Novel agmatine and agmatine-like peptidomimetic inhibitors of the West Nile virus NS2B/NS3 serine protease

Article abstract of DOI:10.1016/j.ejmech.2011.04.055

This communication reports the synthesis and inhibitory activities of novel non-covalent peptidomimetic inhibitors of the West Nile virus NS2B/NS3 protease containing a decarboxylated P1 arginine (agmatine; 4-aminobutylguanidine) and related analogues. On

Full text of DOI:10.1016/j.ejmech.2011.04.055

European Journal of Medicinal Chemistry 46 (2011) 3130e3134
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Novel agmatine and agmatine-like peptidomimetic inhibitors of the West Nile
virus NS2B/NS3 serine protease
*
Huichang Annie Lim, Joma Joy, Jeffrey Hill, Cheng San Brian Chia
*
Experimental Therapeutics Centre, Agency for Science, Technology and Research (A STAR), 31 Biopolis Way, Nanos #03-01, Singapore 138669, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history:
This communication reports the synthesis and inhibitory activities of novel non-covalent peptidomi-
metic inhibitors of the West Nile virus NS2B/NS3 protease containing a decarboxylated P1 arginine
(agmatine; 4-aminobutylguanidine) and related analogues. One agmatine peptidomimetic (4-phenyl-
phenacetyl-Lys-Lys-agmatine; compound 2) was shown to be a competitive inhibitor with a binding
Received 16 February 2011
Received in revised form
5 April 2011
Accepted 20 April 2011
Available online 28 April 2011
affinity of Ki 2.05 ꢀ 0.13
mM and was inactive against thrombin (IC50 > 100 mM). Our results suggest that
peptidomimetics with agmatine at the P1 position could potentially be employed as starting tools in the
design of non-covalent competitive protease inhibitors due to their relative stability and ease of chemical
synthesis compared to inhibitors containing reactive electrophilic warheads.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Agmatine
Arginine mimetic
Serine protease inhibitor
West Nile virus
1. Introduction
A common strategy used for the inhibition of the NS2B/NS3
protease involved covalent inhibitors that compete with the
The West Nile virus (WNV) is a mosquito-borne virus of the
Flaviviridae family. Originating from Africa, it has spread to humans
in different continents, including North America [1,2]. Between
1999 and 2009, the United States Center for Disease Control
reported more than 20,000 people in the United States have been
infected, resulting in 1163 fatalities [3]. Symptoms of infection
include fever, headaches, chills, diaphoresis and lymphadenopathy
which can lead to high fever known as the ‘West Nile Fever’. Some
of these cases progress to meningitis, coma and death [4].
The WNV has a single-strand, positive-sense,11-kb RNA genome
which serves as a messenger RNA for protein synthesis as well as
a template for RNA replication in a mammalian host cell. The viral
genome encodes seven nonstructural proteins (NS1, NS2A, NS2B,
NS3, NS4A, NS4B and NS5) [5] and one attractive target for antiviral
drug development is NS3 [6e8]. In the cytoplasm of a host cell, NS3
complexes to its NS2B cofactor to form a functional trypsin-like
serine protease which cleaves polyprotein precursors into mature
viral proteins [9]. The NS2B/NS3 complex recognizes and selec-
tively cleaves the C-terminal end of two consecutive, highly-
conserved basic residues (Fig. 1) [10]. This unusual specificity is
not shared by many mammalian proteases and could thus be
exploited as an antiviral drug target [6,11].
substrate for the catalytic site. Such peptide-based covalent inhib-
itors have their C-terminal carboxyl group chemically modified into
reactive electrophilic ‘warheads’ (see [11,12] for reviews). A popular
warhead is the aldehyde functional group and peptide aldehydes
have been shown to inhibit the WNV NS2B/NS3 protease at sub-
micromolar potencies [13]. However, warhead peptidomimetics
have several undesirable characteristics, including lack of selec-
tivity over other trypsin-like proteases due to their high reactivity
and low chemical stability, curtailing their potential for drug
development [14]. In this report, we investigated if peptidomi-
metics without electrophilic warheads could inhibit the WNV
NS2B/NS3 protease. These inhibitors do not bind covalently to the
catalytic serine in the active site but instead employ hydrophobic
and/or electrostatic interactions to compete with the natural
substrate for the active site. Such inhibitors should be less reactive
and hence more ‘druggable’ compared to warhead inhibitors.
Our strategy focused on peptidomimetics containing a P1
decarboxylated arginine (agmatine; 4-aminobutylguanidine) and
structurally-related analogues. Agmatine-containing peptidomi-
metics have been reported to inhibit trypsin-like serine proteases
like furin, a mammalian protease responsible for the cleavage of
inactive protein precursors involved in many physiological path-
ways [14] and thrombin, a protease involved in blood-clotting
[15,16]. These inhibitors contained agmatine and agmatine
analogues at the P1 position and were found to be competitive
inhibitors with micromolar to sub-micromolar potencies. On this
* Corresponding author. Tel.: þ65 64070348; fax: þ65 64788768.
E-mail address: cschia@etc.a-star.edu.sg (C. San Brian Chia).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.055

H.A. Lim et al. / European Journal of Medicinal Chemistry 46 (2011) 3130e3134
3131
agmatine-containing peptidomimetics could inhibit the WNV
NS2B/NS3 serine protease.
Asp129
S1
Asp90
Encouraged by this, we synthesized compound 2 where the
N-terminus was capped with a 4-phenyl-phenacetyl (4-Ph-Phac)
group. This group has been reported to be the most potent
N-capping group in a panel of tripeptide aldehyde WNV inhibitors
(IC50 ¼ 32 nM for 4-Ph-Phac-KKR-H) and computer docking studies
using simulated annealing showed that the 4-Ph-Phac group could
squeeze into the S4 WNV protease binding site [13]. Expectedly, the
potency of 2 improved approximately 4-fold compared with 1, with
Tyr161
HN NH2
NH
P1
P3
NH2
O
O
H
N
H
N
N
H
N
H
O
O
R1
an IC50 of 4.7 ꢀ 1.2
2 showed that it acted as a competitive inhibitor with a Ki of
2.05 ꢀ 0.13 M (Fig. 3).
mM (Table 1). A Lineweaver-Burk plot of inhibitor
P1'
P2
NH2
m
We next tested a panel of compounds containing agmatine
homologues with different alkyl chain lengths (compounds 3e5).
Using the recent crystal structure of the tripeptide aldehyde
inhibitor Naphthoyl-KKR-H complexed to WNV NS2B/NS3 protease
(3E90 pdb [18];), the P1 arginine’s guanidino group was observed to
take part in electrostatic attraction with the side-chain carboxylate
anion of Asp129 of NS3 within a distance of 3.2e4.3 Å in the S1
binding site. Altering the length of the agmatine alkyl chain should
alter the guanidino-carboxylate distance and affect binding affinity.
As expected, compounds 3e5 failed to inhibit the NS2B/NS3
Asp82
Asp75
Fig. 1. Schematic diagram of a peptide substrate showing the position of the scissile
bond (arrowed) and the basic residues: Lys (P3, P2) and Arg (P1). The WNV protease
residues in the S1eS3 binding sites participating in substrate binding are numbered
based on the crystal structure 3E90.pdb.
basis, we designed and synthesized ten novel compounds incor-
porating agmatine and its analogues at the P1 position of the WNV
peptide substrate recognition sequence: Lys(P3)-Lys(P2)-Arg(P1)
and tested them for inhibitory activities against WNV NS2B/NS3
serine protease (Fig. 2).
Lastly, we explored the enzyme inhibitory activity of our two
most potent inhibitors against thrombin to gain an insight into
their selectivity against trypsin-like serine proteases.
enzyme (IC50 > 100 mM; Table 1). An intriguing observation was
that the addition or removal of just one methylene group
(compounds 3 and 4 respectively) abrogated all binding affinity,
suggesting that a 4-carbon alkyl chain between the amide and
guanidino group was optimal for binding. Indeed, molecular
modelling of compound 3 using 3E90 pdb as a template showed
that lengthening the alkyl chain by one carbon caused the guani-
dino group of 3 to clash with Asp129 side-chain. Removing one
methylene from the alkyl chain (compound 4) increased the
distance between the guanidino group and the carboxyl side-chain
of Asp129 to approximately 4.1e5.0 Å, possibly disrupting the salt
bridge between them. With this knowledge, we synthesized
2. Results and discussion
2.1. Chemistry
The general synthetic procedure for compounds 1e10 is shown
in Scheme 1: the appropriate diamine was guanylated with N,N-di-
(t-butoxycarbonyl)-S-methylisothiourea based on the method by
Verdini et al. [17] to give the Boc-protected agmatine and its
analogues. These were then coupled with Fmoc-Lys(Boc)-Lys(Boc)-
OH using O-benzotriazole-N,N,N0,N’tetramethyluroniumhexafluoro
phosphate (HBTU) as coupling reagent. After Fmoc removal by 1,8-
diazabicyclo[5.4.0] undec-7-ene (DBU), the deprotected peptido-
mimetics were N-capped with acetic anhydride or 4-biphenylacetic
acid. Finally, the Boc protecting groups were removed by TFA
treatment and the pseudopeptides were purified (HPLC) and
characterized (LCMS).
compound 6 in which the presence of a trans-(b-g) double bond
rigidifies the 4-carbon alkyl chain. Interestingly, the unsaturated
analogue 6 was approximately 13-fold less potent than its saturated
counterpart 2 (IC50 61.2 ꢀ 8.6 and 4.7 ꢀ 1.2
mM respectively), sug-
gesting that a flexible 4-carbon alkyl chain was needed for optimal
positioning of the guanidino group relative to Asp129 and Tyr161 in
the S1 binding site. Based on these results, we could conclude that
a saturated 4-carbon chain was needed for optimal binding.
A
final series of inhibitors containing conformationally
restrained agmatine analogues 7e10 (Table 1) were synthesized
using inhibitor 2 as a template to test the effect of increasing the
agmatine n-butyl chain rigidity on enzyme S1 site binding affinity.
Hence, 5 and 6-membered cyclic agmatine mimetics 7e10 were
synthesized and assayed. Experimental results revealed that
6-membered analogues 7 and 8 did not possess any inhibitory
2.2. Biological activity
Compound 1 showed an inhibitory activity of IC50 ¼ 18.2 ꢀ
4.2
mM against WNV NS2B/NS3 protease (Table 1), proving that
activities (IC50 > 100 mM), suggesting that the rigid 6-membered
rings may have oriented the guanidino group away from its optimal
position in the S1 binding site. This was supported by molecular
modelling studies using the crystal structure 3E90 pdb adopted by
the tripeptide aldehyde inhibitor KKR-H when complexed to the
WNV protease. Our models revealed that replacing the flexible
agamatine n-butyl chain in compound 2 with 6-membered rings
steered the guanidino group away from the S1 binding site which
possibly abrogated all binding affinities (Fig. 4A and B).
a
HN
NH2
NH
b
NH2
NH2
HN
NH2
NH
O
O
H
N
H
N
R'
R'
n
N
N
H
N
H
N
H
H
O
O
NH2
NH2
The assay results for agmatine mimetics containing 5-membered
pyrrolidine rings (compounds 9 and 10) were more interesting;
3
4
R'=4-Ph-Phac,n=5
R'=4-Ph-Phac,n=3
5 R'=4-Ph-Phac,n=2
1
2
R'=Ac
R'=4-Ph-Phac
compound 9 showed no inhibitory activity (IC50 > 100
mM) while 10
exhibited moderate activity (IC50 20.0 ꢀ 5.8
m
M). Our molecular
models suggested that the stereogenic carbon in the pyrrolidine ring
played an important role in orienting the guanidino group either
Fig. 2. Structures of compounds (a) 1 and 2; (b) 3e5. Agmatine is shown in the hashed
box. Structures of compounds 6e10 can be found in Table 1.

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H.A. Lim et al. / European Journal of Medicinal Chemistry 46 (2011) 3130e3134
BocN
(CH2)n
NHBoc
BocN
NHBoc
a
H2
N
NH
H2N
(CH2)n
NH2
+
S
b, c
HN
(CH2)n
NH2
BocN
(CH2)n
NHBoc
d, f
H
N
H
N
H
Lys(Boc)
Lys(Boc)
NH
Ac Lys
Lys
NH
e, f
HN
NH2
O
H
N
CH2
Lys
Lys
(CH2
)
n
NH
Scheme 1. (a) DIPEA, DCM, 25 ꢁC, 16 h; (b) Fmoc-Lys(Boc)-Lys(Boc)-OH, HBTU, DIPEA, DMF, 25 ꢁC, 1 h; (c) DCM, DBU, 25 ꢁC, 1 h; (d) acetic anhydride, DIPEA, DMF, 25 ꢁC, 30 min;
(e) 4-biphenylacetic acid, HBTU, DIPEA, DMF, 25 ꢁC, 1 h; (f) TFA, DCM, 25 ꢁC, 30 min.
away or towards the S1 binding site (Fig. 4C and D respectively). The
Table 1
molecular model of compound 10 showed the guanidino group ori-
enting towards the S1 binding site (Fig. 4D) and was 4-fold less potent
Inhibitory data of WNV peptidomimetic inhibitors with the general sequence:
P4-Lys-Lys-P1. Greek symbols illustrate the relative position of the guanidine
e-nitrogen in relation to the amide nitrogen.
than inhibitor 2 (IC50 20.0 ꢀ 5.8 and 4.7 ꢀ 1.2
mM respectively).
A plausible reason could be that the guanidino group of inhibitor 10
may not be in an optimal position compared to inhibitor 2. Another
reason could be the loss of one H-bond interaction between 10 and
the protease. In fact, by superimposing 10 to the 3E90 pdb crystal
structure, it was observed that the P1 arginine amide proton was
involved inH-bonding toGly151 carbonyl oxygenof theNS3protease.
The removal of the amide proton with a pyrrolidine ring substitution
would result in one less H-bonding interaction between 10 and the
protease (Fig. 5), reducing its binding affinity.
Compound
1
P4
P1
IC50 (mM)
NH
NH
β
β
δ
H
N
ε
α
α
γ
γ
Acetyl
18.2 ꢀ 4.2
4.7 ꢀ 1.2
>100
N
NH2
NH2
H
δ
H
N
ε
2
3
4
5
6
7
4-Ph-Phac
4-Ph-Phac
4-Ph-Phac
4-Ph-Phac
4-Ph-Phac
4-Ph-Phac
N
H
NH
Overall, our experimental results suggested that the WNV NS2B/
NS3 S1 binding site was highly selective for a P1 agmatine.
Substituting 5- or 6-membered rings into the carbon chain severely
abrogated the inhibitory activities of our test compounds.
Finally, we assayed the two most potent inhibitors 1 and 2
against thrombin, a mammalian serine protease involved in blood-
clotting that is selective for peptide substrates with a P1 Arg.
Experimental results showed that both did not inhibit thrombin at
N
H
N
H
NH2
NH
>100
N
H
N
H
NH2
NH
H
N
a concentration of 100 mM, suggesting that the P2 and P3 Lys may
not fit in the thrombin S2 and S3 binding sites respectively,
resulting in selectivity towards the WNV NS2B/NS3 protease.
>100
N
H
NH2
NH
H
N
61.2 ꢀ 8.6
>100
0.006
Legend:
N
H
NH2
Inhibitor concentrations:
μM
μM
= 10 μM
γ
β
NH
H
μM
α
δ
0.004
N
H
N
μM
μM
ε
NH2
α
NH
β
γ
δ
8
4-Ph-Phac
>100
0.002
N
ε
N
NH2
H
NH
δ
δ
ε
N
NH2
9
4-Ph-Phac
4-Ph-Phac
>100
γ
-0.02
0.02
0.04
0.06
0.08
0.10
N
H
β
α
1/S (1/μM)
NH
ε
-0.002
N
NH2
10
20.0 ꢀ 5.8
γ
N
H
Fig. 3. Lineweaver-Burk plot for the inhibition of the WNV NS2B/NS3 protease by
inhibitor 2 using Pyr-RTKR-AMC as substrate.
β
α

H.A. Lim et al. / European Journal of Medicinal Chemistry 46 (2011) 3130e3134
3133
4. Experimental protocols
4.1. Chemistry
All reagents and solvents were obtained from commercial
sources and were used without further purification. Acetonitrile
was purchased from Merck KGaA (Germany). Fmoc-Lys(Boc)-
Lys(Boc)-OH was purchased from GL Biochem (China). Deuterated
solvents were purchased from Cambridge Isotope Laboratories
(USA). All other reagents were purchased from SigmaeAldrich
(USA). Flash chromatography was performed on a automated
system (Teledyne Isco Combiflash RF200) using a silica column
(RediSep RF, 230e400 Mesh, 60 Å average pore size). Crude target
inhibitors were purified using a reverse-phase C18 column (Waters
X-bridge) on a high performance liquid chromatography (HPLC)
system with an ultraviolet detector set at 215 nm (Shimadzu
Prominence). The mobile phase consisted of solvent A (water) and
solvent B (acetonitrile). The gradient started with 1% solvent B for
5 min which was increased to 11% in 40 min. All target compounds
were characterized by electrospray ionization-time of flight-mass
spectrometry (ESI-TOF-MS; Agilent 6224 TOF using Mass Hunter
software). NMR spectra were recorded on a Bruker spectrometer
(400 MHz) in CD3OD. Chemical shifts were expressed as
relative to the solvent peak.
d (ppm)
Fig. 4. Stick-models of compound 2 (grey) superimposed on compounds: (A) 7 (red);
(B) 8 (green); (C) 9 (orange) and (D) 10 (blue) using the crystal structure 3E90 pdb as
a template. The NS2B/NS3 protease and 4-Ph-Phac N-cap have been omitted for clarity.
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
4.1.1. Synthesis of compounds 1e10
The detailed synthetic procedures for compounds 1, 3e10 are
reported in the supplementary notes. The synthetic procedure
for the most potent inhibitor (compound 2) is described herein:
10
2
IC50 =20.0 5.8
M
IC50 =4.7 1.2
M
HN
NH2
NH2
NH2
NH
HN
NH2
O
N
O
O
H
N
N
O
H
N
NH
N
H
N
H
O
H
O
O
O
H
H
NH2
NH2
H
H
O
O
N
N
N
Gly151
N
Gly151
Gly153
Gly153
Fig. 5. Schematic diagrams of inhibitors 2 and 10 showing H-bonding interactions to Gly151 and Gly153 of WNV NS2B/NS3 protease. Hashed lines represent H-bonds. The lost of
one H-bond in 10 may explain the decrease in inhibitory activity. Residue numbers are based on the crystal structure 3E90 pdb. Only Gly151 and Gly153 of the protease are shown
for simplicity.
3. Conclusion
1,4-Diaminobutane (0.5 mmol, 44 mg), N,N-di-(t-butoxycarbonyl)-
S-methylisothiourea (0.4 mmol, 116 mg) and N,N-diisopropyle-
Our results suggested that the S1 binding site of the WNV NS2B/
NS3 protease was highly specific for P1 agmatine and would not
accommodate homologues, 5 and 6-membered alkyl rings. Due to
thylamine (DIPEA; 1 mmol, 175 mL) were dissolved in anhydrous
CH2Cl2 (6 mL). The mixture was stirred at 25 ꢁC, 16 h under N2
atmosphere and the resulting guanylated amine was purified by
flash chromatography using a CH2Cl2/methanol gradient and
monitored using MS. The solvent was removed in vacuo to give
a colourless oil (79 mg, 0.24 mmol, 60%). Fmoc-Lys(Boc)-Lys(Boc)-
OH (0.264 mmol, 184 mg), HBTU (0.48 mmol, 182 mg), DIPEA
the lack of a reactive warhead and a chiral a-carbon at the P1 posi-
tion, the synthesis of agmatine-containing peptidomimetics was
less complex than their electrophilic-warhead counterparts. Hence,
agmatine peptidomimetics could serve as a convenient starting
point to explore the chemistry of the P1 residue of trypsin-like
protease inhibitors, including viral and mammalian protease
inhibitors. We also postulate that decarboxylated P1 peptidomi-
metics can potentially be employed in the design of novel compet-
itive inhibitors against other proteases like aspartyl, cysteine,
threonine and metalloproteases.
(0.72 mmol, 122 mL) and dimethylformamide (DMF, 10 mL) were
added to the oil (79 mg, 0.24 mmol) and the mixture was stirred at
25 ꢁC for 1 h. The contents were dissolved in ethyl acetate (30 mL)
and washed with brine (50 mL) thrice. The organic phase was
removed in vacuo to give a cloudy gel which was dissolved in
CH2Cl2 (10 mL). DBU (0.36 mmol, 54 mL) was added to the mixture

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H.A. Lim et al. / European Journal of Medicinal Chemistry 46 (2011) 3130e3134
and stirred at 25 ꢁC for 1 h. The resulting dipeptide was purified by
flash chromatography using a CH2Cl2/methanol gradient monitored
using MS. The solvent was removed in vacuo to give a colourless oil
(157 mg, 0.20 mmol, 83%). 2-(biphenyl-4-yl)acetic acid (0.22 mmol,
regression fit by selecting the competitive inhibition model in
GraphPad Prism 5 software.
4.2.3. Thrombin inhibition assay
43 mg), HBTU (0.44 mmol, 167 mg), DIPEA (0.66 mmol, 113
m
L) and
The thrombin inhibitory assay was performed using the Anas-
pec SensoLyte AFC thrombin assay kit (USA). Thrombin (40 nM) and
DMF (10 mL) were added to the oil and the mixture was stirred at
25 ꢁC, 30 min. The contents were dissolved in ethyl acetate (30 mL)
and washed with brine (50 mL) thrice. The organic phase was
removed in vacuo to give a cloudy gel. The resulting N-capped
dipeptide was purified by flash chromatography using a CH2Cl2/
methanol gradient and monitored at 254 nm. The solvent was
removed in vacuo to give a colourless oil (141 mg, 0.144 mmol, 72%)
before being dissolved in CH2Cl2 (1 mL) and stirred with TFA (1 mL)
at 25 ꢁC, 30 min. The mixture was dried under a N2 stream, re-
dissolved in methanol (1 mL) and purified by HPLC (water and
acetonitrile solvent) to give the target product (51 mg, 0.088 mmol,
the inhibitor (100 m
M) was incubated in a reaction buffer at 25 ꢁC
for 1 h before addition of the AFC thrombin substrate. The reaction
was monitored at 25 ꢁC by measuring fluorescence intensity
(lex380 nm and lem500 nm) every 45 s for 1 h on a SpectraMax
Gemini XS plate reader. The thrombin inhibitor NAPAP (N-2-
naphthylsulfonyl-glycyl-4-amidinophenylalanine-piperidide) was
used as positive control.
4.3. Molecular modelling software
61%). Spectral data: 1H NMR (400 MHz, CD3OD)
d 1.29e1.96
Molecular modelling and energy minimization were performed
using Chem3D Ultra (v.10) software (ChembridgeSoft, USA).
(16H, m), 2.82e3.23 (8H, m), 3.64 (2H, s, AreCH2eCOe), 4.21e4.31
(2H, m,
CD3OD)
a
d
-Hs), 7.30e7.62 (9H, m, aromatics); 13C NMR (100 MHz,
22.4, 22.5, 25.7, 26.0, 26.5, 26.7, 28.1, 30.0, 30.5, 38.4, 39.1,
Acknowledgements
40.7, 41.6, 53.1, 53.9, 126.4, 126.8, 127.1, 128.6, 129.4, 134.5, 139.8,
140.5, 157.3, 172.7, 173.0, 173.2. ESI-TOF-MS: m/z calc C31H49N8O3
(M þ Hþ) 581.3922, found 581.3918.
We thank A*STAR Biomedical Research Council for financial
support, Dr. Manfred Raida for mass spectrometry analysis,
Dr. Thomas Keller and Dr. Michael Entzeroth for critical reading of
the manuscript.
4.2. Biological activities
4.2.1. WNV NS2B/NS3 enzyme inhibition assay
Appendix. Supplementary material
WNV NS2B/NS3 inhibitory assays were based on published work
[10] and performed in a buffer at pH 8.0 containing TriseHCl
(10 mM), CHAPS (1 mM) and glycerol (20% v/v). The enzyme
(20 nM) and varying concentrations of inhibitor were next added
and pre-incubated at 25 ꢁC for 1 h. The reaction was initiated by the
addition of the fluorogenic peptide substrate Pyr-RTKR-AMC
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.04.055.
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cence (lex355 nm and lem460 nm) every 45 s for 1 h on a Spec-
traMax Gemini XS plate reader (USA). Experiments were done in
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4.2.2. Kinetic measurements for Ki determination
The most potent inhibitor (compound 2) was incubated at
various concentrations (0, 5, 10, 20, 40 and 70 mM) at pH 8.0 with
WNV NS2B/NS3 protease (20 nM), TriseHCl (10 mM), CHAPS
(1 mM) and glycerol (20% v/v) at 25 ꢁC for 1 h. Next, the substrate
Pyr-RTKR-AMC (Bachem, Switzerland) was added at different
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concentrations (10, 20, 40, 60, 100, 250 and 500
mM) and the
reaction components shaken for 5 s. The rate of substrate hydrolysis
was monitored at 37 ꢁC by measuring the increase in fluorescence
(lex355 nm and lem460 nm) every 45 s for 1 h on a SpectraMax
Gemini XS plate reader (USA). The Lineweaver-Burk plot was
generated by plotting the reciprocal of hydrolysis rate (1/V) against
the reciprocal of substrate concentration (1/S) at various inhibitor
concentrations. The Ki for inhibitor 2 was derived by non-linear
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