arena- and Bunyaviridae
Influenza viruses cause significant morbidity and mortality
(Morin et al., 2010; Reguera et al., 2010; Guilligay et al., 2014). In
and vaccination provides only partial protection in some pop-
ulations, such as the elderly (Treanor et al., 2012). Antiviral
therapy comprises the M2 blockers amantadine and rimantadine
the case of influenza virus, the capped 59 end of a cellular pre-
mRNA is bound by PB2 and subsequently cleaved by PA at 10–13
nucleotides from the cap to serve as primer for viral mRNA
and the neuraminidase inhibitors oseltamivir and zanamivir
synthesis (Bouloy et al., 1978; Plotch et al., 1981; Dias et al., 2009;
(Vanderlinden and Naesens, 2014). The global spread of
Yuan et al., 2009). The catalytic site of the PA endonuclease,
amantadine-resistant influenza viruses and growing resistance
to neuraminidase inhibitors (in particular oseltamivir) underline
residing in its N-terminal part (PA-Nter; residues 1–256 or less),
is highly conserved in influenza A and B viruses. Several crys-
the need for novel therapeutics with a different mode of action.
tallographic studies have revealed the protein structure of
An attractive target is the influenza virus RNA polymerase
PA-Nter, which contains two (or possibly one or three) Mn21
complex (Ruigrok et al., 2010) composed of three proteins: PA,
or Mg21 ions in its catalytic core, although it is still unresolved
PB1, and PB2. The RNA-polymerizing activity resides in the
which metal ions are present in native enzyme (Dias et al., 2009;
PB1 subunit. PB2 and PA carry out the first step of the viral
Yuan et al., 2009; Zhao et al., 2009; DuBois et al., 2012; Kowalinski
et al., 2012; Parhi et al., 2013). In biochemical assays, the enzyme’s
A. Stevaert is holder of a PhD grant from the Flemish Agency for Innovation
activity appears higher with manganese, but magnesium appears
more biologically relevant since the intracellular concentration of
free Mg21 is .1000-fold higher than that of Mn21 (Maret, 2010).
For unrelated nucleases, reduced substrate specificity was observed
when tested with manganese instead of magnesium. This is
probably attributable to the less rigid coordination requirements
of manganese compared with magnesium (Yang et al., 2006).
by Science and Technology (IWT). The authors acknowledge financial support
by the Geconcerteerde Onderzoeksacties [GOA/15/019/TBA] from the KU
Leuven; the Emory Pediatric Center for Drug Discovery fund; the Fondazione
Banco di Sardegna; and the Italian Ministero dell’Istruzione, dell’Università e
della Ricerca [PRIN 2010, 2010W2KM5L_003]. S.A.E. Marras is among a group
of inventors who earn royalties for molecular beacon usage.
dx.doi.org/10.1124/mol.114.095588.
s
This article has supplemental material available at molpharm.aspetjournals.
org.
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324
Stevaert et al.
The available crystal structures of PA-Nter enable rational
To validate the inhibition data for PA-Nter, we used two cell
design of PA inhibitors (PAIs). The first class of influenza en- culture methods: an influenza virus ribonucleoprotein (vRNP)
donuclease inhibitors (Tomassini et al., 1994; Hastings et al., reconstitution assay and a virus yield replication assay. A
1996) are 4-substituted-2,4-dioxobutanoic acids with a charac- basic mechanistic study was also performed to verify that the
teristic b-diketo acid (DKA) motif. Among this series, L-742,001 antivirally active DKAs indeed act upon viral RNA synthesis.
(1 in Table 1) was identified as a particularly potent inhibitor,
both in enzyme- and cell-based antiviral assays (Hastings et al.,
1996; Nakazawa et al., 2008). Several other classes of endo-
nuclease inhibitors have been reported (Cianci et al., 1996;
Materials and Methods
Compounds. The synthetic route to obtain 1–6 (i.e., L-742,001
and derivatives) can be found in the Supplemental Data and
Tomassini et al., 1996; Singh and Tomassini, 2001; Iwai
Supplemental Methods. Compound 7 was obtained from Sigma-Aldrich
et al., 2010; Baughman et al., 2012; Sagong et al., 2013; Chen
(St. Louis, MO). The procedures for chemical synthesis of 8–11 and 30–
47 were reported elsewhere (Tomassini et al., 1994; Sechi et al., 2004,
2005, 2006; Maurin et al., 2004; Zeng et al., 2008; Bacchi et al., 2008,
2011; Reddy et al., 2011). Ribavirin (Virazole; ICN Pharmaceuticals,
Costa Mesa, CA) and chloroquine diphosphate salt (Sigma-Aldrich)
et al., 2014). All of these compounds bear chelating motifs able to
bind the bivalent metal ions in the catalytic core of PA-Nter
(Rogolino et al., 2012). A similar concept has led to clinical de-
velopment of HIV integrase (HIV IN) inhibitors, and is also
explored to design inhibitors of HIV RNase H or HCV polymerase. were included as reference compounds. The test compounds were stored
as a 20–100 mM stock solution in dimethyl sulfoxide. In all cellular
assays, the final dimethyl sulfoxide concentration was kept below 0.8%,
a concentration that was free of aspecific effects on cell viability or
luciferase activity.
Pharmacophore Building. Twelve of the more active compounds
(1–6, 10, 16, 34, 39–41) were chosen for generating the pharmacophore
model. For each compound a full minimized model was built in Mo-
lecular Operating Environment software package (MOE, version
2010.11; Chemical Computing Group Inc., Montreal, QC, Canada)
using MMFF94x force field with standard bond length and angles. The
In this research, we investigated a series of DKAs (and DKA-
bioisosteric compounds) incorporating different chemical scaf-
folds that were synthesized as specific PAIs (1–6, Table 1) or
were originally designed toward HIV IN (7–47, Tables 1 and 2)
(Maurin et al., 2004; Sechi et al., 2004, 2005, 2006; Bacchi et al.,
2008, 2011; Zeng et al., 2008; Reddy et al., 2011). Although these
compounds contain relevant scaffolds for PAI development,
information on their activity against PA-Nter is lacking. Hence,
their structure-activity relationship toward PA-Nter was ana-
lyzed to build a plausible pharmacophore model. A selection of obtained database was used as starting point for the pharmacophore
elucidation procedure implemented in MOE. The selected search
scheme was PPCH_All (Planar-Polarity-Charge-Hydrophobicity), and
all settings were kept as default with the exception of conformations
(set to stochastic search). Among the proposed pharmacophore queries
the best one was chosen on the basis of statistical parameters, i.e., high
correlation coefficient and lower root-mean-square deviation value.
Production of Recombinant Influenza Virus PA-Nter Pro-
tein. The coding sequence for PA-Nter (i.e., residues 1–217 from the
PA protein of influenza virus strain A/X-31) was cloned in the pET28a
(1) plasmid (Merck KGaA, Darmstadt, Germany) with an N-terminal
6xHis-tag, and this bacterial expression plasmid was transformed
compounds was also evaluated against untruncated PA.
In the case of HIV IN, the potency of DKAs in enzymatic
assays was found to depend on which bivalent metal ion (Mg21
or Mn21) was used (Marchand et al., 2003). This prompted us to
investigate whether a similar metal ion dependency may occur
when evaluating PAIs in an enzymatic assay, such as the novel
molecular beacon (MB) assay presented here. This real-time
MB method, applicable for high-throughput screening, also
enabled comparison of the kinetic parameters of PA-Nter for
different substrates or metal ions.
ABBREVIATIONS: 1, (Z)-4-(1-benzyl-4-(4-chlorobenzyl)piperidin-4-yl)-2-hydroxy-4-oxobut-2-enoic acid; 2, (Z)-methyl 4-(1-benzyl-4-(4-chlorobenzyl)
piperidin-4-yl)-2-hydroxy-4-oxobut-2-enoate; 3, (Z)-4-(4-(4-chlorobenzyl)-1-(4-fluorobenzyl)piperidin-4-yl)-2-hydroxy-4-oxobut-2-enoic acid; 4,
(Z)-methyl 4-(4-(4-chlorobenzyl)-1-(4-fluorobenzyl)piperidin-4-yl)-2-hydroxy-4-oxobut-2-enoate; 5, (Z)-4-(4-(4-chlorobenzyl)-1-(cyclohexylmethyl)piperidin-4-yl)-
2-hydroxy-4-oxobut-2-enoic acid; 6, (Z)-methyl 4-(4-(4-chlorobenzyl)-1-(cyclohexylmethyl)piperidin-4-yl)-2-hydroxy-4-oxobut-2-enoate; 7, 2,3-dihydroxyfumaric
acid; 8, (Z)-3-hydroxy-1-phenyl-3-(1H-1,2,4-triazol-5-yl)prop-2-en-1-one; 9, (Z)-3-hydroxy-1-(2-hydroxyphenyl)-3-phenylprop-2-en-1-one; 10, (Z)-4-(1-(4-
fluorobenzyl)-1H-pyrrol-2-yl)-2-hydroxy-4-oxobut-2-enoic acid; 11, (Z)-methyl 4-(1-(4-fluorobenzyl)-1H-pyrrol-2-yl)-2-hydroxy-4-oxobut-2-enoate; 12,
(Z)-2-hydroxy-4-oxo-4-phenylbut-2-enoic acid; 13, (Z)-methyl 2-hydroxy-4-oxo-4-phenylbut-2-enoate; 14, (Z)-2-hydroxy-4-(2-methoxyphenyl)-4-
oxobut-2-enoic acid; 15, (Z)-methyl 2-hydroxy-4-(2-methoxyphenyl)-4-oxobut-2-enoate; 16, (Z)-2-hydroxy-4-(4-methoxyphenyl)-4-oxobut-2-enoic
acid; 17, (Z)-4-(4-chlorophenyl)-2-hydroxy-4-oxobut-2-enoic acid; 18, (Z)-4-(2-chlorophenyl)-2-hydroxy-4-oxobut-2-enoic acid; 19, (Z)-methyl 4-(2-
chlorophenyl)-2-hydroxy-4-oxobut-2-enoate; 20, (Z)-4-(3,5-bis(benzyloxy)phenyl)-2-hydroxy-4-oxobut-2-enoic acid; 21, (2Z,5Z)-2,6-dihydroxy-4-oxo-
6-phenylhexa-2,5-dienoic acid; 22, (2Z,5Z)-methyl 2,6-dihydroxy-4-oxo-6-phenylhexa-2,5-dienoate; 23, (2Z,5Z)-2,6-dihydroxy-6-(2-methoxyphenyl)-4-
oxohexa-2,5-dienoic acid; 24, (2Z,5Z)-6-(2-chlorophenyl)-2,6-dihydroxy-4-oxohexa-2,5-dienoic acid; 25, 6-benzyl-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid; 26, 6-benzyl-1-(2-hydroxyethyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid; 27, 1,6-dibenzyl-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid; 28, ethyl 1,6-dibenzyl-4-oxo-1,4-dihydroquinoline-3-carboxylate; 29, N-(4-fluorobenzyl)-5-hydroxy-2-isopropyl-1-methyl-6-oxo-1,6-
dihydropyrimidine-4-carboxamide; 30, (Z)-2-hydroxy-4-(1-methyl-1H-indol-2-yl)-4-oxobut-2-enoic acid; 31, (Z)-4-(1-benzyl-1H-indol-2-yl)-2-hydroxy-
4-oxobut-2-enoic acid; 32, (Z)-methyl 2-hydroxy-4-(1-methyl-1H-indol-2-yl)-4-oxobut-2-enoate; 33, (Z)-methyl 4-(1-ethyl-1H-indol-2-yl)-2-hydroxy-4-
oxobut-2-enoate; 34, (Z)-2-hydroxy-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indol-6-yl)-4-oxobut-2-enoic acid; 35, (Z)-4-(5-ethyl-5H-[1,3]dioxolo[4,5-f]indol-6-
yl)-2-hydroxy-4-oxobut-2-enoic acid; 36, (Z)-4-(5-benzyl-5H-[1,3]dioxolo[4,5-f]indol-6-yl)-2-hydroxy-4-oxobut-2-enoic acid; 37, (Z)-methyl 4-(5-benzyl-
5H-[1,3]dioxolo[4,5-f]indol-6-yl)-2-hydroxy-4-oxobut-2-enoate; 38, (Z)-methyl 2-hydroxy-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indol-6-yl)-4-oxobut-2-
enoate; 39, (Z)-2-hydroxy-4-(1-methyl-1H-indol-3-yl)-4-oxobut-2-enoic acid; 40, (Z)-4-(1-ethyl-1H-indol-3-yl)-2-hydroxy-4-oxobut-2-enoic acid; 41,
(Z)-4-(1-benzyl-1H-indol-3-yl)-2-hydroxy-4-oxobut-2-enoic acid; 42, (Z)-2-hydroxy-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indol-7-yl)-4-oxobut-2-enoic acid;
43, (Z)-methyl 2-hydroxy-4-(5-methyl-5H-[1,3]dioxolo[4,5-f]indol-7-yl)-4-oxobut-2-enoate; 44, 3-(1-ethyl-1H-indol-3-yl)-4-hydroxy-1H-pyrrole-2,5-
dione; 45, (E)-2-hydroxy-2-(2-oxoindolin-3-ylidene)acetic acid; 46, (E)-methyl 2-hydroxy-2-(2-oxoindolin-3-ylidene)acetate; 47, (E)-methyl 2-hydroxy-
2-(2-oxo-1-phenylindolin-3-ylidene)acetate; DFO, Dragonfly Orange dye; DKA, b-diketo acid; DPBA, 2,4-dioxo-4-phenylbutanoic acid; fluc, firefly
luciferase; HIV IN, human immunodeficiency virus integrase; MB, molecular beacon; MDCK, Madin-Darby canine kidney; ML, metal ligator; MTS,
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PAI, influenza virus PA inhibitor; PA-Nter, N-terminal part of
influenza virus PA; PSA, polar surface area; RT-PCR, reverse transcription–polymerase chain reaction; vRNA, viral RNA; vRNP, viral ribonucleoprotein.

Metal-Chelating Inhibitors of Influenza Virus Endonuclease
325
TABLE 1
Inhibition of PA-Nter by different DKA or DKA-bioisosteric compounds as determined in the plasmid-based enzymatic assay
One microgram of PA-Nter was incubated with 1 mg of M13mp18 plasmid substrate and the compounds. After 2 hours incubation, cleavage was
assessed by gel electrophoresis.
a
Scaffold
Compound
R1
R2
IC50
mM
Piperidine scaffold
1 (L-742,001)
H
Me
H
Me
H
Me
Ph
Ph
0.5
0.5
1.4
0.4
0.5
0.6
2
3
4
5
6
p-F-Ph
p-F-Ph
Cy
Cy
Dihydroxybutenedioic acid
7
—
—
.500
1,3-Dioxo-1-phenylpropane scaffold
and triazole bioisostere
8
9
H
77
Ph
o-OH
.500
Pyrrole scaffold
10 (L-731,988)
H
Me
—
—
0.8
5.5
11
2,4-Dioxo-4-phenylbutanoic
acid (DPBA) scaffold
12
13
14
15
16
17
18
19
H
Me
H
Me
H
H
H
Me
H
H
2.7
14
2.2
11
2.0
9.8
9.8
15
o-OMe
o-OMe
p-OMe
p-Cl
o-Cl
o-Cl
20 (L-708,906)
—
—
137
2,4,6-Trioxo-6-
phenylhexanoic acid scaffold
21
22
23
24
H
Me
H
H
H
H
o-OMe
o-Cl
.500
.500
.500
.500
Quinolone scaffold
25
26
27
28
H
H
H
Et
H
.500
.500
.500
.500
CH
Bn
Bn
2
CH OH
2
(continued)

326
Stevaert et al.
TABLE 1—Continued
a
Scaffold
Compound
R1
R2
IC50
Hydroxypyrimidine-
carboxamide scaffold
29
—
—
.500
aIC50, 50% inhibitory concentration. For each compound the IC50 value was calculated using nonlinear regression analysis. Values are the
mean of at least three independent experiments.
into Escherichia coli BL21-CodonPlus cells (Agilent Technologies,
To determine the kinetic parameters for the different MB sub-
Santa Clara, CA). The plasmid was subjected to site-directed mutagen- strates, the substrate concentrations were varied from 25 nM to 5 mM,
esis (QuikChange II; Agilent Technologies) to prepare the K134A-
substituted PA-Nter enzyme.
The bacteria were grown to an optical density of 0.6, when
whereas the enzyme concentration was kept constant at 0.04 mg per
microliter. These tests were performed in the presence of 1 mM MnCl2
and in 10-ml reaction volumes in a 384-well plate format. MB-het1-
isopropylthio-b-galactoside was added at a final concentration of FAM, MB-het2-FAM, and MB-het3-FAM (FAM-labeled) were excited
1 mM to induce expression of recombinant proteins for 5 hours at at 495 nm and emission was recorded at 520 nm, whereas the DFO-
37°C. The bacterial cells were ruptured using a French press, and the labeled MB molecules MB-C15-DFO, MB-A15-DFO, MB-U6A2U7-DFO,
proteins were purified by 6xHis-Ni-NTA chromatography (Qiagen, MB-OH-DFO, and MB-het2-DFO were read at 554-nm excitation and
Valencia, CA), followed by buffer exchange using PD-10 desalting columns 576-nm emission wavelength. The increasing fluorescence signal was
(GE Healthcare, Diegem, Belgium) to keep the proteins in storage buffer measured every 14 seconds for 60 minutes using a spectrofluorometer
(50 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM b-mercaptoethanol, 50% (Safire2; Tecan, Männedorf, Switzerland). All reactions were performed
glycerol). Protein purity was verified by SDS-PAGE with Coomassie Blue
staining, and protein concentration was determined by Bradford assay.
Finally, the purified proteins were divided in aliquots and stored at –80°C.
in duplicate. The initial reaction velocity V0 (determined at concen-
trations at which no substrate inhibition occurred) was calculated
as the slope for the 5- to 15-minute time interval. To estimate the
Plasmid-Based Endonuclease Assay. This enzymatic endonu- Km values, the Michaelis-Menten equation was fitted to these V0 data,
clease activity assay was performed according to a previously published on the basis of nonlinear regression analysis and using GraphPad
method (Carcelli et al., 2014) with minor modifications. One microgram Prism software.
of recombinant PA-Nter was incubated with 1 mg (16.7 nM) of single-
Similar conditions were used for the inhibition assays. Serial
stranded circular DNA plasmid M13mp18 (Bayou Biolabs, Metairie, dilutions of the test compounds were added to the reaction mixture
Louisiana) in the presence of the test compounds and at a final volume containing buffer (with or without added metal ions) and 20 or 100 nM
of 25 ml. The assay buffer contained 50 mM Tris-HCl pH 8, 100 mM MB, and the reaction was started by adding 0.4 mg of PA-Nter. All
NaCl, 10 mM b-mercaptoethanol, and 1 mM MnCl2 (unless stated reactions were performed in duplicate. The initial cleavage velocity was
otherwise). The reaction was incubated at 37°C for 2 hours and then plotted against the compound concentration on a semilogarithmic scale,
stopped by heat inactivation (80°C, 20 minutes). The endonucleolytic and for each compound the IC50 value was calculated using nonlinear
digestion of the plasmid was visualized by gel electrophoresis on a 1%
agarose gel with ethidium bromide staining. The amount of remaining
intact plasmid was quantified by ImageQuant TL software (GE Health-
care). The percentage inhibition of PA endonuclease activity was plotted
regression analysis (GraphPad Prism). Values were the mean of at least
two independent experiments.
The cleavage pattern of the molecular beacons was studied in the
same buffer conditions, but the reaction was stopped at the indicated
against the compound concentration on a semilogarithmic plot, using time points by heat inactivation (80°C, 20 minutes). The samples were
GraphPad Prism software (GraphPad Software, La Jolla, CA). Values
were the mean 6 S.E.M. of three independent experiments. The 50%
inhibitory concentrations (IC50) were obtained by nonlinear least-
squares regression analysis.
loaded on 15% Tris/borate/EDTA–urea polyacrylamide gels (Criterion;
Bio-Rad, Hercules, CA) for denaturing nucleic acid PAGE. The bands
were visualized by fluorescent gel imaging using an Ettan difference
gel electrophoresis imager apparatus (GE Healthcare).
Molecular Beacon-Based Endonuclease Assay. The sequen-
Cells and Media. Madin-Darby canine kidney (MDCK) cells (a
ces of the eight MB substrates are given in Table 3. MB-C15-DFO kind gift from M. Matrosovich, Marburg, Germany) and human
(Dragonfly Orange), MB-A15-DFO, MB-U6A2U7-DFO, MB-OH-DFO, embryonic kidney 293T (HEK293T) cells (purchased from Thermo
and MB-het2-DFO were purchased from Eurogentec (Seraing, Fisher Scientific, Waltham, MA) were grown in Dulbecco’s modified
Belgium), whereas MB-het1-FAM, MB-het2-FAM, and MB-het3- Eagle medium supplemented with 10% fetal calf serum, 1 mM sodium
FAM were synthesized by S.A.E. Marras. The molecular beacons pyruvate, and 0.075% sodium bicarbonate. Virus experiments were per-
were prepared by automated solid-phase DNA synthesis on an Applied formed in MDCK infection medium, consisting of Ultra MDCK
Biosystems (Foster City, CA) ABI 394 DNA/RNA synthesizer. A medium (Lonza, Basel, Switzerland) supplemented with 0.0225%
controlled-pore glass column (Biosearch Technologies, Novato, CA) sodium bicarbonate, 2 mM L-glutamine, and 2 mg/ml tosylphenyla-
was used to incorporate a quencher moiety (dabcyl) at the 39, end and lanylchloromethylketone-treated trypsin (Sigma-Aldrich). The cells
a fluorescein phosphoramidite (Glen Research, Sterling, VA) was used were incubated in a 5% CO2 humidified atmosphere.
to incorporate a fluorophore moiety at the 59 end of the oligodeoxy-
ribonucleotides. The molecular beacons were then purified by high-
pressure liquid chromatography on a Beckman Coulter System
vRNP Reconstitution Assay. The assay to determine the in-
hibitory effect of the compounds on reconstituted influenza virus
vRNPs is described in more detail elsewhere (Meneghesso et al., 2012;
Goldchromatograph (Indianapolis, IN) through a C-18 reverse-phase Stevaert et al., 2013). Four sets of vRNP reconstituting plasmids
column (Waters Corporation, Milford, MA). Concentrations were deter-
mined on a Nanodrop 1000 Spectrophotometer (Thermo Scientific,
Wilmington, DE).
derived from different influenza virus (sub)types were used: 1) four
reverse genetics plasmids derived from influenza A/PR/8/34 (encoded
pVP-PB1, pVP-PB2, pVP-PA, and pVP-NP, which contain the cDNAs
in the bidirectional expression cassette of pHH21), kindly given by
The PA-Nter assay was performed at 37°C in a buffer consisting of
50 mM Tris-HCl pH 8, 100 mM NaCl, and 10 mM b-mercaptoethanol, M. Kim (Korea Research Institute of Chemical Technology, Daejeon)
to which either 1 mM MnCl2, 1 mM MgCl2, or no metal was added. (Kim et al., 2013); 2) four plasmids derived from the avian influenza

Metal-Chelating Inhibitors of Influenza Virus Endonuclease
327
TABLE 2
reporter plasmid, and cotransfected into HEK293T cells using
Lipofectamine 2000 (Meneghesso et al., 2012; Stevaert et al., 2013).
The cells were transferred to a 96-well plate containing serial dilutions of
the test compounds, and after 24 hours incubation at 37°C, luciferase
activity was determined using the ONE-Glo assay system from Promega
(Madison, WI). The 50% effective concentration (EC50) was defined as the
compound concentration causing 50% reduction in the vRNP-driven
firefly luciferase signal, compared with cells receiving medium instead of
compound. These EC50 values were calculated by interpolation assuming
a semi-log dose-response effect. In parallel, compound cytotoxic activity
was determined in untransfected HEK293T cells that had been in-
cubated with serial dilutions of the compounds for 24 hours, using the
MTS cell viability assay (CellTiter 96 AQueous One Solution Cell Pro-
liferation Assay; Promega). These spectrophotometric data were used to
calculate the 50% cytotoxic concentration (CC50), i.e., the concentration
reducing cell viability by 50%, compared with the wells receiving medium
instead of compound. Ribavirin was included as the reference compound.
Virus Yield Assay. To determine anti–influenza virus activity in
infected cell cultures, we performed a virus yield assay (Stevaert
et al., 2013). One day prior to infection, MDCK cells were seeded into
96-well plates at 25,000 cells per well. At day 0, serial dilutions of the
test compounds were added, immediately followed by infection with
influenza A/PR/8/34 virus. The multiplicity of infection was 150 CCID50
per well [50% cell culture infectious dose, determined by the method of
Reed and Muench]. After 24 hours incubation at 35°C, the supernatants
were collected and stored at –80°C. The virus amount in these samples
was estimated by determining the viral genome copy number in a one-
step quantitative real-time reverse transcription (RT-PCR) assay
(CellsDirect One-Step qRT-PCR kit; Invitrogen/Life Technologies,
Gent, Belgium), with influenza virus M1–specific primers and probe
[see Vanderlinden et al. (2010) for all details]. Absolute quantification
of viral RNA (vRNA) copies was performed by including an M1-plasmid
standard. The EC99 and EC90 values were calculated by interpolation
from data of at least three experiments and defined as the compound
concentration causing, respectively, a 2-log10 and 1-log10 reduction in
vRNA copy number, compared with the virus control receiving no
compound. In parallel, the CC50 values after 24 hours incubation with
compounds were determined in uninfected MDCK cells, using the MTS
cell viability assay (CellTiter 96 AQueous One Solution Cell Proliferation
Assay; Promega). Ribavirin was included as the reference compound.
One-Cycle Virus Replication Assay. A simplified time-of-
addition experiment was performed as previously described in
more detail (Stevaert et al., 2013). The compounds were added to
confluent MDCK cells at either 30 minutes before or 1 hour after virus
infection with influenza virus A/PR/8/34, and incubated at 35°C. At
8 hours postinfection, the supernatant was removed and total cellular
RNA extracts were prepared with the RNeasy Mini Kit (Qiagen). To
quantify the negative-sense vRNA, the samples were analyzed by two-
step real-time RT-PCR (Vanderlinden et al., 2010). cDNA synthesis was
performed on 0.5 mg of total cellular RNA using Moloney murine
leukemia virus (M-MLV) reverse transcriptase (Invitrogen/Life Technol-
ogies) and 80 nM M1-FOR primer. Real-time PCR was then performed,
using influenza virus M1-specific primers and probe (Vanderlinden et al.,
2010), and quantitative PCR MasterMix (Eurogentec). All samples were
analyzed in duplicate. The vRNA copy number was quantified by
including an M1-plasmid standard, and the fold increase in vRNA copies
was calculated relative to the viral copy number added at time zero.
Chloroquine was included as the reference compound.
Inhibition of PA-Nter by different DKA or DKA-bioisosteric indole
derivatives as determined in the plasmid-based enzymatic assay
One microgram of PA-Nter was incubated with 1 mg of M13mp18 plasmid substrate
and the compounds. After
electrophoresis.
2 hours incubation, cleavage was assessed by gel
Indole scaffold
Compound
R1
R2
IC50
mM
30
31
32
33
H
Me
Bn
Me
Et
3.4
22
H
Me
Me
10
4.1
34
35
36
37
38
H
Me
Et
Bn
Bn
Me
0.6
5.5
8.9
204
21
H
H
Me
Me
39
40
41
H
H
H
Me
Et
Bn
0.6
0.8
1.6
42
43
H
Me
Me
Me
3.5
22
44
—
Et
.500
45
46
47
H
Me
Me
H
.500
.500
.500
H
Ph
A/turkey/England/50-92/1991 (H5N1) virus, containing the cDNA
sequences in a pol-II–driven expression cassette, generously given by
W. Barclay (Imperial college London, United Kingdom) (Moncorgé et al.,
2010; in this set, the PB2 protein contains the avian influenza Glu627
residue); 3) identical to the previous set, with the exception that the PB2-
expressing plasmid was mutated to encode the human influenza Lys627
residue (Moncorgé et al., 2010); 4) four reverse genetics plasmids derived
from influenza B/Yamanashi/166/98 [designated pAB251-PB1, pAB252-
PB2, pAB253-PA, and pAB255-NP, which contain the cDNAs in a
bidirectional expression cassette, generously donated by J. McCullers
(St. Jude Children’s Research Hospital, Memphis, TN) (Hoffmann et al.,
2002)]. Set 1 was used in combination with an influenza A–specific firefly
luciferase (fluc) reporter plasmid (Kim et al., 2013) that was also given
by M. Kim. For sets 2 and 3, the fluc reporter was a kind gift from
W. Barclay. For set 4, an influenza B–specific fluc reporter plasmid was
used, kindly donated by K. Nagata, University of Tsukuba, Tsukuba,
Ibaraki, Japan (Wakai et al., 2011).
Results
Selection of DKAs and Derivatives. Tables 1 and 2
show the chemical structures of the different series of DKA
derivatives that we evaluated for inhibitory activity against
PA-Nter. In the known PAI, L-742,001 (1), included as the
reference, the b-diketo acid motif is attached to a piperidine
Briefly, the four relevant plasmids (i.e., the expression plasmids for
PB1, PB2, PA and NP) were combined with the corresponding fluc moiety, which carries two cyclic substituents. These two “wings”

328
Stevaert et al.
TABLE 3
Endonucleolytic cleavage by PA-Nter of different MB substrates
Signal-to-noise
a
MB
Sequence
Km 6 S.E.M.
ratiob 6 S.E.M.
mM
MB-C15-DFO
MB-A15-DFO
59‐[DFO]‐GCAGG CCCCCCCCCCCCCCC CCTGC‐[BHQ-2]‐39
59‐[DFO]‐GCAGG AAAAAAAAAAAAAAA CCTGC‐[BHQ-2]‐39
0.09 6 0.004
0.36 6 0.01
8.2 6 1.3
9.4 6 2.3
11.7 6 0.6
MB-U6A2U7-DFO 59‐[DFO]‐GCAGG dUdUdUdUdUdUAAdUdUdUdUdUdUdU CCTGC‐ 0.23 6 0.02
[BHQ-2]‐39
MB-het1-FAM
MB-het2-FAM
MB-het3-FAM
59-[FAM]-GCGAGC TAGGAAACACCAAAGATGATATTT GCTCGC-
[DABCYL]-39
59-[FAM]-CGCACG TTATGCTAAGCAAGTAAC CGTGCG-
[DABCYL]-39
59-[FAM]-CGAGCG GCTGGTAAGGGTTTCCATATAA CGCTCG-
[DABCYL]-39
59-[DFO]‐CGCACG TTATGCTAAGCAAGTAAC CGTGCG‐[BHQ-2]‐39 0.46 6 0.01
59‐[DFO]‐dUdUdUdUdU GGGGGGGGGGGGGGCCCCCCCCCCCC
CC‐[BHQ2]‐39
0.64 6 0.03
0.15 6 0.01
0.46 6 0.06
4.4 6 0.3
5.5 6 0.4
7.0 6 0.6
MB-het2-DFO
MB-OH-DFO
5.7 6 0.8
1.8 6 0.1
0.22 6 0.01
BHQ2, Black Hole Quencher 2 dye (Biosearch Technologies).
aFor each MB, the Michaelis–Menten equation was fitted to the initial reaction rate to estimate the Km values by nonlinear regression analysis.
Values shown are the averages 6 S.E.M. of at least two independent experiments.
bFold increase at 60 minutes using 100 nM MB.
occupy opposite hydrophobic pockets around the catalytic center (and ethyl ester 28), which bear a monoketo acid motif, and
of PA-Nter and are considered crucial for achieving strong the bioisosteric compound 29, where the DKA moiety was
binding to PA-Nter (DuBois et al., 2012; Stevaert et al., 2013). To replaced by a 4,5-dihydroxypyrimidine-4-carboxamide scaffold.
acquire more information about the structural features of this
Activity in the Plasmid-Based PA Endonuclease
prototype, we prepared some closely related analogs (3 and 5), Assay. We used a PA endonuclease assay based on end-point
N-substituted at the piperidine ring, and also considered the gel electrophoretic analysis of substrate cleavage (Fig. 1). Among
diketo methylesters (2, 4, and 6).
the various PA-Nter substrates that were reported to work (Dias
Moreover, on the basis of the assumption that the DKA et al., 2009; DuBois et al., 2012; Kowalinski et al., 2012; Parhi
moiety is essential for anti-PA activity, our aim was to explore et al., 2013; Bauman et al., 2013; Datta et al., 2013; Chen et al.,
this structural motif and the related chemical space. First, we 2014), we chose the M13mp18 single-stranded DNA plasmid
selected a series of DKA analogs carrying an indole scaffold since it is commercially available. The active site substitution
(30–43; Table 2) in which major modifications were made to K134A in the PA-Nter enzyme completely abolished the en-
determine the effect of different substituents on activity. Second, donucleolytic cleavage, in agreement with previous reports
we included other series of compounds where: 1) the b-diketo (Hara et al., 2006; Yuan et al., 2009; Crépin et al., 2010).
acid group was been replaced with a bioisostere (44–47); 2) the
At the time of its discovery (Hastings et al., 1996) L-742,001
aromatic scaffold had been replaced with a second DKA (1) was reported to have an IC50 value of 0.43 mM in an
functionality (7); 3) a triazole bioisostere has been incorporated enzymatic endonuclease assay with a capped mRNA substrate
in place of the carboxyl group (8); 4) an additional phenyl group and influenza virus ribonucleoprotein complexes, which con-
had been introduced on the 1,3-dioxo-phenylpropane framework tain the entire PA protein within the heterotrimeric viral
(9); and 5) the DKA motif had been connected to a different polymerase. We here obtained the same IC50 value (i.e., 0.5 mM;
heterocycle (10, 11). Third, since 2,4-dioxo-4-phenylbutanoic Table 1) against isolated PA-Nter. This supports the view that
acid (DPBA, 12; Table 1) represents a suitable platform to ex- the endonuclease catalytic site has the same structure in
amine structural and biologic features of DKAs, several sub- isolated PA-Nter as in native polymerase, and implicates that
stitutions on the phenyl (13–20), as well as on the diketo moiety enzymatic assays with PA-Nter are relevant for developing
with an additional keto group (21–24), were evaluated.
catalytic site inhibitors (Crépin et al., 2010; Noble et al., 2012).
Finally, inspired by studies on HIV IN (Bacchi et al., 2011), Regarding the direct analogs of L-742,001 that we tested
we considered two additional chemotypes as putative DKA- (Table 1), the IC50 data demonstrate that the activity is
related compounds: the 4-quinolone-3-carboxylic acids 25–27 unchanged when the phenyl moiety of L-742,001 is replaced by
Fig. 1. Illustration of the activity of DKAs in the plasmid-based PA-Nter assay. Recombinant PA-Nter (1 mg) was incubated with 1 mg (16.7 nM) of
single-stranded circular DNA plasmid M13mp18 in the presence of the test compounds. The endonucleolytic digestion of the plasmid was visualized by
gel electrophoresis, and the amount of remaining intact plasmid was quantified. For L-742,001 and 10 (L-731,988), the IC50 values were 0.5 mM and
0.8 mM, respectively. Wild-type (WT) enzyme showed complete cleavage with Mn2+ but was inactive when Mn2+ was omitted from the reaction mixture or
replaced by Mg2+. The endonuclease activity was also completely abolished by the active site substitution K134A.

Metal-Chelating Inhibitors of Influenza Virus Endonuclease
329
a cyclohexyl group (5), and only slightly higher when this phenyl
Pharmacophore Model Generation. To elucidate the
carries a p-fluoro substituent (3). Also, methyl-esterification of structural requirements for novel PAIs, a three-dimensional
the terminal carboxyl in the DKA part did not affect the in- pharmacophore model was built (Fig. 2) on the basis of the
hibitory activity (2, 4, and 6).
efficacy data of a selection of tested compounds; namely, the
A prototypic inhibitor that has been used in several en- training set contained twelve molecules with an IC50 value
zymatic studies with PA-Nter is 2,4-dioxo-4-phenylbutanoic against PA-Nter of 2 mM or less, i.e., the b-diketo acids or esters
acid (DPBA, 12). The IC50 value for DPBA in our plasmid assay 1–6, 10, 16, 34, and 39–41, and these were submitted to
was 2.7 mM, which is comparable to the data reported by others a pharmacophore generation protocol using the Molecular
(Dias et al., 2009; Kowalinski et al., 2012; Noble et al., 2012). Operating Environment software package. With the excep-
This value was unchanged upon addition of a methoxy in ortho tion of 16, 34, and 39, all compounds also showed antiviral
(14) or para position (16). Addition of an ortho-chlorine had no activity in cell culture (see below). Fifteen PAI pharmaco-
effect (18), but a chlorine in para position (17) reduced the phore models were generated and the best hit was evaluated
potency by a factor of 4. We also included three methyl esters in terms of statistical parameters, such as correlation coefficient
(13, 15, and 19); their IC50 values were consistently 4- to 5-fold and root-mean-square deviation value. This model featured five
higher than the corresponding carboxylic acids. Also, replace- pharmacophore elements, including three metal ligator (ML)
ment of the carboxylic functionality with a triazole ring bio- features, one aromatic or hydrophobic element (Hyd-Aro), and
isostere (8) or a phenyl moiety (9) caused a dramatic reduction one cyclic aromatic region (Aro) (Fig. 2, A and B). This five-point
in inhibitory activity. The length of the DKA arm seems optimal pharmacophore was then validated by screening our in-house
in DPBA, since analogs carrying an extra keto group (21–24) database of about 150 compounds, chosen on the basis of their
were devoid of activity. The critical role for the phenyl ring of previously evaluated chelating ability toward metal cofactors
DPBA is evident from the fact that unsubstituted dihydroxy- of other metalloenzymes, and considering compounds having
butenedioic acid (7) was inactive. Introduction of two large molecular diversity. All selected compounds were retrieved from
aromatic substituents (benzyloxygroups) onto this phenyl the database and fitted nicely with the chemical features of
ring [20; known as the HIV IN inhibitor L-708,906 (Hazuda our hypothesis, thus confirming the reliability of the proposed
et al., 2000)] led to a dramatic (50-fold) reduction in activity. model.
The same DKA fragment as in DPBA and L-742,001 is pres-
As shown in Fig. 2A, the overall mapping results indicate
ent in the extended series of indole derivatives that we tested that almost all compounds aligned in a similar spatial arrange-
(Table 2). Several of these had strong activity against PA-Nter ment within the model, highlighting important structural
with IC50 values below 1 mM, which is comparable to the value similarities between compounds bearing piperidine and indole
for L-742,001. The following parameters were found to influence scaffolds. A good fitting was shown for the coplanar diketo acid/
the activity: 1) The N-substituent of the indole increased the ester fragment of all compounds within three points, whereas
IC50 value in the order methyl , ethyl , benzyl (cf., 30–31, 34– the orientation of the substituted heterocyclic portion mapped
36, and 39–41). 2) The location of the DKA arm on the indole into the remaining pharmacophore elements, with the exception
moiety (i.e., at position 2 or 3) had variable impact in relation- of the para-F-benzyl moiety of the pyrrole scaffold (10), which
ship to the size of the indole moiety. Position 3 represents the has a different orientation. Moreover, the N-methylcyclohexyl
optimum geometry in the case of the simple indole ring (compare substituent of the diketo methyl ester 6 is located in an opposite
39 and 41 with 30 and 31, respectively). 3) When the indole part conformation with respect to the same moiety in the correspond-
was enlarged by adding an extra dioxole ring, this extension led ing acid 5. In L-742,001, the two aromatic moieties attached
to increased potency for the 2-diketo derivatives (compare 34 to the piperidine ring are oriented in opposite directions and
and 36 with 30 and 31, respectively). However, for the com- perpendicularly to the DKA motif. Tolerance for various
pounds bearing the diketo motif in 3-position, this extra dioxole orientations of the aromatic and hydrophobic moieties agrees
ring appeared to reduce the activity (cf., 42 and 39). 4) The DKA- with structural data showing that the catalytic core of PA-Nter
methyl esters of these indole derivatives (37 and 43) were mark- is surrounded by different hydrophobic pockets well suited for
edly less active than the corresponding analogs containing a free inhibitor binding (DuBois et al., 2012; Kowalinski et al., 2012).
carboxyl group (36 and 42, respectively).
Hence, it is relevant to design DKA derivatives containing
A favorable IC50 value (0.8 mM; Fig. 1; Table 1) was also hydrophobic substituents in an opportune topological disposi-
noted for the pyrrole derivative 10 (known as the Merck com- tion, so as to occupy these alternative pockets in PA-Nter.
pound L-731,988), which was previously discovered as a potent
With regard to the metal chelating fragment, the three-
inhibitor of HIV IN (Hazuda et al., 2000). As with the data dimensional spatial arrangement and distance constraints
above, its methyl ester proved to be sevenfold less active. This between the chemical features are in agreement with a pre-
pyrrole derivative can be viewed as an analog of indole 31, in viously published minimal pharmacophore model (Parkes et al.,
which the ring system is “size-reduced” to a pyrrole, and the 2003). Both features ML1-ML2 and ML2-ML3 (i.e., the donor
benzyl is substituted with a p-fluoro atom.
triad atoms chemotype; Fig. 2B) are able to coordinate the two
Finally, we evaluated several compounds in which the two- metal ions in the PA-Nter active site. In our pharmacophore
metal–chelating moiety is incorporated into a cyclic system. model, the interfeature distances between ML2 and ML1, and
This strategy proved highly successful for creating a potent between ML2 and ML3, are 2.64 and 2.77 Å, respectively (Fig.
and selective HIV IN inhibitor (Agrawal et al., 2012). In the 2C; distance ML1–ML3 is 4.74 Å). The other two additional
case of PA-Nter, neither of the cyclic diketo analogs tested, features (Aro, Hyd/Aro) would be involved in favorable contacts
i.e., the indole derivatives 44–47, as well as the other DKA with the hydrophobic binding pockets around the active center.
bioisosteres, i.e., the quinolones 25–28 and the hydroxypyrimi- The distances between the key ML1, ML2, and ML3 features
dine carboxamide 29, had any activity in the enzymatic PA-Nter and the aromatic feature Aro are 4.41, 6.12, and 6.46 Å,
assay.
respectively. The distance between the optimally oriented Aro

330
Stevaert et al.
Fig. 2. Three-dimensional arrangement of five pharmacophore features in the b-diketo acid motif. (A) Twelve active compounds 1–6, 10, 16, 34, 39–41
(i.e., b-diketo acids or esters with IC50 values # 2 mM) were aligned and mapped to generate a five-point pharmacophore. (B) The pharmacophore
features are depicted as metal ligators (ML1, ML2, and ML3) in cyan, aromatic (Aro), and hydrophobic or aromatic (Hyd-Aro) features in pink. (C)
Interfeature distances are given in angstroms (Å). (D) These distances are in agreement with both proposed models for interaction between the b-diketo
acid motif and the metal ions. Left: in one model [which is based on the cocrystal structure of PA-Nter in complex with L-742,001 (DuBois et al., 2012)],
the carboxylate and a-hydroxyl functionalities of the DKA motif coordinate one ion, and the third coplanar oxygen at the g-position chelates the second
metal ion together with the a-hydroxyl group. Middle: in an alternative model [previously proposed by us on the basis of L-742,001 docking within the
holo form of PA-Nter, and assuming that L-742,001 mainly binds in its monodeprotonated form (Stevaert et al., 2013)], the g-oxygen of the DKA motif is
not involved. Instead, one metal is coordinated by the lone oxygen pair of the a-hydroxyl group and one oxygen atom of the carboxylate group, whereas
the second metal ion interacts with both oxygens of the carboxylate moiety. Right: overlay of both models showing the intermetal distances.
and Hyd/Aro features is 2.55 Å, whereas that between ML1 and which is required to achieve antiviral activity in cell culture.
Hyd/Aro is 2.98 Å.
The calculated atom-based values (Supplemental Table 1) for
In Fig. 2D, two proposed models for interaction of the b-diketo almost all selected compounds (1–6, 10, 16, 34, and 39–41)
acid motif with the metal ions are shown (see figure legend for fall within the desirable range for good absorption and mem-
all details). Importantly, the ML1-ML2-ML3 interfeature dis- brane permeability (i.e., logP , 5, molecular weight , 500,
tances in our pharmacophoric model are coherent with favorable H-bond acceptor and donor counts of ,5 and ,10, respectively).
ligand-metal interactions and in agreement with both binding However, three compounds (i.e., 16, 34, and 39) showed un-
models (DuBois et al., 2012; Stevaert et al., 2013). Namely, the favorable predicted logP values, which may (at least partially)
Mn21 to Mn21 distance is 3.82 Å in the crystal-based and 3.31 Å explain their inactivity in cellular assays (see below). Other
in the docking-based model (Fig. 2D). In thermodynamic models studies suggest that compounds with a polar surface area
using Mg21 as the cofactor, the Mg21 to Mg21 distance was (PSA) , 60 Å2 would probably be well absorbed (.90%), whereas
predicted to decrease from 4.91 to 3.85 Å upon RNA binding compounds with PSA . 140 Å2 are predicted to be poorly
(Xiao et al., 2014). These changes in the intermetal distance absorbed (,10%) (Lipinski et al., 2001). All twelve compounds
along the catalytic pathway were already demonstrated for had an intermediate PSA value well below 140 Å2.
Bacillus halodurans RNase H (Yang et al., 2006) and EcoRV
(Horton and Perona, 2004).
Development of the Molecular Beacon–Based Endo-
nuclease Assay. The plasmid-based endonuclease method
The twelve compounds selected for pharmacophore gener- has the disadvantages of being discontinuous and time consum-
ation were also submitted to basic physicochemical calcula- ing, which makes it less suitable for screening large series of
tions to try to predict their membrane-permeating capability, compounds. We therefore developed an alternative real-time

Metal-Chelating Inhibitors of Influenza Virus Endonuclease
331
assay that uses a molecular beacon as the enzyme substrate
When analyzing the MB cleavage by gel electrophoresis
and is amenable to high-throughput format. An MB is a single- (Fig. 3C), we observed a difference in the cleavage pattern,
stranded DNA probe that contains a fluorophore moiety at one depending on which metal ion was used. This is in accordance
end and a nonfluorescent quencher moiety at the other. When with the results obtained by Noble et al. (2012). When using
intact, the MB forms a stem-and-loop structure, bringing the Mn21 as a cofactor, PA-Nter appears able to cleave shorter
fluorophore and quencher in very close proximity, which oligonucleotides than in the presence of Mg21 or without
results in superior quenching efficiency compared with a linear addition of a bivalent metal (i.e., in the presence of the copurified
dual-labeled probe. MB cleavage by recombinant PA-Nter metal). The Mn21 condition gave rise to cleavage products which
(depicted in Fig. 3A) separates the fluorophore from the increased in function of time and appeared as an extra band on
quencher, and the evolving fluorescence reveals the cleavage the gel, which migrated more slowly than the intact beacon. Most
process in real-time (Marras et al., 2006).
probably, migration of the shorter oligonucleotides is slowed
First, the real-time MB method was used to determine the down by the presence of the positively charged fluorophore. The
kinetic parameters of PA-Nter versus Mn21 or Mg21, and endonuclease activity resulting from the copurified metal was
eight different oligonucleotide substrates (i.e., an MB with completely abrogated upon addition of EDTA to the reaction
a heterogeneous sequence, or containing oligo-C, -A, or -U). mixture (Fig. 3C, right). The observation that the metal cofactor
The Km values determined for the eight different MB changes the cleavage pattern suggests that the structure of the
substrates were in the range of 0.09–0.64 mM (see Fig. 3B active site depends on which metal ions are present. Since this
and Table 3, which also contains the signal-to-noise ratio at may influence the binding properties of PAIs, we used the MB
100 nM MB). This indicates that, under the conditions of this assay to evaluate a series of representative DKA inhibitors with
MB assay, PA-Nter does not possess much preference for activity toward PA-Nter in the plasmid-based assay. The MB
specific dinucleotide linkages. In this context, it is noteworthy assay with these compounds was performed in the presence of
that the MB-het2-DFO substrate, which contains two 59-GC- Mn21, Mg21, or without addition of a bivalent metal ion, and
39 motifs, did not prove to be a better substrate than the using either MB-U6A2U7-DFO or MB-het2-DFO as the sub-
dU-rich MB-U6A2U7-DFO. On the contrary, we noted a 6-times strate. As shown in Table 4, the metal ion and MB substrate had
higher Vmax with MB-U6A2U7-DFO than with MB-het2-DFO. very little, if any, impact on the IC50 values for L-742,001 (1). In
This contrasts with the observation by another group that Fig. 3E, the fluorographs for this compound and derived dose-
PA-Nter has a preference for 59-GC-39 in RNA substrates response curve are shown for the experiments with Mn21
;
(Datta et al., 2013). However, efficient cleavage of U-rich RNA similar curves were obtained in the Mg21 or no-added-metal
substrates was already demonstrated by Dias et al. (2009). In conditions. In contrast, other PAIs, i.e., 10, 31, 34, 39, 40, and
these two studies, PA-Nter was evaluated against RNA sub- 41, had up to 61-fold lower IC50 values in the presence of Mn21
strates, whereas our MB assay uses DNA-oligonucleotides compared with Mg21 (Table 4). When the data obtained in the
(which were preferred because of their higher chemical stability). plasmid and molecular beacon assay (both with Mn21) are being
Other groups have successfully developed fluorescent assays for compared, both assays gave the same IC50 values for L-742,001,
PA endonuclease using totally different DNA or RNA probes 5, 10, 34, 40, and 41. For some compounds, we observed
(Kowalinski et al., 2012; Noble et al., 2012; Bauman et al., 2013; markedly higher IC50 values when using the MB instead of the
Datta et al., 2013; Chen et al., 2014), confirming that PA-Nter plasmid substrate (i.e., factor increase of 35 for 39 and 57 for
can cleave different DNA or RNA sequences in enzymatic 12). On the other hand, for 31, a sevenfold lower IC50 value was
assays. From a technical viewpoint, our MB assay is superior in obtained in the MB assay than in the plasmid assay.
having a higher signal-to-noise ratio than fluorescent assays
using nonbent oligonucleotide substrates.
Finally, we verified that assays using truncated PA-Nter
are adequate to investigate PAIs interacting with the catalytic
By using gel electrophoresis with fluorescence detection (Fig. site. Namely, the inhibitory activity of a few DKA compounds
3C), we confirmed that the fluorescence enhancement observed was determined in an enzymatic assay with untruncated PA
by real-time fluorometry was attributable to MB cleavage and and Mn21 (Noble et al., 2012). The IC50 values for compounds 1,
not to mere opening of the stem structure upon binding of the 30, and 39 (Table 4, right column) were very similar to the
MB to the active site of PA-Nter. An intriguing and unexpected values obtained with PA-Nter using the plasmid/Mn21 assay.
finding relates to the impact of the bivalent metal ion on
Anti–Influenza Virus Activity in Cell Culture. Besides
cleavage efficiency by PA-Nter. While using the 7-kb plasmid evaluation in the PA-Nter enzymatic assay, all DKA analogs
substrate (M13mp18), we observed no endonuclease activity were examined for anti–influenza virus activity in cell culture.
when Mn21 was omitted from the reaction mixture or replaced We first performed the influenza vRNP reconstitution assay in
by Mg21 (second and third from last lanes in Fig. 1), in agree- HEK293T cells. Using L-742,001, we previously demonstrated
ment with a previous report (Dias et al., 2009). In contrast, that this method is well suited for determining the activity and
PA-Nter was found to efficiently cleave the MB substrate when selectivity of PAIs in cell culture (Stevaert et al., 2013). In the
magnesium instead of manganese was added, and even without present study, we further expanded this vRNP assay by in-
addition of a metal ion, which is probably attributable to cluding four different vRNP complexes, i.e., derived from a
copurification of a metal ion (probably Mg21) (Xiao et al., human influenza A or B virus, an avian H5N1 virus containing
2014) during preparation of the PA-Nter enzyme. Significant the PB2-627Glu avian signature, or the PB2-627Lys human
substrate cleavage in the absence of added salt was also ob- adaptation residue (Moncorgé et al., 2010). As shown in Table 5,
served by Noble et al. (2012) in experiments with untruncated L-742,001 and its close analogs 2–6 had strong and consistent
PA. The effect of the metal ion depended on which MB sequence activity against all four vRNP complexes, which agrees with our
was used, since one MB was more rapidly cleaved in the pres- previous analysis that the key residues for binding of L-742,001
ence of Mg21 than Mn21, whereas for another sequence, the in PA-Nter show little variation among influenza A (human or
opposite was seen (see Fig. 3D for all details).
avian) or B viruses (Stevaert et al., 2013).

332
Stevaert et al.
Fig. 3. Utility of the MB assay for determination
of PA-Nter enzyme properties and activity of PA
inhibitors. (A) Schematic representation of the
MB assay. MB cleavage by recombinant PA-Nter
(which can occur at different sites) separates
the fluorophore (F) from the quencher (Q). The
evolving fluorescence, indicating enzymatic activ-
ity, can be monitored in real-time. (B) Michaelis-
Menten curve for MB-U6A2U7-DFO. This MB was
incubated with PA-Nter and the evolving fluores-
cence was recorded as a function of time and MB
substrate concentration. The initial cleavage rate
(V0) was obtained from the slope (fluorescence
units per minute) of the best-fit line derived from
the 5–15-minute interval of the reaction. Values
shown are the averages 6 S.E.M. of three in-
dependent experiments. (C) Gel electrophoretic
analysis of the MB cleavage pattern. The amount
of intact MB-U6A2U7-DFO substrate decreased
over time when incubated with PA-Nter. The first-
appearing cleavage products migrated faster
than the intact MB substrate, whereas the late-
cleavage products showed slower migration through
the gel. When using Mn2+ as a cofactor, PA-Nter
appears able to cleave shorter oligonucleotides
than in the presence of Mg2+ or without addition
of a bivalent metal (i.e., in the presence of the
copurified metal). Addition of 100 mM EDTA
completely abrogated the endonuclease activity.
(D) Influence of the metal ion on the initial
cleavage rate of MB-U6A2U7-DFO and MB-het2-
DFO. The initial cleavage rate (V0; on the basis of
the real-time fluorographs) for MB-U6A2U7-DFO
(left graph) was 1.4-fold and significantly higher
with Mg2+ than with Mn2+, and still increased
when no metal ion was added. An opposite effect
was seen with MB-het2-DFO (right graph): In this
case, the reaction rate was 2.2-fold higher in the
presence of Mn2+ compared with Mg2+
. The
statistical significance (****P , 0.0001; ***P ,
0.001; ns, not significant) was calculated by one-
way analysis of variance followed by a Tukey’s
multiple comparison test using GraphPad Prism.
(E) Dose-dependent inhibition of MB cleavage by
L-742,001. MB-U6A2U7-DFO (at 20 nM) was
incubated with 1 mg PA-Nter and increasing
concentrations of L-742,001. The initial cleavage
rate (V0) was obtained from the slope (fluorescence
units per minute) of the best-fit line derived from
the 5–15-minute interval of the reaction. These
V0-values were used to calculate the percentage
of inhibition. The inset shows the resulting dose-
response curve.
Among our series of DKA analogs with an indole scaffold derivatives were not toxic at the highest concentration tested
(Tables 2 and 6), the following were shown to inhibit influenza (200 mM). Importantly, although these compounds displayed
vRNP activity: 33 and 40, which have an N-ethyl group; and 31, relatively low potency in the vRNP assay [i.e., average EC50
36, 37, and 41, which carry an N-benzyl substituent (introduced values ranging from 14 mM (36) to 118 mM (40)], the same
to modulate lipophilicity, thus assumed to improve cellular up- values were obtained for all four vRNP complexes, whether
take). Of these, 31, 36, 40, and 41 are unesterified DKAs, derived from influenza A (human or avian) or B viruses.
whereas 33 and 37 are methyl esters. The highest potency was
Three other analogs in Table 1 had noticeable activity in the
obtained for 36. With the exception of 41, these indole vRNP assay: 10 (L-731,988) and its methyl ester 11 (both are

Metal-Chelating Inhibitors of Influenza Virus Endonuclease
333
TABLE 4
Inhibitory activity of a selection of DKA derivatives in the MB versus plasmid assay with PA-Nter, or an assay using
untruncated PA
IC50 (mM), 50% inhibitory concentration. For each compound the IC50 value was calculated using nonlinear regression analysis. Values are the
mean of at least two independent experiments.
PA-Nter, molecular beacon assay
Plasmid assayc
Compound
MB-U6A2U7-DFOa
MB-het2-DFOb
Untruncated PAd Mn2+
Mn2+
Mn2+
Mg2+
No metal
Mn2+
3.9
Mg2+
7.1
No metal
3.5
1
5
1.5
3.9
4.2
154
26
3.2
3.6
21
3.1
6.3
177
.200
41
44
59
2.5
9.1
0.5
0.5
0.8
2.7
3.4
22
0.6
0.6
0.8
1.6
3.5
0.5
10
12
30
31
34
39
40
41
42
100
.200
41
2.4
.200
.200
1.1
1.1
54
180
.200
174
98
.200
.200
86
5.2
1.4
23
65
72
aMB-U6A2U7-DFO, 59‐[DFO]‐GCAGG (dU)6A2(dU)6 CCTGC‐[BHQ2]‐39.
bMB-het2-DFO, 59-[DFO]‐CGCACG TTATGCTAAGCAAGTAACC GTGCG‐[BHQ2]‐39.
cSame data as in Tables 1 and 2.
dSee Noble et al. (2012) for a description of the fluorescence-based enzymatic assay with untruncated PA expressed in insect cells.
strong inhibitors in the enzymatic test) and 20 (L-708,906;
a substituted DPBA derivative with weak activity in the
enzymatic test). Several DKAs with potent activity in the
PA-Nter enzymatic assay (i.e., IC50 values below 5 mM; Tables 1
and 2) were devoid of any activity in the vRNP cell culture
assay. This might be explained by insufficient cellular uptake
owing to the anionic charge of the DKA moiety. On the other
hand, the activity of 9 in the vRNP assay appears unrelated to
inhibition of the endonuclease since this compound was inactive
in the enzymatic assay.
Discussion
The potential of influenza virus endonuclease inhibitors as
an entirely novel concept in antiviral therapy was recognized
about twenty years ago, when two prototype inhibitors,
L-742,001 and flutimide were initially described (Hastings
et al., 1996; Tomassini et al., 1996). The fact that these com-
pounds were discovered in a screening approach combining
enzymatic and cell-based methods probably explains why these
molecules still serve as the two main lead PAIs today. Their
presumed binding mode in the catalytic center of the enzyme
was only recently revealed in cocrystallization experiments
with PA-Nter (DuBois et al., 2012; Kowalinski et al., 2012).
These studies further revealed several hydrophobic pockets
surrounding the catalytic core of PA-Nter, and that the binding
of flutimide, L-742,001, and its congener, DPBA, involves an
induced-fit mechanism. On the basis of these structural in-
sights, several groups embarked on in silico design of novel
PAIs with unrelated chemical structures (Ishikawa and Fujii,
2011; Bauman et al., 2013; Chen et al., 2014).
We followed another strategy by evaluating, in com-
plementary enzyme- and cell-based biological assays, available
scaffolds of DKA derivatives, which were originally developed
toward HIV integrase or other metal-dependent enzymes. By
determining their inhibitory activity against PA-Nter, we aimed
at identifying which structures (and their structural determi-
nants) could serve as the basis for future design of PAIs. For this
purpose, we created a three-dimensional pharmacophore model
defining the optimal distances between the metal chelating
parts of the ligands and the aromatic/hydrophobic moieties.
Efficient development of novel PAIs requires an enzymatic
assay that is amenable to high-throughput screening. The
plasmid-based electrophoretic assay used here for evaluating
the DKA analogs is quite robust but too labor-intensive to be
used in screening programs. In contrast, our real-time MB
assay can be performed in miniaturized plate format and at
low cost. Although similar fluorescent assays have been described
by others (Kowalinski et al., 2012; Noble et al., 2012; Bauman
et al., 2013; Datta et al., 2013; Chen et al., 2014), our MB assay is
unique in having a superior signal-to-noise ratio. Besides this
In a second stage, the compounds showing activity in the
vRNP assay were submitted to a virus yield assay in influenza
virus–infected MDCK cells. For L-742,001 and its close analogs
(1-6), and 10, 20, 31, 40, and 41, the inhibitory activity noted in
the vRNP reconstitution assay was confirmed in influenza
virus–infected cells. Although their inhibitory potency was
quite moderate (i.e., antiviral EC99 values of ∼80 mM), our
cell culture data indicate that these compounds may rep-
resent some novel and cell-permeable PAIs. To validate this
assumption, we performed a basic time-of-addition experi-
ment (Fig. 4) in which viral RNA synthesis is measured after
one virus replication cycle. We previously demonstrated
(Stevaert et al., 2013) that the polyphenolic compound
epigallocatechin gallate, which acts as a PAI in enzymatic
assays (Kuzuhara et al., 2009; Kowalinski et al., 2012), has
an unrelated antiviral mode of action in cell culture. Figure 4
shows that the reference compound chloroquine, which
inhibits influenza virus entry by increasing the endosomal
pH (Vanderlinden et al., 2012), was devoid of activity
when added postentry, i.e., at 1 hour after virus addition.
L-742,001 kept its full inhibitory activity when added at
11 hour, confirming that its action point is situated after virus
entry. For 10, 40, and 41, the highest inhibition was seen
(i.e., at least 500-fold reduction in viral RNA synthesis) when
the compounds were added 30 minutes prior to the virus.
However, the finding that these compounds still produced
∼60- to ∼300-fold reduction in viral RNA copy number when
added at 11 hour, indicates that they do inhibit viral RNA
synthesis quite potently, which fits with their designation as
PAIs.

334
Stevaert et al.
TABLE 5
Activity of compounds 1–29 in two influenza virus cell–based assays
Data shown are the mean 6 S.E.M. of at least three independent tests.
Influenza virusa vRNP reconstitution assay in HEK293T cellsb
Virus yield assay in MDCK cellsb
Antiviral activity
Human A/PR/8/34
antiviral activitye
c
Compound
EC50
Cytotoxicity
Cytotoxicity
d
CC50
CC50
Human
A/PR/8/34
Human
Avian 50-92/
PB2-627E
Avian 50-92/
PB2-627K
EC99
EC90
B/Yamanashi
mM
1
3.6
2.8
3.1
6.9
0.8
0.4
4.4
11
4.6
8.5
6.6
7.6
3.9
7.2
ND
ND
105
88
11
20
$104
$102
$103
$93
$106
$94
.200
.200
117
.200
.200
.200
.200
.200
.200
.200
.200
.200
.200
.200
.200
.200
.200
.200
.200
.200
.200
.200
.200
.200
8.4
6.0
5.4
3.9
181
.100
.100
.100
.100
.100
ND
2
3
1.8
17
2.9
2.0
4
2.3
15
8.8
15
ND
ND
29
5.6
3.8
5
1.7
1.5
1.0
6
2.3
2.7
1.9
7
8
.100
.100
57
NDf
ND
25
ND
ND
.100
74
ND
ND
.100
58
ND
9
.200
.200
$184
ND
10
84
132
116
49
95
65
11
47
.100
ND
ND
ND
ND
ND
ND
ND
ND
79
ND
ND
ND
ND
ND
ND
ND
ND
ND
10
$90
ND
ND
ND
ND
ND
ND
ND
ND
55
ND
ND
ND
ND
ND
ND
ND
ND
ND
6.7
12
.100
.100
.100
.100
.100
.100
.100
.100
72
.100
.100
.100
.100
.100
.100
.100
.100
.100
16
ND
ND
ND
ND
ND
ND
ND
ND
97
ND
ND
ND
ND
ND
ND
ND
ND
ND
3.1
ND
ND
ND
ND
ND
ND
ND
ND
32
ND
ND
ND
ND
ND
ND
ND
ND
ND
50
ND
ND
ND
ND
ND
ND
ND
ND
37
ND
ND
ND
ND
ND
ND
ND
ND
ND
45
13
ND
14
ND
15
ND
16
ND
17
ND
18
ND
19
ND
20
$194
ND
21
22
ND
23
ND
24
ND
25
ND
26
ND
27
ND
28
ND
29
ND
Ribavirin
.200
aReconstituted vRNP complexes from: human influenza A virus, strain A/PR/8/34; human influenza B virus, strain B/Yamanashi/166/98; and avian influenza A virus, strain
A/turkey/England/50-92/1991 with a PB2-627E or PB2-627K residue.
bHEK293T cells, human embryonic kidney 293T cells; MDCK cells, Madin-Darby canine kidney cells.
cEC50, 50% effective concentration (mM), i.e., compound concentration producing 50% reduction in vRNP-driven firefly reporter signal, estimated at 24 hours after
transfection.
dCC50, 50% cytotoxic concentration (mM) at 24 hours determined by MTS cell viability assay.
eCompound concentration (mM) causing a 1-log10 (EC90) or 2-log10 (EC99) reduction in virus yield at 24 hours postinfection, as determined by real-time RT-PCR.
fND, Not done.
analytical aspect, we obtained some unanticipated findings that biologically relevant cofactor, given that its intracellular concen-
further broaden the relevance of the MB assay. First, the mere tration is at least 1000-fold higher than that of Mn21 (Zhao et al.,
observation that a bent molecular beacon is a very efficient 2009; Xiao et al., 2014). This may implicate that PA-Nter assays
substrate for PA-Nter is reminiscent of what has been described using Mg21 are more stringent for evaluating potential PAIs. For
for the type II endonuclease EcoRV, which requires bending of most of the DKA inhibitors tested here, the inhibitory activity
the nucleic acid substrate for catalysis to occur (Horton and against PA-Nter was far less with Mg21 than with Mn21. A
Perona, 2004; Xiao et al., 2014). Second, PA-Nter proved to have notable exception is L-742,001 (and its cyclohexyl analog 5), for
low cleavage site specificity, since it was able to cleave molecular which the activity was similar for both metal ions. It is tempting
beacons with different DNA sequences, cutting not only the to speculate that this metal ion–independent behavior of
beacon itself but also subsequently formed smaller fragments. L-742,001 (or, at least, its strong activity versus Mg21) may
Third, whereas most enzymatic studies with PA-Nter have contribute to its superior efficacy in cell culture. For comparison,
indicated that the enzyme is considerably more active in the compound 10 had a low IC50, similar to that of L-742,001, when
presence of Mn21 compared with Mg21 (Dias et al., 2009; Crépin evaluated against PA-Nter with Mn21 but was 40-fold less active
et al., 2010; Datta et al., 2013), our molecular beacon assay works in the presence of Mg21. In influenza virus–infected cells, 10 was
equally well with Mg21 as with Mn21. We speculate that the only moderately active (despite the fact that this molecule is
intrinsic bending of this substrate may bring the scissile a strong inhibitor of human immunodeficiency virus in infected
phosphodiester group in a prereactive conformation and drive cells, indicating that it enters the cells quite efficiently) (Hazuda
catalysis; hence, cleavage occurs with the less reactive Mg21. On et al., 2000). The issue of cell penetration may also explain our
the other hand, with more flexible substrates (such as the observation that the anti–influenza virus activity in cell culture
M13mp18 plasmid), the softer metal Mn21 seems to be required was higher for the DKA-indole derivatives carrying an N-benzyl
for optimal enzymatic activity. Mg21 has been proposed as the (compared with ethyl or methyl).

Metal-Chelating Inhibitors of Influenza Virus Endonuclease
335
TABLE 6
Activity of compounds 30–47 in two influenza virus cell–based assays
Influenza virusa vRNP reconstitution assay in HEK293T cells
Virus yield assay in MDCK cellsb
Antiviral activitye
Human A/PR/8/34
c
Antiviral activity EC50
Compound
Cytotoxicity
d
Cytotoxicity CC50
CC50
Human A/
PR/8/34
Human B/
Yamanashi
Avian 50-92/
PB2-627E
Avian 50-92/
PB2-627K
EC99
EC90
mM
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
.100
92
.100
77
NDf
59
ND
18
ND
.200
ND
39
ND
87
ND
62
.200
.200
.200
.200
.100
.200
.200
.200
.200
.200
.200
136
.200
.200
.200
.200
.200
.200
ND
85
ND
55
ND
126
ND
.25
ND
ND
.12.5
.25
ND
ND
95
ND
.25
ND
ND
.12.5
.25
ND
ND
69
ND
$169
ND
ND
.100
.100
16
ND
ND
11
ND
ND
17
ND
ND
12
.200
$184
ND
48
29
54
30
.100
.100
166
ND
ND
65
ND
ND
121
96
ND
ND
121
62
ND
$194
.200
ND
45
43
64
38
.100
.100
.100
.100
.100
.100
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
aReconstituted vRNP complexes from: human influenza A virus, strain A/PR/8/34; human influenza B virus, strain B/Yamanashi/166/98; and avian influenza A virus, strain
A/turkey/England/50-92/1991 with a PB2-627E or PB2-627K residue.
bHEK293T cells, human embryonic kidney 293T cells; MDCK cells, Madin-Darby canine kidney cells.
cEC50, 50% effective concentration (mM), i.e., compound concentration producing 50% reduction in vRNP-driven firefly reporter signal, estimated at 24 hours after
transfection.
dCC50, 50% cytotoxic concentration (mM) at 24 hours determined by MTS cell viability assay.
eCompound concentration (mM) causing a 1-log10 (EC90) or 2-log10 (EC99) reduction in virus yield at 24 hours postinfection, as determined by real-time RT-PCR.
fND, Not done.
We hypothesize that the less rigid coordination requirements studies, this remains speculative. The finding that several
of Mn21 may create flexibility and allow binding of structurally DKA inhibitors prefer Mn21 to exert their effect on PA-Nter is
diverse inhibitors (Bock et al., 1999). In contrast, Mg21 may reminiscent of what was described for HIV IN (Marchand et al.,
result in a tighter catalytic site that only accepts the best fitting 2003). In this case, both the aromatic and DKA part were
compounds. Noble et al., (2012) showed a change in circular shown to play a role in this metal selectivity, which was
dichroism spectrum when comparing PA with Mn21 to PA with explained by a metal-dependent binding orientation of the
Mg21, which correlates with an increase in helical content. If inhibitor. Unfortunately, the understanding of how PAIs bind
the secondary structure of PA-Nter changes depending on the to PA-Nter is still quite limited. For a selection of PAIs
metal, it is possible that the metal ion also affects which (including L-742,001), the binding mode was recently revealed
hydrophobic pockets are available for binding of the inhibitor. by cocrystallographic analyses (DuBois et al., 2012; Kowalinski
Without crystallographic data of PA-Nter crystallized with et al., 2012). For L-742,001, our resistance studies in cells
inhibitors in the presence of only Mg21 and/or more docking infected with a series of mutant influenza viruses established
Fig. 4. Compounds 10 (L-731,988), 40, and 41 inhibit viral
RNA synthesis besides affecting the virus entry process.
Light gray bars: the test compounds L-742,001 (20 mM), 10
(200 mM), 40 (150 mM), 41 (150 mM), or chloroquine (80 mM)
were added to MDCK cells, and after 30 minutes incubation
at 35°C, influenza virus A/PR/8/34 was added. Dark gray
bars: virus was added first and allowed to enter during
1 hour incubation, after which the compounds were added at
the same concentrations as above. In both conditions, total
cellular RNA was extracted at 10 hours postinfection (VC,
untreated virus control). The number of vRNA copies was
quantified by two-step real-time RT-PCR. On the y-axis, the
fold increase in vRNA copies is shown relative to the viral
copy number added at time zero. L-742,001 remains fully
effective when added after virus entry. In contrast, the
reported entry inhibitor chloroquine (Vanderlinden et al.,
2012) is inactive when added at
1 hour postinfection.
Compounds 10, 40, and 41 have a dual effect by acting both
upon virus entry and viral RNA synthesis. Data shown are
the mean 6 S.E.M. of two independent tests.

336
Stevaert et al.
K. Nagata for the generous donation of plasmids. M.S. thanks
Dr. Andrea Brancale for the use of software for molecular modeling
calculation.
the role of several residues in the catalytic center of PA or
surrounding hydrophobic pockets in positioning this inhibitor
in the active site of PA-Nter (Stevaert et al., 2013).
Our enzyme inhibition data for PA-Nter convincingly demon-
strate that the setup of the enzymatic assay (i.e., substrate,
metal cofactor, and type of readout) should be carefully chosen
when performing a compound screening for PAI development.
We provide evidence that inhibitors targeting the catalytic site
have similar activity whether assessed with PA-Nter or un-
truncated PA. Given the uncertainty on which metal ions
(i.e., Mg21 or Mn21) are present in the active site, evaluation of
potential PAIs against both metals (as possible with our MB
assay) seems recommended. A number of recent reports have
described novel PAIs with DKA-unrelated structures (Iwai
et al., 2010; Baughman et al., 2012; Bauman et al., 2013; Chen
et al., 2014). Only a few of these molecules were subsequently
proven to have activity in influenza virus–infected cell cultures.
The importance of proof-of-concept cell culture evaluation
during early hit discovery of potential PAIs also appears from
our cell culture studies using influenza virus vRNP reconstitu-
tion and replication assays. The fact that hit compounds with
excellent activity against PA-Nter (i.e., comparable to that of
L-742,001) may not survive proof-of-concept evaluation in cell
culture can be related to different factors. As suggested above,
an enzymatic PA-Nter assay using Mg21 might be more
stringent and have a better predictive value. Second, the
hydrophilic/lipophilic balance for the chelating fragment
and aromatic framework of DKA-based compounds may play an
important role in explaining their antiviral potency. Besides, we
previously demonstrated that the catechol compound epigallo-
catechin gallate, which is nicely active in an enzymatic PA-Nter
assay, owes its excellent activity in virus-infected cells entirely
to inhibition of virus entry (Stevaert et al., 2013). To avoid this
confusion, we here evaluated the active DKA compounds in
a basic time-of-addition experiment. Our data indicate that the
pyrrole compound 10 and the indole compounds 40 and 41 do
inhibit viral RNA synthesis, besides having a secondary effect
on virus entry.
Authorship Contributions
Participated in research design: Stevaert, Naesens, Sechi, Rogolino,
Kim.
Conducted experiments: Stevaert, Nurra, Pala, Carcelli, Shepard,
Domaoal.
Contributed new reagents or analytic tools: Marras.
Performed data analysis: Stevaert, Naesens, Sechi, Rogolino, Kim,
Alfonso-Prieto.
Wrote or contributed to the writing of the manuscript: Stevaert,
Naesens, Sechi, Alfonso-Prieto.
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In conclusion, we demonstrated that diverse DKA scaffolds
can lead to potent inhibition of PA-Nter in enzymatic assays,
which agrees with the notion that the catalytic center of
PA-Nter is quite spacious and flexible, and subject to induced-
fit inhibitor binding (DuBois et al., 2012; Kowalinski et al.,
2012). Our pharmacophore model defines strict structural re-
quirements along with chemical features, and serves as
a basis for design of novel PAIs with DKA-based structures
or bioisosteres thereof. For high-throughput compound screen-
ing, the novel molecular beacon assay appears well suited. This
method enables determination of the activity of potential PAIs
versus either Mn21 or Mg21, the latter probably being the
biologically relevant cofactor. Any hit-to-lead process on novel
PAIs requires rapid progression of potential hit compounds to
relevant cell culture assays, such as the vRNP reconstitution,
virus yield, and time-of-addition methods, which we used to
identify some relevant scaffolds for further design of PAIs.
Successful development of this new class of anti–influenza
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Address correspondence to: Dr. Lieve Naesens, Rega Institute for Medical
Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
E-mail: lieve.naesens@rega.kuleuven.be