DNA-linked inhibitor antibody assay (DIANA) as a new method for screening influenza neuraminidase inhibitors

Article abstract of DOI:10.1042/BCJ20180764

Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes. DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing the evaluation of inhibition constants from a single inhibitor concentration. Here, we report the design, synthesis, and structural characterization of a tamiphosphor derivative linked to a reporter DNA oligonucleotide for the development of a DIANA-type assay to screen potential influenza neuraminidase inhibitors. The neuraminidase is first captured by an immobilized antibody, and the test compound competes for binding to the enzyme with the oligo-linked detection probe, which is then quantified by qPCR. We validated this novel assay by comparing it with the standard fluorometric assay and demonstrated its usefulness for sensitive neuraminidase detection as well as high-throughput screening of potential new neuraminidase inhibitors.

Full text of DOI:10.1042/BCJ20180764

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10.1042/BCJ20180764
DNA-linked inhibitor antibody assay (DIANA) as a new method for screening influenza
neuraminidase inhibitors
1
*
1
1
1
Milan Kožíšek , Václav Navrátil , Kateřina Rojíková , Jana Pokorná , Carlos Berenguer
1
,2
1
1
1,2
1
2
Albiñana , Petr Pachl , Jitka Zemanová , Aleš Machara , Pavel Šácha , Jason Hudlický ,
3
1,4
1,5*
Ivana Císařová , Pavlína Řezáčová , Jan Konvalinka
1Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Czech
Republic, Gilead Sciences and IOCB Research Center,Flemingovo n. 2, 16610 Prague 6, Czech
2
Republic; Department of Organic Chemistry, Faculty of Science, Charles University, Hlavova
3
8
, 12800 Prague 2, Czech Republic; Department of Inorganic Chemistry, Faculty of Science,
4
Charles University, Hlavova 8, Prague 2, 12843, Czech Republic; Institute of Molecular
Genetics of the Academy of Sciences of the Czech Republic, Vídeňská 1083, 14000 Prague 4,
5
Czech Republic; Department of Biochemistry, Faculty of Science, Charles University,
Hlavova 8, 12800 Prague 2, Czech Republic
*Corresponding authors:
Milan Kožíšek and Jan Konvalinka
fax : +420 220 183 578; tel : +420 220 183 218
e-mails : milan.kozisek@uochb.cas.cz, konval@uochb.cas.cz
Use of open access articles is permitted based on the terms of the specific Creative Commons Licence under 1
which the article is published. Archiving of non-open access articles is permitted in accordance with the
Archiving Policy of Portland Press (
http://www.portlandpresspublishing.com/content/open-access-policy#Archiving).

ABSTRACT
Influenza neuraminidase is responsible for the escape of new viral particles from the
infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or
stockpiled for emergencies. However, the increasing development of viral resistance against
approved inhibitors has underscored the need for development of new antivirals effective
against resistant influenza strains. A facile, sensitive, and inexpensive screening method would
help achieve this goal.
Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay
(
DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes.
DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing
evaluation of inhibition constants from a single inhibitor concentration.
Here, we report the design, synthesis, and structural characterization of a tamiphosphor
derivative linked to a reporter DNA oligonucleotide for the development of a DIANA-type
assay to screen potential influenza neuraminidase inhibitors. The neuraminidase is first
captured by an immobilized antibody, and the test compound competes for binding to the
enzyme with the oligo-linked detection probe, which is then quantified by qPCR. We validated
this novel assay by comparing it with the standard fluorometric assay and demonstrated its
usefulness for sensitive neuraminidase detection as well as high-throughput screening of
potential new neuraminidase inhibitors.
Abbreviations
AU, asymmetric unit; CCP4, Collaborative Computational Project Number 4; CuAAC,
copper(I)-catalyzed azide alkyne cycloaddition; PDBCO, dibenzocyclooctyne group; DCM,
dichloromethane; DIANA, DNA-linked inhibitor antibody assay; DIPEA, N,N-
diisopropylethylamine; DMAP, 4-dimethylaminopyridine; DMS, dimethyl sulfate; DMSO,
dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; FDA, Food and Drug
Administration; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HOTT,
N,N,N',N'-tetramethyl-S-(1-oxido-2-pyridyl)thiuronium hexafluorophosphate; HPLC, high-
performance liquid chromatography; K
d
, dissociation constant; K , inhibition constant; LC-MS,
i
liquid chromatography-mass spectrometry; 4-MUNANA, 2´-(4-methylumbelliferyl)-α-D-N-
acetylneuraminic acid; NA, neuraminidase; NHS, N-hydroxysuccinimide; PDB, Protein Data
Bank; PEG, polyethylene glycol; qPCR, quantitative polymerase chain reaction; RMSD, root-
mean-square deviation; SPAAC, strain-promoted alkyne-azide cycloaddition; TBS, Tris-
buffered
tetrafluoroborate;
Tris, tris(hydroxymethyl)aminomethane; TMF, tamiphosphor.
saline;
TBTU,
N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium
TFA,
trifluoroacetic
acid;
THF,
tetrahydrofuran;
2

INTRODUCTION
Influenza virus causes severe respiratory infections associated with significant
morbidity and mortality. Worldwide, annual influenza epidemics result in three to five million
cases of severe acute viral infection, and roughly 290,000 to 650,000 deaths (1). Several
influenza pandemics have occurred in the last century, each caused by a new strain of the virus
in humans. Two types of influenza antivirals, which target different steps in the viral lifecycle,
have been approved by the Food and Drug Administration (FDA). The first type blocks the
viral ion channel formed by the M2 protein, preventing the virus from infecting the cell.
However, use of M2 inhibitors leads to rapid development of drug resistance, and these drugs
are no longer routinely used. The second type inhibits neuraminidase (NA).
NA is an influenza virus membrane glycoprotein that is essential for release of new
virions from an infected cell. NA inhibitors are the first line treatment for patients in need of
anti-influenza drug therapy. Nevertheless, the increasing development of viral resistance
against approved NA inhibitors has underscored the need for new anti-influenza drugs active
against resistant viral strains and different NA subtypes. While several methods for NA
inhibitor characterization based on enzymatic activity measurements have been described, most
of them are time-consuming, sensitive to interference, and expensive (2-5).
Recently, our group developed a multiwell plate-based DNA-linked inhibitor antibody
assay (DIANA) as a simple and reliable technique for enzyme detection and screening of small-
molecule inhibitors (6). First, the target enzyme is captured by an antibody immobilized on the
well. The enzyme then binds a detection probe consisting of a DNA oligonucleotide covalently
linked to a known competitive inhibitor. The bound probe is measured by quantitative PCR
(
qPCR, Figure 1A). DIANA showed a several-order-of-magnitude higher sensitivity toward a
model enzyme when compared to sandwich ELISA and enabled detection of target enzymes in
complex biological matrices. DIANA also has been used to screen inhibitors of two clinically
relevant enzymes: glutamate carboxypeptidase II and carbonic anhydrase IX (6). The assay
enables inhibition constants (K ) to be calculated directly from single-well measurements.
i
Here, we propose DIANA as a new reliable and rapid method for screening influenza
NA inhibitors. We report preparation of an active-site-directed detection probe, and structurally
characterize the interactions of the inhibitor incorporated in the probe with recombinant NA.
Additionally, we compare inhibition constants for various compounds tested by DIANA with
results from the standard fluorometric assay.
3

EXPERIMENTAL
Cloning, expression, and purification of recombinant NA2009wt
DNA encoding the neuraminidase ectodomain (residues 82 to 469) from the
A/California/07/2009 (H1N1) influenza virus was prepared by GenScript USA Inc. (Genbank
Source Sequence CY121682). DNA was inserted into the pMT/BiP/V5-HisA vector
(
Invitrogen) with an N-terminal tag containing two Strep-tags, a FLAG-tag and a thrombin
cleavage site. This construct was used to transfect Drosophila Schneider S2 cells (Invitrogen),
and large-scale expression was performed as previously described (7). Recombinant
neuraminidase expressed into the cell culture medium was subsequently purified using one-step
purification on Strep-Tactin agarose resin (IBA GmbH) (8, 9). First, Strep-Tactin resin was
equilibrated in buffer W (100 mM Tris-HCl, pH 8.0, 150 mM NaCl), and medium with added
BioLock biotin blocking solution (IBA GmbH) was applied. The matrix with bound tagged
protein was thoroughly washed with buffer W, and elution was performed with 10 mM
desthiobiotin in buffer W. The resin was regenerated with buffer W containing 1 mM 2-(4-
hydroxyphenylazo)benzoic acid (Sigma-Aldrich) and stored at 4 ˚C for later use. The
purification process was monitored by SDS PAGE and Western blot using murine monoclonal
Anti-FLAG M2-peroxidase antibody clone M2 (Sigma-Aldrich). The N-terminal tag was
removed by cleavage with thrombin protease immobilized on agarose beads (Sigma-Aldrich).
Synthesis of inhibitors
The compounds presented here were either prepared by the same procedures described
previously (10) (compounds 2-7) or as outlined in Figures 2 and 3. For preparation of ω-
hexylazido tamiphosphor 1, we used our previously described approach to a key Barton ester
based on a modified version of Gunasekera’s procedure (Figure 2). Briefly, the phosphonate
salt of ethyl oseltamivir carboxylate was free-based with aqueous bicarbonate and Boc-
protected to provide 12. The ethyl ester moiety was hydrolyzed, and acid 13 was treated with
HOTT (N,N,N’,N’-tetramethyl-S-(1-oxido-2-pyridyl)thiuronium hexafluorophosphate) reagent
to yield the key Barton thioester. Upon irradiation with a flood lamp in a solution of
bromotrichloromethane, bromide 14 was obtained. Palladium-catalyzed Hirao coupling of 14
with dimethyl phosphite afforded dimethyl phosphonate 15 in very good yield. The dimethyl
ester was subjected to selective mono-O-demethylation with sodium hydroxide, yielding
intermediate 16. Surprisingly, alkylation of 16 at 60 °C furnished alkylated product 17, which
lacks the methyl ester moiety. Apparently, under these conditions, the desired alkylation was
4

followed with demethylation mediated by halide ions (11). This serendipitous finding led to a
shortcut in the synthesis of ω-hexylazido tamiphosphor that resulted not only in omission of the
problematic second O-demethylation by thiophenolate but also improvement of overall yield.
The last step of the reaction sequence was Boc deprotection with trifluoroacetic acid (TFA).
Compound 2 was prepared by the same procedure and was isolated as a mixture of
diastereomeric monoalkyl esters because the phosphorus atom is an additional stereogenic
center. As expected, we were not able to observe separation of these diastereomers on analytical
HPLC, and thus the material was used as such.
Oseltamivir derivatives with an alkyl moiety at the C-5 amino functionality were also
prepared from oseltamivir phosphonate. In this case, the starting compound was treated with
ethyl 2-bromopropionate to yield a mixture of diastereomers 18 and 19. After separation
followed by saponification and Boc deprotection with TFA (Figure 3), diastereomers 8 and 9
were produced in 38% and 34% yield, respectively. To determine the absolute configuration of
the introduced polar moiety, we performed crystallization attempts in different solvent systems.
However, our attempts to obtain a monocrystal of 19 suitable for X-ray analysis failed, and we
decided to introduce 4-nitrobenzoyl moiety to the structure of 19 to facilitate crystallization.
Diastereomer 19 was treated with 4-nitrobenzoyl chloride in the presence of
triethylamine. Crystallization of the resulting derivative 20 eventually yielded a suitable
monocrystal, the X-ray structure of which is shown in Figure S1. This structural information
allowed us to assign an (R) configuration to the newly formed stereocenter in 20. We surmise
that the stereogenic center in 9 has the same configuration since it is not altered by N-acylation.
Alkylation of the same starting material with dimethyl maleate produced a
diastereomeric mixture of triester 21 in good yield (Figure 3). Cleavage of alkylesters followed
by preparative HPLC resulted in 10, which was used as a mixture of diastereomers. Amide 11
has an intentionally impaired basic functionality at C-5. All potent NA inhibitors possess either
basic amino or guanidino functionalities at C-5, as their interaction with three acidic residues
of NA contributes significantly to strong inhibitor-NA binding. Therefore, the binding affinity
of 11, which has a non-basic moiety at C-5, should be greatly diminished. This compound was
prepared to demonstrate the viability of DIANA assay on a broader range of K
acetate was introduced by standard amide coupling mediated with TBTU, yielding ethyl ester
2, which was subsequently hydrolyzed to give sialylmimetic 11 in 65% overall yield.
i
values. An
2
Preparation of DIANA detection probe
5

The detection probe was prepared by copper-free click-chemistry (12). The probe
consists of an oligonucleotide of sequence 5´-CCT GCC AGT TGA GCA TTT TTA TCT GCC
ACC TTC TCC ACC AGA CAA AAG CTG GAA A-3´ with the terminal 3´- phosphate moiety
modified by an 6-amino-2-(hydroxymethyl)hexyl group (Generi-Biotech, OPC purification).
The modified oligonucleotide was reacted with NHS-DBCO (dibenzocyclooctyne-N-
hydroxysuccinimidyl ester, Sigma Aldrich) in a 1:50 ratio. The product (designated as
DNA_DBCO) was conjugated with a NA inhibitor containing an azido group (1). The
conjugation was performed at a 1:10 ratio of DNA_DBCO conjugate to 1. The DNA conjugates
were purified from the unconjugated small molecule by ultrafiltration with a 10 kDa cutoff and
then analyzed by LC-MS.
Determination of inhibition constants (K
Enzyme inhibition constants (K ) were determined in 0.1 M MES, pH 6.15, 150 mM
NaCl and 10 mM CaCl at 37 °C by fluorometric assay using 2´-(4-methylumbelliferyl)-α-D-
i
) by fluorometric assay
i
2
N-acetylneuraminic acid (4-MUNANA, Sigma-Aldrich) as a substrate (2). Substrate cleavage
was monitored with an Infinite M1000 fluorescence reader (TECAN) using an excitation
wavelength of 355 nm and an emission wavelength of 450 nm.
Each 40 μl reaction contained 17 nM (34 ng) NA2009wt and 500 μM 4-MUNANA. The
reactions were performed for 20 min at 37 ˚C in black fluorescence 96-well plates and
terminated by addition of 40 μl of 1 M sodium carbonate. Inhibition constants were determined
by measuring the reduction in fluorescence of the product 4-MU in the presence of different
inhibitor concentrations. The data were analyzed using the equation for competitive inhibition
according to Williams and Morrison or Dixon analysis when the K value was expected to be
i
above 100 nM (13, 14).
General DIANA protocol
The protocol described as “general assay conditions” by Navrátil et al. (6) was used with
the following modifications. A sheep antibody against NA of influenza A virus H1N1 (cat. no.
AF4858, R&D systems) was used as the capture antibody and was immobilized onto the plate
by applying 5 μl antibody solution (10 ng/μl in TBS: 20 mM Tris-HCl, 150 mM NaCl) to the
bottom of the wells. Then, the immobilized antibody was blocked with casein blocker and
incubated overnight, followed by washing using a microplate washer (405™ Microplate
Washer LS, BioTek). Next, 2 ng NA in 5 μl Q1 buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1%
(
w/v) Tween 20, 5 mM CaCl
2
) was applied to the bottom of the wells and incubated for 2 h,
6

followed by another wash in the microplate washer. Afterward, 5 μl of detection probe at a
concentration of 200 pM diluted in Q2 buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% (w/v)
Tween 20, 5 mM CaCl
2
, 0.0055% (w/v) casein) mixed in a 9:1 ratio with inhibitor dissolved in
1
00% DMSO was applied to the bottom of the wells (final concentration of 10% DMSO) and
incubated for 1 h. The plate was again washed in the microplate washer to remove unbound
probe. Finally, the amount of bound probe was determined by qPCR as described (6).
Determination of inhibition constants by DIANA
Inhibitors were tested at concentrations ranging from 10 nM to 10 μM for tight-binding
inhibitors and 316 nM to 316 μM for weak inhibitors. The model for determination of K
presence of detection probe with serial dilutions of inhibitor was described previously (6). K
was determined as follows: the ΔC values for each inhibitor were obtained as the difference in
between the well(s) incubated with inhibitor and the mean of wells without inhibitor
typically 12 wells per experiment). The K values of the inhibitors were computed from their
ΔC values and concentration of inhibitor according to the formula K
= (2-ΔCq / (1 - 2-ΔCq)) × Itot
)), where the Itot is the total inhibitor concentration, Ptot is the total concentration
of the probe (200 pM) and K is the dissociation constant of the probe determined by incubating
serial dilutions of the probe with a constant amount of enzyme (K = 3.9 nM). The final K value
for each inhibitor was calculated as the average of K values determined from each inhibitor
i
in the
i
q
C
q
(
i
q
i
/
(1 + (Ptot / K
d
d
d
i
i
concentration and corresponding C .
q
Protein crystallography
Enzyme-inhibitor complexes for crystallization were prepared by mixing NA2009wt in
5
mM Tris-HCl, pH 8.0, with 1 and 3. Mixtures were concentrated by ultrafiltration to a final
concentration of 8 mg/ml (3-fold molar excess of inhibitor in the mixture). Crystals were grown
using the hanging-drop vapor diffusion method at 19 ⁰C. Drops consisted of 1 μl NA-inhibitor
complex and 1 μl reservoir solution. The reservoir solutions for NA2009wt-1 and NA2009wt-3
were 100 mM HEPES, pH 7.5, 10% PEG 8000 and 100 mM HEPES, pH 7.0, 8% PEG 8000,
respectively. All crystals were transferred into a cryoprotectant consisting of reservoir solution
supplemented with 20% (v/v) ethylene glycol and flash-cooled in liquid nitrogen.
Data collection and structure determination
Diffraction data were collected at 100K on BL14.1 at the BESSY II electron storage
ring operated by the Helmholtz-Zentrum Berlin MX14.1 of BESSY, Berlin, Germany (15). The
7

dataset was processed using XDSAPP (16). The crystal parameters and data collection statistics
are listed in Table 1. The structure was determined by molecular replacement with the program
Molrep (17) using the crystal structure of NA2009wt complexed with tamiphosphor (10). Model
refinement was performed using the program REFMAC 5.7.0032 (18) from the CCP4 package
(
19) in combination with manual adjustments in Coot software (20). Compounds were modeled
after complete refinement of the protein chains and solvent model. The Molprobity server (21)
was used to evaluate the final model quality. The final refinement statistics are summarized in
Table 1. The structures were analyzed using the programs lsqkab (superpose) (22), baverage,
and contact from the CCP4 package (19).
PDB accession codes
Atomic coordinates and structure factors have been deposited in the PDB database under
accession codes 6G01 and 6G02.
8

RESULTS
Design of the detection probe
The FDA-approved drug oseltamivir carboxylate is the most widely used NA inhibitor,
and we aimed to prepare our detection probe by linking this compound or a derivative to a DNA
oligonucleotide. First, we sought to determine an appropriate way to link the oligonucleotide
with the inhibitor without compromising inhibitor binding to NA. The main features of
oseltamivir carboxylate that mimic the sialic acid substrate of influenza NA are as follows: (i)
a negatively charged carboxylate at C-1 interacting with three arginine residues (also known as
the arginine triad conserved in sialidases), (ii) a C-3 pentyloxy moiety accommodated in the
hydrophobic pocket, (iii) the C-4 acetamide and basic amino group at carbon C-5 that interact
with an aspartic acid and two glutamic acids. According to the crystal structure of NA in
complex with oseltamivir (PDB 3TI6) (23), the C-1 carboxylate group is suitable site for
attachment of a linker. However, a negative charge at C-1 is indispensable for inhibitor tight
binding, as demonstrated on a series of oseltamivir derivatives substituted at the carboxylate
moiety and a series of phospha-congeners (tamiphosphor derivatives) (24-26). As attachment
of a linker to the carboxylate moiety via an ester bond would lead to loss of the negative charge,
we used an oseltamivir derivative with a negatively charged C-1 phosphonate group, which
maintains its negative charge after linker.
In 2009, Streicher and co-workers demonstrated that replacement of the C-1 carboxylate
with a monoalkyl phosphonate moiety does not diminish inhibitory activity (27, 28). This so-
called tamiphosphor binds NA with similar potency as oseltamivir carboxylate, as we recently
confirmed by protein microcalorimetry (10). Moreover, the same group in 2011 demonstrated
immobilization/conjugation of the ω-azidohexyl ester of tamiphosphor by CuAAC click
chemistry. Subsequently, conjugates of this modified tamiphosphor with biotin and fluorescein
were designed for fluorometric detection and quantification of influenza viruses. Both
conjugates displayed selective, high-affinity binding to influenza NA and showed promise for
development of various diagnostic tools for biological research (29, 30). Together, these
findings indicate that monoalkylated tamiphosphor derivatives are suitable sialylmimetics.
Moreover, the phosphonate functionality is well-suited chemical handle for further
modification to develop tethered and very potent NA inhibitors.
Construction of DIANA detection probes requires a linker of appropriate length
equipped with bioorthogonal functional groups. In this particular case, ligation of the
9

sialylmimetic inhibitor to the oligonucleotide was performed by strain-promoted alkyne-azide
cycloaddition (SPAAC). Tamiphosphor was modified with a clickable ω-azidoalkyl moiety
(
yielding 1) suitable for conjugation to a DNA oligonucleotide equipped with a
dibenzocyclooctyne moiety, and the probe was prepared by click-chemistry (Figure 1B) (12).
The quality and quantity of the probe were monitored by LC-MS.
To determine the effect of linker attachment to tamiphosphor on enzyme binding, we
determined the inhibition potencies of 1, oseltamivir carboxylate (4), and tamiphosphor (2)
using a standard kinetic assay. All three compounds bound NA with comparable affinity (K
i
values of 24 nM, 24 nM and 26 nM, respectively), supporting use of a tamiphosphor-
oligonucleotide conjugate as a DIANA detection probe (Figure 1B).
X-ray structures of neuraminidase in complex with tamiphosphor derivatives
We recently reported the structure of tamiphosphor in complex with NA at 1.8 Å
resolution (10). The structure revealed that the O3 atom of the phosphonate functionality is
oriented out of the active site and does not engage in direct interactions with protein residues
(
10). This observation led us to prepare a modified inhibitor for the DIANA detection probe by
substituting one hydroxyl in tamiphosphor with a large linker connected to an oligonucleotide.
To analyze the binding of tamiphosphor derivatives to NA, we solved X-ray crystal
structures of complexes of 1 (ω-azidoalkyl ester) and 3 (methyl ester) with NA (strain
NA2009wt) at high resolution. These structures revealed that the compounds bind into the active
site with a pose very similar to that of tamiphosphor (10) (Figure 4A, B). The RMSD values
for superposition of tamiphosphor with the corresponding atoms in 1 and 3 were 0.22 Å. The
methyl ester of 3 was modeled into well-defined electron density map in a position pointing out
of the active site (Figure 4A). Most of the atoms of 1 could be traced in the electron density
map, with the exception of the three terminal nitrogen atoms of the tamiphosphor extension.
The azide group does not interact with the protein and is fully exposed to the solvent, and thus
its electron density map was of lower quality. Of the linker atoms, only C24 appears to interact
with NA. This atom is within van der Waals distance (3.7 - 4.0 Å) of residues Pro431 and Ile149
(
Figure 4C). This interaction may explain the slight improvement in K value observed for 1 (24
i
nM) compared to 3 (39 nM) (Table 2). The RMSD value for superposition of the corresponding
atoms of 1 and 3 was 0.15 Å.
1
0

Development of DIANA for neuraminidase inhibitor screening
We captured recombinant NA with an immobilized polyclonal anti-NA antibody and
detected it with the DNA probe based on 1 (Figure 1). Using this set-up, potential inhibitors
can be screened by incubating the test compounds with the captured enzyme in the presence of
the probe. The amount of bound probe is quantifiable by qPCR, and the inhibitory potency of
the compounds can be calculated from the difference in qPCR cycle number between wells
incubated with and without the test compound (ΔC ) (6).
q
2
+
2+
We found that it is important to include Ca in the assay buffer, as Ca is essential for
NA activity and stabilization of the active site (31, 32). Because small-molecule libraries are
typically dissolved in DMSO, we also tested the influence of including up to 10% DMSO in
the assay and found no significant effect.
Using serial dilutions of NA and the probe, we determined optimal working
concentrations of 2 ng NA and 200 pM probe per well. These concentrations led to a signal-to-
background ratio of approximately 7 qPCR cycles (over 2 orders of magnitude) and a Z´-score
of approximately 0.83 (average value calculated from several experiments with known
inhibitors). According to JH Zhang et al. (1999), a Z´-score between 0.5 and 1.0 is indicative
of an excellent assay for high-throughput screening (33).
We next assessed whether we could use this novel assay to determine the K value from
i
a single inhibitor concentration. We determined inhibition constants from single-well
measurements, as described in Materials and Methods, with serial dilutions of tamiphosphor.
The resulting K
i
values were constant within the range of the assay (i.e., over tamiphosphor
was 52 ±
nM, in good agreement with the value of 26 ± 4 nM obtained from standard enzyme kinetics.
concentrations spanning 43 nM to 12 μM; see Figure 5A). The average calculated K
i
9
We conclude that DIANA is suitable for accurate K determinations from single-well
i
measurements.
Comparison of DIANA with standard enzymatic assay
To compare the ability of DIANA and a standard enzymatic assay to determine
inhibition constants of novel compounds, we prepared a series of eleven modified oseltamivir
and tamiphosphor derivatives with various inhibition potencies. Compounds 2-4, known
inhibitors of NA with nanomolar K
tight-binding inhibitors. Compounds 5, an ethyl ester prodrug of oseltamivir phosphate, and 10-
1 represent inhibitors with several-order-of-magnitude lower inhibition potency and were
chosen to demonstrate the ability of the assay to identify and characterize weakly binding
i
values, served as standards for validation of the assay for
1
1
1

compounds. Guanidylated compounds 6 and 7 also have been described previously, with
contradictory data on their inhibitory activity compared to oseltamivir (10, 34, 35). In addition
to these known inhibitors, we prepared and tested three new compounds that feature stereogenic
centers adjacent to the former C-5 amino functionality. Compounds 8 and 9 are diastereomers,
allowing us to validate the assay on two very structurally similar inhibitors.
We first used an established fluorometric assay to determine the inhibition constants of
all compounds (see Table 2). This analysis was performed at pH 6.15, which is optimal for
NA2009wt activity and is necessary to achieve a sufficient signal-to-background ratio, using the
fluorescent substrate 4-MUNANA.(2, 36) The K values of 1-11 ranged from 20 nM to 40 μM.
i
As expected, 1-4 exhibited the highest potency. Oseltamivir ethylester (5) was a roughly 100-
fold inhibitor than oseltamivir carboxylate (4), which corroborates the necessity of a negatively
charged moiety in the C-1 position. Interestingly, substitution of the basic amine moiety with
the basic and bulkier guanidium group led to a several-fold decrease in potency (6 and 7). On
the other hand, the effect of modifying the C-5 amino group with a propanoate moiety was
dependent on the configuration of the stereogenic center: the S-configuration in 8 led to
inhibitory potency comparable to that of oseltamivir carboxylate, whereas the R-configuration
in 9 led to an approximately 10-fold decrease in potency. These results also indicate that an
acidic substituent is tolerated at the C-5 position. Finally, 10 and 11 showed the lowest
inhibitory activity of all compounds, with binding constants in the micromolar range.
We next evaluated the inhibition constants of all 11 compounds using DIANA. Because
there was no requirement for acidic pH, this assay was performed in at physiological pH (pH
7
.4). We tested serial dilutions of each inhibitor (7 concentrations spanning a three-order-of-
magnitude range) in duplicate in two independent experiments and calculated K values as the
average K determined from each well. However, only wells with C -values at least 2 cycles
lower than the background C and at least 1 cycle higher than C in wells incubated without
inhibitor were considered for K determination (note that cycle number is indirectly proportional
to the logarithm of the probe concentration, i.e., the higher the probe concentration, the lower
the cycle number). These cutoff values were applied to avoid potential errors in K values caused
by considering C values near the limits of the assay window. Indeed, we found that the K
values for individual compounds were constant over the concentrations tested (Table 2).
i
i
q
q
q
i
i
q
i
1
2

DISCUSSION
We previously described DIANA as an ultrasensitive tool with a large dynamic range
for determination of enzyme inhibition constants (6). Here, we adapted the protocol for
evaluation of NA inhibitors. In our setup, NA was captured by an immobilized antibody, and
tested compounds competed with an active-site-directed detection probe, which was then
quantified by qPCR (Figure 1A). The detection probe was prepared by linking a tamiphosphor
derivative 1 to a reporter DNA oligonucleotide (Figure 1B). The crystal structure of complex
of ω-azidoalkylester of tamiphosphor (compound 1) with NA2009wt revealed that modification
of the phosphate moiety of tamiphosphor does not affect its interactions with active site
residues. The esterification results in very minor interactions with residues at the edge of the
active site of the enzyme, and the terminal azide group is fully exposed to solvent and thus
available for further modifications. We thus concluded that modification of the azide group
with a DNA oligonucleotide in the DIANA probe will not significantly affect binding of 1 to
the NA2009wt active site.
We then characterized and validated the assay setup using oseltamivir carboxylate as a
reference inhibitor. We synthesized a series of novel oseltamivir derivatives and showed that
DIANA faithfully reproduced the structure-activity relationship of the series determined by
standard kinetic assay (Figure 5B). The absolute K values obtained by DIANA are, however,
i
on average roughly 4-fold higher than those obtained by standard kinetics (ranging from
equivalent to 12-fold higher). The most pronounced differences were observed for inhibitors
containing substituents targeting 150-loop identified as very dynamic part of NA sensitive to
protonation (37). These inconsistencies may thus result from the different protonation of the
enzyme under different conditions used for individual methods, such as use of different pH and
buffers. The advantage of DIANA assay is that it uses the experimental conditions that are
similar to the physiological ones while the kinetic assay has to be performed in artificial
conditions needed for efficient cleavage of the substrate.
In order to analyze whether our method is suitable for the testing of inhibitors of known
resistant mutants of NA, we determined Ki values of the compound 1 with the wild-type NA
and resistant variants containing oseltamivir major resistance mutations H275Y and
H275Y/I223V . K values for these mutants were 1700- and 11100-fold higher than for the wt
i
NA, respectively (data not shown). Due to these unfavourable binding characteristics of
compound 1, the DNA-probe would not be suitable for sensitive DIANA testing with resistant
NA mutants.
1
3

In conclusion, our results demonstrate that DIANA is useful for determining inhibition
constants of wt NA inhibitors and consumes only a small amount of enzyme (the kinetic assay
consumes an amount more than ten-fold higher of NA per measurement). Moreover, the multi-
well plate format, the possibility to determine inhibition potency from a single point
measurement, and the possibility to use up to 10% DMSO make this assay a promising choice
for screening small-molecule libraries for new influenza NA inhibitors.
Supplementary Material
Figure showing crystal structure of compound 20, experimental details and spectra of prepared
compounds.
Acknowledgments
The authors wish to thank Jana Starková and Iva Flaisigová for technical assistance and
Hillary Hoffman for proofreading of the manuscript. Diffraction data were collected on BL14.1
at the BESSY II electron storage ring operated by the Helmholtz-Zentrum Berlin.
Declaration of interest
The Authors declare that there are no competing interests associated with this manuscript.
Funding information
This work was supported by the project InterBioMed LO1302 from the Ministry of Education
of the Czech Republic.
Author contribution statement
MK - conceived idea, designed and performed experiments, analyzed results, wrote the paper
and communicated; VN - designed experiments and wrote the paper; KR - performed
experiments and analyzed data; JP - analyzed and validated data; CBA - designed and
performed experiments; PP - analyzed and validated data; JZ - designed and performed
experiments; AM - designed experiments; PŠ – designed experiments; JH - performed
experiments; IC - performed experiments; PŘ - analyzed data, visualized results, and wrote the
paper; JK - supervised the studies and revised the paper
1
4

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1
7

Table 1: Crystal data and diffraction data collection and refinement statistics.
NA2009wt/compound 3
NA2009wt/compound 1
PDB code
6G01
6G02
Data collection statistics
Space group
C222
1
P4
1
18.88 137.64 118.81
114.89 114.89 63.93
90 90 90
Cell parameters (Å; ˚)
9
0 90 90
2
Number of molecules in AU
Wavelength (Å)
2
0.9184
0.9184
Resolution (Å)
44.89-1.61 (1.71-1.61)
121,339 (19,127)
3.12 (2.79)
42.72-1.86 (1.97-1.86)
70,090 (11,223)
3.75 (3.58)
99.8 (99.6)
0.136 (0.738)
9.24 (1.86)
99.4 (61.4)
24.1
Number of unique reflections
Redundancy
Completeness (%)
98.2 (96.4)
a
R
meas
0.095 (0.645)
10.29 (1.88)
99.7 (75.8)
Average I/V(I)
CC1/2 (%)
Wilson B (Å2)
22.3
Refinement statistics
Resolution range (Å)
No. of reflections in working set
No. of reflections in test set
R value (%)b
44.89-1.61 (1.656-1.614)
119,520 (8,443)
1,821 (129)
42.71-1.84 (1.891-1.84)
70,147 (5,127)
1,799 (131)
17.8 (31.7)
15.3 (27.9)
1
8

R
free value (%)c
20.5 (30.3)
0.014
1.637
7,153
6056
19.7 (28.9)
0.015
1.670
6,995
6006
RMSD bond length (Å)
RMSD angle (q)
Number of atoms in AU
Number of protein atoms in AU
Number of water molecules in AU
825
809
Mean B value
protein/waters/compounds (Å2)
17.7/28.7/14.8
20.0/31.0/22.6
Ramachandran plot statisticsd
Residues in favored regions (%)
Residues in allowed regions (%)
96.4
3.6
96.1
3.9
The data in parentheses refer to the highest-resolution shell.
a
1/2
n
R
meas = 6hkl(n/(n-1)) 6
i
I
i
(hkl) - ¢I(hkl)²|/6hkl
6 I (hkl), where the
i
i
I
i
(hkl) is an individual intensity of the ith observation of reflection hkl
and ¢I(hkl)² is the average intensity of reflection hkl with summation
over all data.
b
o c
R-value = ||F | - |F ||/|F
o
|, where F
o
and F are the observed and calculated
c
structure factors, respectively.
c
Rfree is equivalent to R-value but is calculated for 5 % of the reflections
chosen at random and omitted from the refinement process (38).
d
as determined by Molprobity (39).
1
9

Table 2. Inhibition constants determined by standard kinetic assay and DIANA. The chemical
formulas of the inhibitors are also shown. The “fold K ” parameter represents the ratio K
DIANA, pH 7.4)/K (kinetic assay, pH 6.15). Substitutions of the moieties in oseltamivir
i
i
(
i
carboxylate (4) are highlighted in blue. The K values for both assays were calculated from two
i
independent experiments.
K
(
i
(kinetic assay)
pH 6.15) [nM]
K
i
(DIANA)
fold
Compound Chemical formula
(pH 7.4) [nM]
K
i
1
24 ± 5
33 ± 8
1.4
2
3
26 ± 4
39 ± 1
52 ± 9
2.0
2.6
100 ± 20
4
5
24 ± 4
31 ± 6
1.3
1.0
2 100 ± 200
2 000 ± 500
6
140 ± 30
570 ± 180
4.1
7
8
46 ± 12
15 ± 2
570 ± 200
170 ± 60
12
11
2
0

9
250 ± 40
960 ± 260
3.8
1.3
5
10
13 000 ± 1 000
43 000 ± 11 000
16 000 ± 5 000
210 000 ± 110 000
11
2
1

FIGURE LEGENDS
Figure 1. (A) Schematic representation of evaluation of neuraminidase inhibitors using
DIANA. The detection probe binds to the active site of neuraminidase captured on the well by
an immobilized anti-NA antibody. The amount of bound detection probe is determined by
qPCR. The inhibition potency of the tested compound can be determined from the difference
in the amount of bound probe after incubation of neuraminidase in the presence and absence of
the compound. (B) Preparation of a covalent conjugate consisting of a reporter DNA
oligonucleotide and a neuraminidase inhibitor (compound 1) used as a DIANA detection probe.
Only one regioisomer of SPAAC product is shown.
Figure 2. Synthesis of ω-azidohexyl tamiphosphor derivative 1. The following reagents and
conditions were used: (a) NaHCO
5%; (d) S-(1-oxido-2-pyridyl)-N,N,N',N'-tetramethylthiuronium hexafluorophosphate, Et
DMAP, THF; (e) bromotrichloromethane, DCM, hv, 78%; (f) dimethyl phosphite,
tetrakis(triphenylphosphine)palladium, Et N, toluene, 86%; (g) 1-azido-6-bromohexane, NaI,
3
/H
2
O; (b) Boc
2
O, Et
3
N, 98%; (c) 0.5 M NaOH, 1,4-dioxane,
9
3
N,
3
DIPEA, DMF, 60 °C, 56%; (h) trifluoroacetic acid.
Figure 3. Synthesis of oseltamivir derivatives. The following reagents and conditions were
used: (a) Ethyl 2-bromopropionate, NaHCO
chloride, Et N; (d) i. NaHCO , ii. dimethyl maleate; (e) 2-[2-(2-methoxyethoxy)ethoxy]acetic
acid, TBTU, Et N, DMF.
3
; b) 0.5 M NaOH, 1,4-dioxane; (c) 4-nitrobenzoyl
3
3
3
Figure 4. Crystal structure of NA2009wt in complex with 3 (A) and 1 (B). Compounds are
represented as sticks with carbon atoms colored yellow (3) or maroon (1), oxygen atoms red,
nitrogen atoms blue, and phosphorus atoms orange. The tamiphosphor molecule from the
previously reported structure (10) is overlaid and colored black. The protein is shown in green
with residues forming polar interactions highlighted as sticks. The 2F
O
-F electron density
C
maps are contoured at 1.0 V. In panel B, van der Waals interactions are represented by dashed
lines. (C) Overlay of 1 and 3. The protein is represented by its solvent accessible surface (gray),
and Pro431 and Ile149 are highlighted in green.
2
2

Figure 5. A) Determination of inhibition constants from a single inhibitor concentration by
DIANA: serially diluted tamiphosphor (TMF, x-axis) was tested using DIANA, and K values
were calculated from each data point (y-axis). The dashed horizontal line shows the average K
calculated as the mean of K values determined from concentrations of tamiphosphor in the 43
nM to 12 μM range. Error bars indicate standard error of mean from two independent
experiments. Both axes are shown as log-scales. B) Plot of K values of 11 neuraminidase
inhibitors determined either by kinetic measurement (fluorometric assay; x-axis) or by DIANA
y-axis). Error bars indicate standard error of mean from two independent experiments. Both
axes are shown as log-scales. The diagonal solid line represents a 1:1 ratio of values from both
methods. The dashed line is the linear regression of the K values determined by both methods
values determined by DIANA were on average 4-fold higher than those determined by
kinetic assay).
i
i
i
i
(
i
(
K
i
2
3

Supplementary Material
for the article
DNA-linked inhibitor antibody assay (DIANA) as a new method for screening influenza
neuraminidase inhibitors
1
*
1
1
1
Milan Kožíšek , Václav Navrátil , Kateřina Rojíková , Jana Pokorná , Carlos Berenguer
1
,2
1
1
1,2
1
2
Albiñana , Petr Pachl , Jitka Zemanová , Aleš Machara , Pavel Šácha , Jason Hudlický , Ivana
3
1,4
1,5*
Císařová , Pavlína Řezáčová , Jan Konvalinka
1
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Czech
Republic, Gilead Sciences and IOCB Research Center, Flemingovo n. 2, 166 10 Prague 6, Czech
2
Republic; Department of Organic Chemistry, Faculty of Science, Charles University, Hlavova
3
8
, 128 00 Prague 2, Czech Republic; Department of Inorganic Chemistry, Faculty of Science,
4
Charles University, Hlavova 8, Prague, 128 43, Czech Republic; Institute of Molecular Genetics
of the Academy of Sciences of the Czech Republic, Vídeňská 1083, 140 00 Prague 4, Czech
5
Republic; Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, 128
00 Prague 2, Czech Republic
*
Corresponding authors:
Milan Kožíšek or Jan Konvalinka, Institute of Organic Chemistry and Biochemistry of the Czech
Academy of Sciences, Czech Republic, Gilead Sciences and IOCB Research Center, Flemingovo
n. 2, 166 10 Prague 6, Czech Republic
Fax : +420 220 183 578; Tel : +420 220 183 218; e-mails : milan.kozisek@uochb.cas.cz,
konval@uochb.cas.cz
S1

Contents:
Figure S1. Crystal structure of compound 20.
S3
S5
Experimental procedures
1
13
Copies of H and C spectra
References
S25
S51
S2