Effects of detergents on the West Nile virus protease activity

Article abstract of DOI:10.1016/j.bmc.2009.03.050

Detergents such as Triton X-100 are often used in drug discovery research to weed out small molecule promiscuous and non-specific inhibitors which act by aggregation in solution and undesirable precipitation in aqueous assay buffers. We evaluated the effe

Full text of DOI:10.1016/j.bmc.2009.03.050

Bioorganic & Medicinal Chemistry 17 (2009) 3278–3282
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Effects of detergents on the West Nile virus protease activity
à
*
Manolya D. Ezgimen , Niklaus H. Mueller , Tadahisa Teramoto, R. Padmanabhan
Department of Microbiology and Immunology, Georgetown University School of Medicine, 3900 Reservoir Road Med-Dent Bldg. Room SW309, Washington DC 20057, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Detergents such as Triton X-100 are often used in drug discovery research to weed out small molecule
promiscuous and non-specific inhibitors which act by aggregation in solution and undesirable precipita-
tion in aqueous assay buffers. We evaluated the effects of commonly used detergents, Triton X-100,
Tween-20, Nonidet-40 (NP-40), Brij-35, and CHAPS, on the enzymatic activity of West Nile virus
(WNV) protease. Unexpectedly, Triton X-100, Tween-20, and NP-40 showed an enhancement of
in vitro WNV protease activity from 2 to 2.5-fold depending on the detergent and its concentration.
On the other hand, Brij-35, at P0.001% enhanced the protease activity by 1.5-fold and CHAPS had the
least enhancing effect. The kinetic analysis showed that the increase in protease activity by Triton X-
100 was dose-dependent. Furthermore, at Triton X-100 and Tween-20 concentrations higher than
0.001%, the inhibition of compound B, one of the lead compounds against WNV protease identified in
Received 13 December 2008
Revised 23 March 2009
Accepted 25 March 2009
Available online 1 April 2009
Keywords:
Promiscuous inhibitors
False positive hits
Flavivirus
Drug discovery
Antiviral compounds
Triton X-100
CHAPS
Tween-20
Flavivirus
Serine protease
Fluorescence assay
a high throughput screen (IC50 value of 5.7 2.5 lM), was reversed. However, in the presence of CHAPS,
compound B still showed good inhibition of WNV protease. Our results, taken together, indicate that non-
ionic detergents, Triton X-100, Tween, and NP-40 are unsuitable for the purpose of discrimination of true
versus promiscuous inhibitors of WNV protease in high throughput assays.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
(C, prM, E) and seven non-structural (NS) proteins arranged in
the order NH2-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-
The family of Flaviviridae contains more than 70 viruses includ-
ing Yellow fever virus (YFV), Dengue virus (subtypes 1–4) (DENV1–
4), West Nile virus (WNV), Kunjin, and Japanese encephalitis virus
primarily transmitted by arthropods.1 The mosquito-borne flavivi-
ruses such as Dengue and West Nile viruses have recently emerged
in major epidemics causing severe and lethal diseases such as den-
gue hemorrhagic fever and WNV encephalitis with significant mor-
bidity.2,3 Currently, there is no antiviral therapy or vaccines
available for treatment or prevention of DENV and WNV infections.
Flaviviruses are composed of a positive-sense RNA which is
translated into a single polyprotein comprising three structural
COOH. The polyprotein is processed by the host proteases in the
endoplasmic reticulum at the C-prM, prM-E, and E-NS1 as well
as NS4A-NS4B sites and by the viral serine protease at specific sites
containing two basic amino acid residues at P1 and P2 positions of
the substrate followed by Ala, Ser, or Gly at the P10 position within
the nonstructural regions.1
The NS3 protein was identified as a multifunctional protein
exhibiting enzymatic activities of a serine protease within the N-
terminal region as well as an NTPase, 50-RNA triphosphatase, and
RNA helicase within the C-terminal region.4–12 The serine protease
domain (NS3-pro; ꢀ184 aa) consists of three highly conserved
amino acids, His51, Asp75, and Ser135, in the catalytic triad.13,14
In previous studies, it was shown that NS3-pro requires the NS2B
(130 aa) cofactor for protease activity in processing of the polypro-
tein at NS2A-NS2B, NS2B-NS3, NS3-NS4A, and NS4B-NS5 sites.15–21
NS2B consists of three hydrophobic regions flanking a conserved
ꢀ40 residue hydrophilic domain (NS2BH) which is sufficient for
protease activity in vitro.20,22,23 The role of flavivirus protease in
polyprotein processing, which is a prerequisite for viral RNA repli-
cation, renders the protease as an attractive target for development
of inhibitors as potential therapeutics.
Abbreviations: AMC, 7-amino-4-methylcoumarin; CHAPS, (3-[(3-Cholamidopro-
pyl)dimethylammonio]-1-propanesulfonate); CMC, critical micelle concentration;
DENV, dengue virus; DMSO, dimethylsulfoxide; IC50, 50% inhibitory concentration;
Km
protein 2B; NS3-pro, protein domain of non-structural protein 3; RFU, relative
fluorescence unit; max, maximum enzyme velocity; WNV, West Nile virus.
, Michaelis–Menten constant; NS2BH, hydrophilic domain of non-structural
v
* Corresponding author. Tel.: +1 202 687 2092; fax: +1 202 687 1800.
E-mail address: rp55@georgetown.edu (R. Padmanabhan).
Thesis research was conducted towards Diploma degree from Technical Univer-
sity of Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany.
à
Present address: University of Colorado Denver School of Medicine, Department
One of the frequently used experimental approaches in identifi-
cation of small molecule lead compounds is high throughput
of Neurology, Mail Stop B182 Research Complex 2, 12700 E. 19th Avenue, Aurora, CO
80045, United states.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.050

M. D. Ezgimen et al. / Bioorg. Med. Chem. 17 (2009) 3278–3282
3279
screening (HTS) based on robust in vitro as well as cell-based as-
says. Using the HTS, hundreds of thousands of compounds can be
screened within a short time. However, potential false-positive
‘hits’ could arise due to compounds having undesirable properties
such as aggregation leading to nonspecific inhibition. Compounds
which exhibit these properties are known as ‘promiscuous’ inhibi-
tors, which could inhibit a variety of targets in a nonspecific man-
ner.24,25 The aggregates of compounds may envelop the protein
and block the binding site for the substrate inhibiting the protein’s
function.24,26 To identify such compounds and eliminate them
from the list of true inhibitors, previous studies have reported
the use of detergents that modulate surface properties and cause
disaggregation of the compounds.24,27 Thus, if a compound shows
inhibition in the presence of a detergent, it is then considered as
a true lead compound that warrants further analysis. Triton X-
100 is commonly used for distinguishing ‘false positive hits’ from
the true inhibitors.25 In this study, we demonstrate that Triton X-
100 and Tween-20, and NP-40, the three nonionic detergents are
not suitable for discrimination of true from ‘promiscuous’ inhibi-
tors in HTS because they enhance the WNV protease activity by
2- to 2.5-fold. However, a zwitterionic detergent, CHAPS (3-[(3-
cholamidopropyl)dimethylammonio]-1-propanesulfonate), has no
significant effect on protease activity per se and hence could be
used to distinguish false positive hits from true inhibitors. A lead
inhibitor of WNV protease previously identified by HTS shows
good inhibition in the presence of CHAPS but not in the presence
of Triton X-100.
(RFU) were converted to micromolar (
l
M) concentrations of
AMC using a standard curve generated from the fluorescence val-
ues versus micromolar concentrations of free AMC. Kinetic values
were determined using Microsoft Excel and GraphPad Prism 5 (La
Jolla, CA). RFUs, obtained in a standard assay in the absence of
detergents, were taken as 100% (control).
To assess the effects of inhibitors in the presence and ab-
sence of detergents, protease assays were performed as above
except in the presence of various concentrations of an inhibitor
(in the range, 0.3125–100 lM), serially diluted in DMSO. In
these assays, the inhibitor–enzyme complex was allowed to
form by preincubation for 15 min at room temperature before
addition of the substrate (100 lM final concentration). After five
minutes incubation, fluorescence was measured as described
above. From the initial velocities of the reactions, the IC50 val-
ues were determined using GraphPad Prism 5. The lowest and
highest fluorescence values were set as 100% and 0% inhibition,
respectively.
2.3. Effect of Triton X-100 on kinetic constants of WNV
protease
In the standard assay, the WNV substrate concentrations were
varied from 0 to 2 mM to determine the kinetic parameters, Km
and Vmax, of the WNV protease in the presence of DMSO (2%). Time
course of WNV protease reaction, monitored by Gemini EM, spec-
trofluorometer was analyzed by linear regression to obtain the
slopes in RFU/min. This value was then converted to lM/min using
a standard curve of free AMC as described above. Using GraphPad
Prism 5 software, initial rates (v) of enzyme activity were fit by
2. Materials and methods
2.1. Materials
non-linear regression analysis into Michaelis–Menten equation
Vmaxꢁ½sꢂ
v
¼
where the Vmax and Km are the maximum velocity and
½sꢂþkm
Michaelis–Menten constant, respectively, and [S] is the substrate
Triton X-100 was purchased from ICN Biomedicals. The puri-
fied WNV protease (NS2B(H)/NS3-pro) was obtained as reported
previously.28 The fluorogenic WNV substrate, t-butyl-oxycarbonyl
(Boc)-Gly-Lys-Arg-7-amino-4-methylcoumarin (AMC), was pur-
chased from Bachem. Compound B was purchased from I.F. Lab
(vendor ID, F0842-0004) (Life Chemicals Inc., Burlington, ON, Can-
ada). CHAPS was purchased from Pierce, Rockford, IL.
concentration.
3. Results
3.1. Triton X-100 enhances the WNV protease activity in a dose-
dependent manner
2.2. In vitro protease assay
Since Triton X-100 is often used to discriminate between the
‘false positives’ hits and the true inhibitors in the in vitro assays,
we first tested the effect of Triton X-100 on the activity of WNV
protease. The non-ionic detergents, Tween-20, NP-40, and Brij-35
as well as the zwitterionic detergent, CHAPS, were also used for
comparison. As shown in Figure 1, Triton X-100, Tween-20, and
NP-40 enhanced the relative activity of the protease at concentra-
tions P0.001%. The level of enhancement reached about 2- to 2.3-
fold when these detergents were present in the range of 0.01–
0.1%. This stimulatory effect of Triton X-100 is not unique to
the tripeptide substrate (Boc-Gly-Lys-Arg-AMC) used in the prote-
ase assay. The same enhancement effect of Triton X-100 was also
seen when the tetrapeptide substrate, 4-methoxy-naphthalamide,
was used (Fig. 1C). Another non-ionic detergent, Brij-35, en-
hanced the protease activity only about 1.5-fold in the same
range of detergent concentration. On the other hand, the protease
activity was not significantly affected by the zwitterionic deter-
gent, CHAPS (Fig. 1).
All assays were done in triplicate in black 96-well plates. The
in vitro protease assays were performed as described before.28,29
The reaction mixture of 100 ll/assay contained 200 mM Tris pH
9.5, 30% glycerol, 27 nM WNV protease, 2% DMSO, and fluoro-
genic WNV substrate Boc-Gly-Lys-Arg-AMC at concentrations that
varied from 15.5 lM to 2 mM or at 100 lM for fixed substrate
concentrations. In some assays, the effect of Triton X-100 or
CHAPS on the WNV protease activity was verified using a tetra-
peptide substrate, N-carbobenzyloxy-Val-Lys-Lys-Arg-4-Meth-
oxy-b-naphthylamide. Aprotinin (bovine pancreatic trypsin
inhibitor, BPTI) was used as a positive control for inhibition of
WNV protease (Ki = ꢀ162 nM)28 at
a final concentration of
10 lM, and dimethylsulfoxide (DMSO) for the no-inhibitor control
at a concentration of 2%. In vitro protease assays in presence of
Triton X-100 were performed by adding the detergent to the pro-
tease buffer (31 nM WNV protease/200 mM Tris–HCl, pH 9.0/30%
glycerol). The fluorescence of the AMC, the product of the prote-
ase activity on the tripeptide substrate was measured at excita-
tion and emission wavelengths of 385 and 465 nm, respectively.
The hydrolysis of the tetrapeptide substrate to the product, 4-
methoxy-b-naphthylamine, was monitored at excitation and
emission wavelengths of 290 and 420 nm, respectively. Fluores-
cence was measured using the SpectraMax Gemini EM spctroflu-
orometer (Molecular Devices). The relative fluorescence units
3.2. Effect of Triton X-100 on the Km and the Vmax of the WNV
protease
The Km and Vmax values were determined for the WNV protease
in presence and absence of Triton X-100 to examine whether the
detergent affects the affinity of the enzyme for the substrate. As
shown in Figure 2, the Km values do not differ significantly in the

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M. D. Ezgimen et al. / Bioorg. Med. Chem. 17 (2009) 3278–3282
Figure 2. Effect of Triton X-100 on Km and Vmax values of WNV protease. Different
WNV substrate concentrations (0–2000 M) were used to determine WNV protease
l
activity in the presence and absence of Triton X-100 in standard protease assays as
described under Section 2. The experiments were repeated three times with similar
results.
Table 1
Effect of Triton X-100 on the Km and vmax values of WNV protease
kcat (sꢃ1
)
kcat/Km 10ꢃ3
Mꢃ1 sꢃ1
a
Triton X-100
concd (%)
Km
(l
M)
Vmax
M/min)
(
l
(
l
)
0
172 60
182.6 45
187.1 15
240 30
0.97 0.18
0.84 0.11
1.25 0.25
1.65 0.20
1.70 0.01
0.60 0.11
0.52 0.07
0.77 0.15
1.02 0.12
1.05 0.01
3.5 0.6
2.8 0.4
4.1 0.5
4.3 0.1
4.8 0.4
0.0001
0.001
0.01
0.1
220 20
The protease assays were performed as described under Section 2 in the presence of
indicated concentrations of Triton X-100. The Km and Vmax values were calculated
using the GraphPad Prism 5.0 software.
a
Assuming a total enzyme concentration of 27 nM.
3.3. Effect of Triton X-100 on the inhibition of WNV protease by
compound B
Compound B was identified as a lead inhibitor against WNV
protease with a Ki of 3.4
l
M
0.6 lM in the in vitro assay and
selectivity index of 100 in a replicon-based assay in mammalian
(Vero) cells.29 Therefore, we sought to examine the inhibitory ef-
fect of the lead compound in the presence of Triton X-100. As
shown in Figure 3, when Triton X-100 was included at concentra-
tions well below its critical micelle concentration (up to 0.001%)
(CMC = ꢀ0.02%), compound B still retained its inhibitory activity.
However, at 0.01% Triton X-100 (ꢀ0.5 CMC) in the assay, the per-
cent inhibition of the WNV protease activity by compound B was
essentially abolished (Fig. 3A and B). However, the inhibitory activ-
ity of bovine pancreatic trypsin inhibitor (BPTI), also known as
aprotinin, which is a potent inhibitor of DENV and WNV proteases
with Ki values of 26 and 162 nM, respectively28, was reduced only
by about 20% in the presence of Triton X-100 (Figs. 1C and 3A).
Figure 1. WNV protease activity in presence of different detergents. Panel A.
Standard protease assays were carried out using the tripeptide substrate, t-butyl-
oxycarbonyl (Boc)-Gly-Lys-Arg-7-amino-4-methylcoumarin (AMC) as described
under Section 2 in the absence of any detergent or in the presence of Triton-
X100, Tween-20, or CHAPS at the indicated concentrations of 10ꢃ6, 10ꢃ5, 10ꢃ4, 10ꢃ3
,
10ꢃ2, 10ꢃ1, or 1% in the assay mixture. Panel B. The experimental conditions for the
assay were the same as in Panel A except that NP-40, Brij-35, or CHAPS was used at
the indicated concentrations. Panel C. The conditions of the assay were the same
except that the effects of Triton X-100 and CHAPS at indicated concentrations on
the WNV protease activity using a tetrapeptide substrate, N-carbobenzyloxy-Val-
Lys-Lys-Arg-4-methoxy-b-naphthylamide as described under Section 2. The
percent protease activity was plotted compared to that of the no-detergent control
set at 100%. The experiments were repeated three times and the error bars
represent the standard deviation of the mean.
4. Discussion
Triton X-100, polyethylglycol tert-octylphenyl ether, is a non-
ionic detergent, and is used extensively for solubilization of mem-
brane proteins and their biochemical characterization. In drug
discovery field, this detergent is used to discriminate the ‘promis-
cuous’ inhibitors from true inhibitors of a target protein or enzyme
in the in vitro assays.24,25,30,31 Compounds that inhibit unrelated
enzyme targets in the in vitro assays often do so by aggregation
and/or formation of colloidal particles which could bind to the tar-
gets nonspecifically and interfere with substrate or inhibitor bind-
ing leading to false ‘hits’. The presence of a detergent below its
CMC, provided it does not interfere with the assay, prevents aggre-
presence and absence of Triton X-100 whereas the Vmax values are
increased with increasing concentrations of Triton X-100 (Table 1)
in a dose-dependent manner. This experiment was repeated three
times with similar values for Km and Vmax supporting our
conclusion.

M. D. Ezgimen et al. / Bioorg. Med. Chem. 17 (2009) 3278–3282
3281
Figure 3. Effect of Triton X-100 on the inhibition of WNV protease by compound B. (A) The assays were performed as described under Section 2 in the presence and absence
of Triton X-100 and 2 M aprotinin (light grey bars) or 20 M compound B (dark grey bars). At Triton X-100 concentrations at 0.5 CMC (CMC = 0.02%), the inhibition of
l
l
compound B is reduced ꢀ85%. (B) The concentrations of compound B were varied as indicated. The protease assays were performed at two different Triton X-100
concentrations (0.001%-grey bars and 0.01%-white bars) along with no-detergent control (black bars). The experiments in panels A and B were repeated at least three times
with similar results. The error bars represent standard deviation of the mean.
gation of the inhibitor compound and allows interaction between
the inhibitor and the target.
potent inhibition of WNV protease activity in vitro (P. Viswanathan
et al. unpublished results; data not shown). (3) Compound B inhib-
ited WNV RNA replication in a cell-based assay with an EC50 value
However, the findings of the present study using the WNV pro-
tease as the enzyme target indicate that Triton X-100 interferes
with the inhibitory activity of compound B (Fig. 3) and other com-
pounds tested (data not shown). However, inclusion of a zwitter-
ionic detergent such as CHAPS did not reverse the inhibitory
activity of compound B (Fig. 1A). There are other examples of Tri-
ton X-100, Nonidet P-40 (NP-40) and/or Tween-20 interfering with
target assays and those that describe that CHAPS or Brij-35 could
also be used as a detergent to distinguish between true inhibitors
from false-positive ones.32–35 For example, it was previously re-
ported that Triton X-100 may act as a nonspecific activator of chy-
motrypsin.36 In the investigation of small molecule inhibitors of
Rce1p protease, which is required for Ras GTPase maturation, Tri-
ton X-100, NP-40, and CHAPS at their CMC inactivated the prote-
ase. Tween-20 at its CMC, on the other hand, was also ineffective
for this purpose. Therefore, these detergents could not be used
for distinguishing promiscuous versus true inhibition by the com-
pounds.37 Moreover, Triton X-100 and NP-40 decreased the inhibi-
tion of reverse transcriptase (RT) activity by inophyllum B whereas
CHAPS had no effect.38 In this regard, it was noted that Triton X-
100 is an activator of HIV-1 RT activity and reverses the inhibition
of TIBO R82150.39
of 1.4 0.4 l
M.29 Moreover, in the presence of Triton X-100 at
0.001% compound B showed good inhibition of WNV protease with
no change in the IC50 value compared to no-detergent control
(Fig. 3B). In the presence of Triton X-100 greater than 0.001% and
Tween-20, the inhibition of WNV protease by compound B is re-
duced by up to 70% whereas the ionic detergent, CHAPS, did not
have any affect. This study is the first to report on the enhance-
ment of trypsin-like WNV serine protease activity by Triton X-
100, Tween-20, and NP-40 whereas CHAPS shows no significant ef-
fect on the activity. In fact, the lack of appreciable stimulation by
CHAPS up to 0.5% (CMC = 0.48% or 7.4 mM)40 is not due to high
pH of the assay buffer because it has been reported that CHAPS
maintains its zwitter ionic property in a wide pH range of 2–
12.41 CHAPS was a component of the purification/assay buffer used
in the characterization of the WNV protease activity in an earlier
study23; however, its property in the context of enhancing effects
of detergents on the WNV protease activity was not investigated
earlier to this study.
The mechanism by which Triton X-100 enhances the WNV pro-
tease activity is unknown at present. However, our kinetic analysis
suggests that the detergent increases the Vmax values of the en-
zyme in a dose-dependent manner without affecting the affinity
of the enzyme (Km values) for the substrate. One possibility is that
Triton X-100, Tween-20, and NP-40 prevent the protease sticking
to the wall of the 96-well plate thereby increasing the local con-
centration of the enzyme compared to the well without the deter-
gent. Alternately, Triton X-100 may prevent aggregation of the
The possibility that compound B is a promiscuous inhibitor
rather than a specific inhibitor of WNV protease is unlikely because
of the following properties of compound B as an inhibitor of WNV
protease. (1) Compound B shows some specificity as it did not have
any inhibitory effect on cellular serine proteases such as trypsin
and factor Xa.29 (2) Other derivatives of compound B also exhibit

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M. D. Ezgimen et al. / Bioorg. Med. Chem. 17 (2009) 3278–3282
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enzyme thereby increasing the concentration of the active enzyme
which would only influence the Vmax but not the Km. This possibil-
ity is supported by our data that Km values for WNV protease re-
main almost the same, whereas Vmax slightly increases with
higher Triton X-100 concentrations (Table 1). However, the result
that increasing the Triton X-100 concentration from 0.001 to 0.01
reduced the inhibition of the compound B by 70% (Fig. 3B), is not
consistent with this interpretation because the total amount of
the enzyme in each assay was only 2.7 pmol, the substrate 10 nmol
and the inhibitor 2 nmol and therefore, increasing the concentra-
tion of the detergent is not expected to have any effect on the inhi-
bition of compound B. Moreover, Triton X-100 does not have any
effect on the substrate in the absence of WNV protease (data not
shown). Another possibility should be considered that Triton X-
100 at higher concentrations not only enhances the Vmax of the
hydrolysis of the substrate without significant change of Km, but
may also cause a conformational change at a different site that af-
fects the binding of the compound B to the enzyme thereby atten-
uating its inhibition.
Therefore, our study emphasizes the need to test the effect of
the chosen detergent on the enzyme (or any molecular target)
prior to using it in the HTS for identification and elimination of
false positive hits.
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Acknowledgements
This work was supported by a Grant from NIAID/NIH (AI-
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