Phosphonate inhibitors of West Nile virus NS2B/NS3 protease

Article abstract of DOI:10.1080/14756366.2018.1506772

West Nile virus (WNV) is a member of the flavivirus genus belonging to the Flaviviridae family. The viral serine protease NS2B/NS3 has been considered an attractive target for the development of anti-WNV agents. Although several NS2B/NS3 protease inhibitors have been described so far, most of them are reversible inhibitors. Herein, we present a series of α-aminoalkylphosphonate diphenyl esters and their peptidyl derivatives as potent inhibitors of the NS2B/NS3 protease. The most potent inhibitor identified was Cbz-Lys-Arg-(4-GuPhe)^P(OPh)₂ displaying K_i and k₂/K_i values of 0.4 μM and 28 265 M^?1s^?1, respectively, with no significant inhibition of trypsin, cathepsin G, and HAT protease.

Full text of DOI:10.1080/14756366.2018.1506772

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Phosphonate inhibitors of West Nile virus NS2B/
NS3 protease
Marcin Skoreński, Aleksandra Milewska, Krzysztof Pyrć, Marcin Sieńczyk &
Józef Oleksyszyn
To cite this article: Marcin Skoreński, Aleksandra Milewska, Krzysztof Pyrć, Marcin Sieńczyk &
Józef Oleksyszyn (2019) Phosphonate inhibitors of West Nile virus NS2B/NS3 protease, Journal of
Enzyme Inhibition and Medicinal Chemistry, 34:1, 8-14, DOI: 10.1080/14756366.2018.1506772
To link to this article: https://doi.org/10.1080/14756366.2018.1506772
© 2018 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 34, NO. 1, 8–14
https://doi.org/10.1080/14756366.2018.1506772
RESEARCH PAPER
Phosphonate inhibitors of West Nile virus NS2B/NS3 protease
a
b,c
a
a
ꢀ
ꢀb,c
ꢀ
ꢀ
Marcin Skorenski , Aleksandra Milewska , Krzysztof Pyrc , Marcin Sienczyk and Jozef Oleksyszyn
aFaculty of Chemistry, Division of Medicinal Chemistry and Microbiology, Wroclaw University of Science and Technology, Wroclaw, Poland;
c
bFaculty of Biochemistry, Biophysics and Biotechnology, Microbiology Department, Jagiellonian University, Krakow, Poland; Laboratory of
Virology, Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland
ABSTRACT
ARTICLE HISTORY
Received 22 September 2017
Revised 17 July 2018
Accepted 25 July 2018
West Nile virus (WNV) is a member of the flavivirus genus belonging to the Flaviviridae family. The viral
serine protease NS2B/NS3 has been considered an attractive target for the development of anti-WNV
agents. Although several NS2B/NS3 protease inhibitors have been described so far, most of them are
reversible inhibitors. Herein, we present a series of a-aminoalkylphosphonate diphenyl esters and their
peptidyl derivatives as potent inhibitors of the NS2B/NS3 protease. The most potent inhibitor identified
was Cbz-Lys-Arg-(4-GuPhe)P(OPh)2 displaying Ki and k2/Ki values of 0.4 mM and 28 265 Mꢀ1sꢀ1, respect-
ively, with no significant inhibition of trypsin, cathepsin G, and HAT protease.
KEYWORDS
NS2B/NS3 protease;
aminophosphonates;
serine proteases; enzyme
inhibitors; West Nile virus
Introduction
demonstrated that inactivation of NS2B/NS3 protease catalytic
centre blocks viral replication8. To become fully functional, the
NS3 segment requires a short co-factor, NS2B. The WNV protease
contains the classical serine protease catalytic triad Asp-His-Ser.
The protease binding site exists as a shallow groove composed of
7 subsites (S4-S3’, according to the Schechter and Berger nomen-
clature)9. An analysis of the substrate preference of WNV NS2B/
NS3 protease revealed that the natural substrates contain a highly
conserved arginine residue in the P1 position. Further studies
showed that basic amino acids were also preferred in P2 as well
The West Nile virus (WNV) belongs to the flavivirus genus
(Flaviviridae family) and is a mosquito-borne human pathogen of
global occurrence. WNV was first isolated from humans in 1937 in
the West Nile district of Uganda1. In 1953, it was identified in
birds of the Nile delta region. Until 1997, WNV was not considered
pathogenic to birds when a more virulent strain appeared in Israel
and caused fatal disease with signs of encephalitis and paralysis in
various bird species. In 1999, a pathogenic WNV strain was trans-
ferred to New York leading to its rapid spread throughout the
USA, Canada and in the following years, the virus further spread,
reaching northern countries of South America2. The virus also
became a relevant human pathogen in Eurasia, causing large out-
breaks in Greece, Israel, Romania, and Russia3–6. Although the life-
cycle of WNV involves the transmission of viruses between birds
and mosquitoes, various mammalian species, including humans,
and horses, are susceptible to the virus. However, mammals are
generally dead-end hosts, being infected through the bites of
infected mosquitoes7. Although infections with WNV are mainly
asymptomatic, one-fifth of the infected humans develops symp-
toms of the milder West Nile fever or more severe neuroinvasive
diseases (meningitis and encephalitis). Unfortunately, no vaccine
or effective antiviral therapy against WNV is available8.
as in the P3 positions10,11
.
Until now the most potent inhibitors of NS2B/NS3 protease
have been reported by Stoermer et al.11. These compounds are
tripeptide aldehydes (1,2) with a modified N-capping group
(Figure 1). Although inhibitors 1 and 2 displayed low Ki values of
6 and 9 nM, respectively, due to the high reactivity of an aldehyde
group, low stability and tendency to form hemiaminals, their
application as potential therapeutics is limited12. Hammamy et al.
presented a series of decarboxylated substrate analogues contain-
ing chlorophenylacetyl (3) or phenylacetyl moiety as an N-capping
group which are one of the most potent reversible NS2B/NS3
inhibitors reported thus far13. Recently, Bastos et al. presented an
interesting group of novel peptide-hybrids reversible inhibitors
based on 2,4-thiazolidinedione scaffold (4)14. An interesting revers-
ible inhibitor of NS2B/NS3 was described by Behnam et al.15 com-
pound 5 containing a benzyloxyphenylglycine residue at P1
position showed a significant reduction of Dengue and WNV titres
in cell-based assays of virus replication (EC50 ¼ 15.5 mM).
The flaviviral genome is a positive-sense single strand RNA. The
viral replication process occurs in the cytoplasm where the RNA
serves as a template for production of a large polyprotein, which
is further processed by host and viral proteases. This proteolytic
maturation yields structural (C, prM, and E) and non-structural pro-
teins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). NS3 plays a
key role during the polyprotein processing. This protein is com-
posed of an N-terminal protease domain (1–179 amino acids) and
Herein, we present the synthesis and application of a-aminoal-
kylphosphonates and their peptidyl derivatives as NS2B/NS3 WNV
protease inhibitors. These compounds belong to a class of irre-
versible inhibitors that specifically and exclusively react with the
a C-terminal helicase domain (residues 180–618). It has been active site serine residue leading to the formation of a slow
ꢀ
CONTACT Jozef Oleksyszyn
jozef.oleksyszyn@pwr.edu.pl
Faculty of Chemistry, Division of Medicinal Chemistry and Microbiology, Wroclaw University of
Science and Technology, Wybrzeze Wyspianskiego 27, Wroclaw 50-370, Poland
Supplemental data for this article can be accessed here.
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
9
Figure 1. Inhibitors of the West Nile virus NS2B/NS3 protease.
Figure 2. Mechanism of serine proteases inhibition by a-aminoalkylphosphonate diphenyl esters. Residue numbering according to the West Nile virus NS2B/
NS3 protease.
hydrolysing protease-inhibitor complex (Figure 2)16. One of the purchased from IRIS Biotech (Marktredwitz, Germany). All other
major advantages of a-aminoalkylphosphonate diphenyl esters is
reagents, catalysts and solvents were purchased either from
ꢀ
their lack of reactivity with cysteine, aspartyl, and metallopro-
Sigma-Aldrich (Poznan, Poland), Merck (Warszawa, Poland), or Alfa
teases as well as good stability in buffer and human plasma17,18
.
Aesar (Karlsruhe, Germany).
In this work, we present a series of lysine, arginine, and peptidyl
diphenylphosphonate derivatives which could be considered as
starting templates for further structure optimisation studies as
NS2B/NS3 protease inhibitors.
Inhibitors synthesis
The synthesis of simple Cbz-protected a-aminoalkylphosphonate
diphenyl esters (6–27) was performed as previously reported19,20
.
For the overall synthetic strategy as well as the spectroscopic data
of the obtained key intermediates and new products, see the
Supplementary material associated with the manuscript. The spec-
troscopic data for already known compounds fully agreed with
the literature data. Briefly, the synthesis of ornithine (9) and lysine
(6) diphenylphosphonates started with the preparation of N-
Chemistry
Chemical reagents
(Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophos-
phate (PyBOP), CbzLys(Boc)-OH, Fmoc-Arg(Pbf)-OH, 2-chlorotrityl
resin, trifluoroacetic acid, triisopropylsilane (TIPS), N,N-diisopropy-
lethylamine (DIPEA), and di-tert-butyl dicarbonate (Boc2O) were phthalimide-protected amino aldehydes, which were further used

ꢀ
M. SKORENSKI ET AL.
10
Figure 3. Synthesis of peptidyl a-aminoalkylphosphonate diphenyl esters (35–38) of general formula Cbz-Lys-Arg-AaaP(OPh)2, where AaaP is a phosphonate ester
analogue of arginine.
in the a-amidoalkylation reaction with benzyl carbamate and tri- the original a-amidoalkylation reaction procedure with substituted
phenyl phosphite (Scheme S1.A and S1.B)20. The phthalimide
benzaldehydes obtained from 4-fluorobenzaldehyde and appropri-
group of resulting orthogonally protected derivatives was
removed with hydrazine hydrate, which led to the target
compounds21. Their subsequent guanidinylation with N,N’-di-Boc-
S-ethyl isothiourea in the presence of HgCl2 and triethylamine
followed by Boc deprotection (50% TFA in DCM) resulted in
diphenylphosphonate analogues of arginine (7) and homoarginine
(10)22. In order to synthesise phosphonate analogue of thio-argin-
ine (8), 4-chlorobutylaldehyde obtained under Swern conditions
from 4-chlorobutane-1-ol was used in the amidoalkylation reaction
with benzyl carbamate and triphenyl phosphite with Cu(CF3SO3)2
as a catalyst (Scheme S2)23. The obtained product was treated
with thiourea in refluxing ethanol yielding target compound
which crystallised as white solid from diethyl ether. The synthesis
of diphenylphosphonate glutamine from 4-oxo-N-tritylbutanamide
followed the procedure Ewa et al. (Scheme S3)24. The synthesis of
4-amino (12) and 4-guanidine (13) derivatives of diphenylphosph-
onate phenylalanine started from the esterification of 4-nitrophe-
nylacetic acid with methanol followed by the reduction of nitro
group (Scheme S4). After Boc protection of the amino group, the
ester function was reduced to alcohol and then oxidised, by
means of the Swern method. Subsequently, a-amidoalkylation
with benzyl carbamate and triphenyl phosphite under mild condi-
tions with Cu(CF3SO3)2 as catalyst produced Cbz-(4-N-
Boc)PheP(OPh)2. Boc deprotection with 50% TFA in DCM gave 12,
which was further transformed into 13 as described above (for
derivatives 7 and 10)25. A similar approach was applied for the
synthesis of diphenylphosphonate phenylglycine derivatives
(14–19) with the exception of nitrobenzaldehyde that was used
for phosphonates synthesis, followed by nitro group reduction
with SnCl2 prior to the guanylation step (Scheme S5)26. The syn-
thesis of amidines 21 and 23 from the corresponding nitriles (20
and 22) followed the protocol developed by Oleksyszyn et al.
(Scheme S6 and S7)20,27. Heterocyclic derivatives of diphenyl-
ate heterocyclic secondary amine (Scheme S8): pyrazole (24), ben-
zimidazole (25), N-methylpiperazine (26), or morpholine (27)20,28
.
For the Cbz-protected derivatives that are most active against
NS2B/NS3 protease, we extended their structure with a dipeptidyl
Cbz-Lys-Arg fragment. The synthetic strategy, outlined in Figure 3,
started with the removal of the Cbz protecting group in orthogon-
ally protected phosphonates via hydrogenation over 10% Pd/C,
yielding target derivatives (32–35) containing a free amino group.
In parallel, the Cbz-Lys(Boc)-Arg(Pbf)-OH was synthesised using
solid phase peptide synthesis approach on 2-chlorotrityl resin.
Next, the obtained phosphonate analogues of arginine were
coupled to Cbz-Lys(Boc)-Arg(Pbf)-OH using PyBOP as the coupling
agent in the presence of DIPEA. The reaction was performed in
DMF for 12 h. The reaction mixture was then diluted five times
with ethyl acetate and washed with 5% citric acid, 5% NaHCO3,
and brine. The organic phase was dried over anhydrous MgSO4,
filtered and evaporated to dryness. The obtained crude product
was treated with cleavage solution (95% TFA, 2.5% TIPS, 2.5%
H2O; v/v/v) for 2 h at room temperature prior to the precipitation
of deprotected phosphonate peptides with diethyl ether. The final
compounds were purified on the HPLC (Varian ProStar 210 with a
R
dual k absorbance detector system equipped with the DiscoveryV
BIO Wide Pore C8 HPLC Column 250 mm ꢁ 212 mm, 10 mm) with a
15 ml/min flow rate using a gradient 5–95% (0.05% TFA/aceto-
R
nitrile) in (0.05% TFA/H2O) over 15 min (Method A) or DiscoveryV
BIO Wide Pore C8 HPLC Column (250 mm ꢁ 46 mm, 10 mm) with a
0.9 ml/min flow rate using a gradient 0 ꢀ 100% (0.05% TFA/aceto-
nitrile) in (0.05% TFA/H2O) over 15 min (Method B)). The nuclear
magnetic resonance spectra (1H and 31P) were recorded on either
1
a Bruker Avance DRX-300 (300.13 MHz for H NMR, 121.50 MHz for
31P NMR) or Bruker Avance 600 MHz (600.58 MHz for 1H NMR,
243.10 MHz for 31P NMR) spectrometer. Chemical shifts are
phosphonate phenylglycine analogue (24–27) were obtained via reported in parts per million (ppm) relative to a tetramethylsilane

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
11
internal standard. High-resolution mass spectrometry was acquired (Bachem,
on Waters Acquity Ultra Performance LC, LCT Premier, XE.
Bubendorf,
kem ¼ 440 nm, for human cathepsin G) or Phe-Ser Arg-AMC
(Bachem, Bubendorf, Switzerland; 40 mM,
Switzerland;
40 mM,
kex ¼ 340 nm,
kex ¼ 340 nm,
kem ¼ 440 nm, for HAT protease). The progress of the reactions
was monitored continuously at 37 ꢂC on a Spectra Max Gemini
XPS spectrofluorometer for 20 min. Control curves in the absence
of inhibitor were linear. The rate of the tested protease inhibition
was calculated from the linear range of the plot.
In vitro NS2B/NS3 protease inhibition
For the initial screening, Cbz-protected aminophosphonates were
assayed in 96 well microplates (NuncTM F96 MicroWellTM White
Polystyrene Plate) in the following protease buffer: 50 mM Tris,
1 mM Chaps, 20% glycerol, pH 8.5. WNV NS2B/NS3 protease
(AnaSpec, Liege, Belgium; 20 nM) was pre-incubated with tested
Molecular docking
ꢂ
inhibitor (100 mM) in the protease buffer at 37 C for 10 min prior
In order to evaluate the binding mode of the obtained phospho-
nate inhibitors into the NS2B/NS3 active site, we have performed
a molecular docking simulation (AutoDock Vina 1.1.2) using the
WNV NS2B/NS3 protease (2fp7.pdb) as a receptor31. The coordi-
nates of the Bz-Nle-Lys-Arg-Arg-H inhibitor molecule as well as
water molecules were removed from the structure (Figure 4)32.
Since numerous studies have shown that a-aminoalkylphospho-
nates inhibitors complex serine proteases as phosphonic acids, we
docked energy minimised (MM2 force field) inhibitor 38 in the
chemical form of (Cbz-Lys-Arg-(4-GuPhe)P(OH)2)33,34. The centre of
the grid box was defined at the catalytic Ser hydroxyl oxygen
with the grid box size 100 ꢁ 100 ꢁ 100 Å. Pictures were prepared
in Pymol35.
an addition of the fluorescent substrate Pyr-RTKR-AMC (AnaSpec,
Liege, Belgium; 20 mM). The progress of the reaction was moni-
tored continuously (kex ¼ 354 nm, kem ¼ 442 nm) at 37 ꢂC on a
Spectra Max Gemini XPS spectrofluorometer (Molecular Devices,
Sunnyvale, CA) for 30 min. For compounds which exhibited more
than 50% of inhibition, the Ki and k2/Ki values were calculated. In
96 well microplates narrowed concentration of inhibitors and
constant substrate concentration (Pyr-RTKR-AMC, C ¼ 20 mM,
KM ¼ 59 mM) were prepared. The enzyme solution was added and
enzymatic reaction was monitored (Figure S1). Using a model for
irreversible inhibition, in which the first order inactivation rate
constant kobs is hyperbolic dependent from the inhibitor concen-
tration Ki and k2/Ki values were calculated (equations (1–4) in
supplementary material)29. Control progress curves in the absence
of inhibitor were linear. The standard deviation for the presented
values was calculated using the mean of two independent experi-
ments and did not exceed 10%.
Results and discussion
Inhibitor P1 position screening
Compounds presented in this study are categorised into three
groups: (I) compounds 6–11 are simple diphenyl phosphonate
analogues of lysine, arginine, glutamine, ornithine, homoarginine,
and thioarginine; (II) compounds 12–23 are aromatic analogues
bearing a basic moiety, and (III) derivatives of diphenylphosphona-
tec phenylglycine (24–27) with different heterocyclic substituents.
From all tested simple Cbz-N-capped derivatives, the highest
potency of action toward NS2B/NS3 protease was observed for
compounds 6, 7, 13, and 16 (Table 1). The most potent com-
Control proteases inhibition assay
ꢀ ꢀ
Bovine b-trypsin (AppliChem, Łodz, Poland), human cathepsin G
ꢀ
(Biocentrum, Krakow, Poland) and Human Airway Trypsin-like
Protease (HAT) (R&D Systems, Minneapolis, MN) were used as
control proteases to screen the activity of NS2B/NS3 protease
inhibitors (36–39) obtained in this study. Inhibitors were assayed
in 96-well microplates in the 0.1 M HEPES, 0.5 M NaCl, 0.03%, pH
7.5 Triton X-100 (for bovine b-trypsin and cathepsin G). Bovine
b-trypsin (15 nM), human cathepsin G (150 nM) or HAT protease
(0.001 mg) was pre-incubated with tested inhibitor (25 mM) in the
protease buffer at 37 ꢂC for 10 min prior to the addition of the
fluorescent substrate: Cbz-Arg-AFC (synthesised in-house accord-
ing to the procedure described by Bissell30; 50 mM; kex ¼ 400 nm,
pound was observed to be 13 with k2/Ki value of 200 Mꢀ1sꢀ1
.
Replacing the guanidine moiety in 13 with amino group (12)
resulted in a dramatic drop in the inhibitory activity (11% of inhib-
ition at 100 mM). In general, derivatives substituted at the meta
position showed weaker inhibition levels as compared to their
analogues substituted at para position of the phenyl ring (14, 15,
kem ¼ 505 nm, for bovine b-trypsin); MeO-Suc-Ala-Ala-Pro-Val-AMC 16 vs. 17, 18, 19). Compounds with heterocycles (24–27) showed
Figure 4. (A) Docking conformation of Cbz-Lys-Arg-(4-GuPhe)P(OH)2 (38, green) in the binding site of NS2B/NS3 protease. The peptidyl inhibitor is shown in red (Bz-
Nle-Lys-Arg-Arg-H) is present in the original crystal structure of WNV protease (2fp7.pdb). (B) Interaction of Cbz-Lys-Arg-(4GuPhe)P(OH)2 (38, orange) with the NS2B/NS3
active site. The hydrogen bond network is indicated with yellow dashed lines.

ꢀ
M. SKORENSKI ET AL.
12
Table 1. Continued.
Table 1. Activities of simple Cbz N-capped phosphonates against the NS2B/NS3
WNV protease.a
No
R
Ki [lM]b
k2/Ki [Mꢀ1sꢀ1
]
25
4%
3%
26
27
No
R
Ki [lM]b
k2/Ki [Mꢀ1sꢀ1
]
6
22 2 lM
80
7
8
13 1 lM
154
1%
22%
aMean values standard deviation of two experiments conducted in duplicates.
bpercent of inhibition was calculated for compounds which displayed low activ-
ity toward NS2B/NS3 protease after 30 min incubation at 37 ꢂC; substrate used:
Pyr-RTKR-AMC (C ¼ 20 mM, KM ¼ 59 mM). Bold values indicate the most
active compounds.
9
12%
12%
10
11
12
13
15%
11%
Table 2. Activities of the peptide phosphonates against the NS2B/NS3
WNV proteasea
No.
Compound
Ki (lM)
k2/Ki (Mꢀ1sꢀ1
)
36
37
38
39
40
Cbz-Lys-Arg-LysP(OPh)2
Cbz-Lys-Arg-ArgP(OPh)2
Cbz-Lys-Arg-(4-GuPhe)P(OPh)2
Cbz-Lys-Arg-(4-GuPhg)P(OPh)2
Cbz-Lys-Arg-Arg-H
8
3
0.9
0.3
5 520
10 725
28 265
24 890
0.4 0.03
0.7 0.2
0.12 0.02
4
0.3 lM
200
Not determined
aMean values standard deviation of two experiments conducted in duplicates.
Bold value indicates the most active compound.
14
15
16
3%
16%
very weak (1–5%) inhibitionagainst NS2B/NS3 protease when used
at 100 mM concentration. This was probably because the hetero-
cyclic group could not fit into the P1 binding pocket. The phenyl
(21) and naphthyl (23) amidines were slightly more active against
the tested protease. Nevertheless, among the tested series of
a-aminoalkylphosphonate diphenyl esters we selected the most
active compounds for further modifications. In summary, we iden-
tified lysine (6) and arginine (7) as the most favourable P1 resi-
dues, whereas for non-proteinogenic amino acid analogues we
selected guanidine derivatives (13) and p (16)23.
10 1 lM
87
17
18
4%
8%
19
11%
Influence of peptide chain elongation
The structure of the most active inhibitors identified in the initial
screening step was elongated with a P2 Arg and P3 Lys. The
resultant compounds (36–39) showed significantly increased
inhibitory potencies against NS2B/NS3 protease (Table 2). The
introduction of the additional two residues into the structure
resulted in a similar (69-fold) improvement in their inhibitory
potency, leading to 36 (k2/Ki ¼ 5 520 Mꢀ1sꢀ1) and 37 (k2/Ki ¼
10 725 Mꢀ1sꢀ1). The most potent NS2B/NS3 protease inhibitor
identified in the presented studies was compound 38, which dis-
played a k2/Ki value of 28 265 Mꢀ1sꢀ1. The highest (ꢃ290-fold)
increase of inhibitory potencies was observed for the peptidyl
derivative of the phosphonate analogue of 4-guanidinephenylgly-
cine (39) which showed a k2/Ki value of 24 890 Mꢀ1s. The inhib-
ition data observed for 36–39 is in agreement with the reported
X-ray structure of NS2B/NS3 protease, thus highlighting the signifi-
cant role of P2 and P3 residues in binding molecules (substrates
and inhibitors) to the enzyme32. As reference compound we syn-
thesised peptide aldehyde inhibitor (40). This reversible inhibitor
20
21
8%
22%
22
23
2%
12%
24
5%
(continued)

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
13
showed Ki lower (ꢃ4 times) than our most potent phosphonate
inhibitor. However, it is difficult to compare the Ki values between
reversible and irreversible inhibitors. Noteworthy, all of the
obtained compounds were tested as diastereoisomeric mixtures
thus their separation into single isomers will lead to significantly
more potent inhibitors as observed previously36. Further investiga-
tion into the design and synthesis of phosphonate inhibitors of
NS2B/NS3 protease might lead to discovering more potent and
selective inhibitors. Future work should involve structure-activity
relationship studies of the P2 and P3 residues as well as ring sub-
stituents. The next challenge is a more comprehensive structure-
activity relationship study aiming to optimise the peptidyl
fragment of the inhibitor as well as the structure of the aromatic
ring substituent prior to the in vivo studies of most potent
derivatives.
Acknowledgements
The authors would like to thank Dr. Ewa Burchacka and Maciej
Walczak for the kind gift of Cbz-GlnP(OPh)2 and Dr. Renata Grzywa
for guidance in the inhibitors docking analysis.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Ministry of Science and Higher
Education’s Iuventus Plus Programme [IP2012 0556 72]. The
funder had no role in the study design, data collection and ana-
lysis, decision to publish, or preparation of the manuscript. MS
and JO are grateful to Wroclaw University of Technology for sup-
port [Statute Funds 0401/0195/17]. MS is grateful to National
Science Centre [UMO-2013/09/N/ST5/02439]. AM and KP are grate-
ful to National Science Centre [UMO-2016/21/B/NZ6/01307].
Project supported by Wroclaw Centre of Biotechnology, pro-
gramme The Leading National Research Centre (KNOW) for
years 2014–2018.
Protease selectivity assay
The selectivity of inhibitors 36–39 were determined by means of
serine proteases of a similar substrate recognition pattern such as
bovine b-trypsin, human cathepsin G, and HAT protease. The
results clearly showed that the inhibition levels observed for all of
the investigated compounds at a concentration of 25 mM did not
exceed 10% after a 30 min incubation period (Table S2). These
results indicate that the obtained peptidyl inhibitors are not sig-
nificantly active against members of proteases with a trypsin-like
activity. However, the selectivity toward other members of this
family will be further examined.
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10. Chappell KJ, Stoermer MJ, Fairlie DP, Young PR. West Nile
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Molecular docking
The analysis of inhibitor 38 docked to the active site of NS2B/NS3
protease revealed docking conformation to be very similar to the
one observed for Bz-Nle-Lys-Arg-Arg-H present in the crystal struc-
ture reported by Erbel et al. (2fp7.pdb)32. The reactive phospho-
nate warhead of the inhibitor is in close proximity (1.6 Å) to the
catalytic serine residue allowing the formation of a covalent bond
between the protease and inhibitor (Figure 4(A), Figure S3). The
basic side chains of the amino acids in P1 and P3 positions are
responsible for the formation of an extensive hydrogen bonding
network with the protease (Figure 4(B)) including the P1 4-guani-
dinephenylalanine electrostatic interaction with the side chain of
Asp129; P2 arginine with the carbonyl oxygen of Gly83, and Asp82
and the side chain of Asn84; and lysine in P3 position with Phe85
carbonyl oxygen. Additionally, two tyrosine residues (Tyr150,
Tyr161) could be responsible for interaction with aromatic ring of
4-guanidinephenylalanine inhibitor residue (Figure 4(A), Figure S4).
This interaction provides explanation for the improved activity of
inhibitors 38 and 39 over simple lysine and arginine analogues
(36,37).
11. Stoermer MJ, Chappell KJ, Liebscher S, et al. Potent cationic
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Conclusions
In this work, we present a series of phosphonate diphenyl esters
with low micromolar inhibitory activities against the WNV NS2B/
NS3 protease. This class of inhibitors has never been reported to
inhibit the NS2B/NS3 protease. The rigid 4-guanidinephenylalanine
and 4-guanidinephenylglycine moieties at the P1 position were
found to be more potent than a P1 arginine. Future work should
involve more structure-activity relationship studies at the P2 and
P3 residues and changing the phosphonate ester moieties.

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