Published online 27 June 2012
Nucleic Acids Research, 2012, Vol. 40, No. 17 8607–8621
doi:10.1093/nar/gks623
Identification and analysis of hepatitis C virus NS3
helicase inhibitors using nucleic acid binding assays
Sourav Mukherjee1, Alicia M. Hanson1, William R. Shadrick1, Jean Ndjomou1, Noreena
L. Sweeney1, John J. Hernandez1, Diana Bartczak1, Kelin Li2, Kevin J. Frankowski2,
Julie A. Heck3, Leggy A. Arnold1, Frank J. Schoenen2 and David N. Frick1,*
1
Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53211,
University of Kansas Specialized Chemistry Center, University of Kansas, 2034 Becker Dr., Lawrence,
KS 66047 and 3Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla,
NY 10595, USA
2
Received March 26, 2012; Revised May 30, 2012; Accepted June 4, 2012
Typical assays used to discover and analyze small
molecules that inhibit the hepatitis C virus (HCV)
NS3 helicase yield few hits and are often confounded by compound interference. Oligonucleotide
binding assays are examined here as an alternative.
After comparing fluorescence polarization (FP),
homogeneous time-resolved fluorescence (HTRF! ;
Cisbio) and AlphaScreen! (Perkin Elmer) assays,
an FP-based assay was chosen to screen Sigma’s
Library of Pharmacologically Active Compounds
(LOPAC) for compounds that inhibit NS3-DNA
complex formation. Four LOPAC compounds inhibited the FP-based assay: aurintricarboxylic acid
(ATA) (IC50 = 1.4 kM), suramin sodium salt (IC50 =
3.6 kM), NF 023 hydrate (IC50 = 6.2 kM) and tyrphostin
AG 538 (IC50 = 3.6 kM). All but AG 538 inhibited
helicase-catalyzed strand separation, and all but
NF 023 inhibited replication of subgenomic HCV replicons. A counterscreen using Escherichia coli
single-stranded DNA binding protein (SSB) revealed
that none of the new HCV helicase inhibitors were
specific for NS3h. However, when the SSB-based
assay was used to analyze derivatives of another
non-specific helicase inhibitor, the main component
of the dye primuline, it revealed that some primuline
derivatives (e.g. PubChem CID50930730) are up to
30-fold more specific for HCV NS3h than similarly
potent HCV helicase inhibitors.
INTRODUCTION
All cells and viruses need helicases to read, replicate and
repair their genomes. Cellular organisms encode
numerous specialized helicases that unwind DNA, RNA
or displace nucleic acid binding proteins in reactions
fuelled by ATP hydrolysis. Small molecules that inhibit
helicases would therefore be valuable as molecular
probes to understand the biological role of a particular
helicase, or as antibiotic or antiviral drugs (1,2). For
example, several compounds that inhibit a helicase
encoded by herpes simplex virus (HSV) are potent drugs
in animal models (3,4). Despite this clear need, relatively
few specific helicase inhibitors have been reported, and the
mechanisms through which the most potent compounds
exert their action are still not clear. Although HSV
helicase inhibitors have progressed furthest in pre-clinical
trials (5), the viral helicase that has been most widely
studied as a drug target is the one encoded by the hepatitis
C virus (HCV). The uniquely promiscuous HCV helicase
unwinds duplex DNA and RNA in a reaction fuelled by
virtually any nucleoside triphosphate (6). The ability of
HCV helicase to act on DNA is particularly intriguing
because the HCV genome and replication cycle are
entirely RNA-based. There is no convincing evidence
that HCV helicase ever encounters DNA in host cells.
Compounds that disrupt the interaction of the helicase
and DNA, therefore, would be useful to understand why
an RNA virus encodes a helicase that acts on DNA. They
also might be useful antivirals because HCV needs a functional helicase to replicate in cells (7) and helicase inhibitors halt HCV replication in cells (8).
*To whom correspondence should be addressed. Tel: +1 414 229 6670; Fax: +1 414 229 5530; Email: [email protected]
Present address:
Julie A. Heck, Department of Biology, College of Wooster, Wooster, OH 44691, USA.
" The Author(s) 2012. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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ABSTRACT
8608 Nucleic Acids Research, 2012, Vol. 40, No. 17
both DNA and RNA is intriguing because other similar
enzymes typically prefer DNA or RNA and because NS3
likely never encounters DNA. The RNA virus replicates in
the cytoplasm and has no DNA stage in its replication
cycle. Numerous NS3 crystal structures show how the
protein binds DNA (26–28) or RNA (29), both in the
absence or presence of ATP analogs (28,29). These structural studies reveal that amino acid side chains in the
NS3h nucleic acid binding cleft do not directly contact
the 20 -hydroxyl of RNA, explaining the enzyme’s
unusual promiscuity, and justifying the use of DNA oligonucleotides as surrogates for RNA to probe the enzyme’s
functions. HCV helicase binds nucleic acids with low
nanomolar affinity (30,31) and NS3h preferentially interacts with polypyrimidine tracts like those found in the 30
untranslated region of the virus genome (10,32). While it is
clear that one strand of DNA (or RNA) binds in a cleft
separating the two conserved helicase motor domains
from a third helical domain, it is not clear where else on
the protein nucleic acids might bind. Based on modeling
studies, some groups have suggested that RNA might bind
in the positively charged cleft separating the protease from
the helicase (12,33), and more recent evidence suggests the
protease region binds certain sequences in the internal
ribosome entry site of the HCV RNA genome (34).
We show here how DNA binding assays can be used
to identify new helicase inhibitors and how DNA binding
assays with unrelated proteins can be used to screen a
library of helicase inhibitors for specific compounds. A
truncated NS3 lacking the protease domain (i.e. NS3h)
is used because it is still unclear exactly how the
protease region affects NS3–RNA interactions, and
DNA is used here instead of more costly RNA, because
the nucleic acid-binding site on NS3h does not differentiate between the DNA and RNA. First, we compare
various DNA binding assays for their screening utility.
Next, we use a fluorescence-polarization (FP)-based
binding assay to identify three new HCV helicase
inhibitors. Binding assays with the unrelated E. coli
single-stranded DNA binding protein (SSB) are then
used to reveal that the new compounds, like helicase inhibitors discovered in a prior screen of the NCI
Mechanistic Set (21), are not specific for HCV helicase.
In the final part of this study, we use a library of compounds derived from a scaffold identified in the prior
screen (21) to show that binding assays can be used to
differentiate specific inhibitors from non-specific HCV
helicase inhibitors.
MATERIALS AND METHODS
Materials
DNA oligonucleotides were obtained from Integrated
DNA Technologies (Coralville, IA). HCV NS3h was
expressed and purified as described (6). Helicase substrates were prepared by combining DNA oligonucleotides (Integrated DNA Technologies, Coralville, IA) at a
1:1 molar ratio to a concentration of 20 mM in 10 mM
Tris–HCl pH 8.5, placing in 95! C water, and allowing
them to cool to room temperature for 1 h. The partially
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The HCV helicase resides in the C-terminal two-thirds of
the viral multifunctional non-structural protein 3 (NS3),
which is also a protease. The NS3 protease and helicase
are covalently associated during HCV replication for
unknown reasons. HCV and related viruses encode the
only proteins known that are both proteases and helicases.
Recombinant DNA technology can be used to separate the
two NS3 functional domains, and express the proteins separately in Escherichia coli or other model organisms. Both
mono-functional, recombinant, truncated NS3 proteins
(called NS3p and NS3h) retain their activities in vitro.
Although NS3h retains a helicase function, its ability to
unwind RNA is somewhat diminished (9). The NS3
helicase was one of the first HCV enzymes to be characterized, and crystal structures of NS3h were first solved in
the mid 1990s (10,11). However, helicase inhibitor development has been far slower than it has been for other HCV
drug targets (2,12). To date, only a few classes of helicase
inhibitors have been reported to slow HCV RNA replication in cells. HCV helicase inhibitors reported to act as antivirals include nucleoside mimics (13), triphenylmethanes
(14), acridones (8,15), amidinoanthracyclines (16), tropolones (17), symmetrical benzimidazoles (18–20) and primuline derivatives (21).
One reason that so few molecular probes targeting HCV
helicase are available is because high throughput screens
for helicase inhibitors yield few hits. For example, a sensitive molecular beacon-based helicase assay (MBHA) (22)
has been used to screen 290 735 compounds in the
NIH Molecular Libraries Small Molecule Repository
by the Scripps Research Institute Molecular Screening
Center (PubChem Project: http://pubchem.ncbi.nlm.nih.
gov/assay/assay.cgi?aid=1800, 14 June 2011, date last
accessed), and only 500 compounds (0.2%) were confirmed as hits upon retesting (PubChem Project: http://
pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=1943, 14
June 2011, date last accessed). The most potent hits in
the NIH screen did not, however, directly inhibit
helicase action, but instead they interfered with the assay
(PubChem Project: http://pubchem.ncbi.nlm.nih.gov/
assay/assay.cgi?aid=485301, 14 June 2011, date last
accessed). They also did not inhibit HCV replication in
cells (PubChem Project: http://pubchem.ncbi.nlm.nih.
gov/assay/assay.cgi?aid=463235, 14 June 2011, date last
accessed) (23).
Screens for helicase inhibitors typically rely on assays
that monitor either helicase catalyzed strand separation or
ATP hydrolysis. Both assays are relatively complex, and
inhibitory compounds might act through the enzyme,
ATP, nucleic acid or other required cofactors (24). Both
assays monitor a helicase’s motor action, and it is possible
that the protein conformational changes that take place in
these assays, or some other unknown factor, obfuscates
inhibitor identification in large screens. Here, we test
whether simpler DNA-binding assays might be more
useful for HCV helicase inhibitor discovery.
Hepatitis C virus helicase binds single stranded DNA
and RNA with similar high affinities in the absence of
ATP. When ATP is present, it fuels helicase movements
and the subsequent separation of both DNA and RNA
duplexes (25). The ability of HCV helicase to separate
Nucleic Acids Research, 2012, Vol. 40, No. 17 8609
duplex helicase substrates possessing a 30 ssDNA tail
were then purified of free oligonucleotides by mixing
DNA 6:1 with 6X loading buffer (0.25% bromophenol
blue, 0.25% xylene cyanol FF, 40% sucrose) and separating with 20% non-denaturing PAGE at a constant
200 V for 1 h.
Electrophoretic mobility shift assay
FP-based DNA-binding assay
For screening, assays were performed in a total volume of
20.2 ml in 384-well, flat-bottom, low volume, black microplates (Greiner Bio-One, catalog #784076). First, 20 ml of a
FP-assay solution (5 nM Cy5-TTTTTTTTTTTTTTT-30
(Cy5-dT15), 15 nM NS3h, 25 mM MOPS, pH 7.5,
1.25 mM MgCl2, 0.0025 mg/ml BSA, 0.005% (v/v)
Tween20 and 0.025 mM DTT) was dispensed in each
well, then 0.2 ml of dimethylsulfoxide (DMSO) or
compound dissolved in DMSO was added by pin
transfer, such that the final concentration of DMSO was
1% (v/v) in each assay.
For confirmation and IC50 value determination, assays
were performed in half area 96-well microplates (Corning
Life Sciences, catalog #3694). First, 47.5 ml of a FP-assay
solution was dispensed in each well, then 2.5 ml of DMSO
or compound dissolved in DMSO was added, such that
the final concentrations in each assay were 5 nM Cy5dT15, 15 nM NS3h, 25 mM MOPS, pH 7.5, 1.25 mM
MgCl2, 0.0025 mg/ml BSA, 0.005% (v/v) Tween20,
0.025 mM DTT and 5% DMSO (v/v).
Polarization was monitored with a TECAN Infinite
M1000 PRO multi-mode microplate reader by exciting
at 635 nm (5 nm bandwidth) and measuring total fluorescence intensity, parallel and perpendicular polarized light
at 667 nm (20 nm bandwidth). G-factors were calculated
from wells with Cy5-dT15 alone. Inhibition (%) was
calculated by normalizing data to values obtained with
positive controls (200 nM dT20 or 100 mM primuline)
and negative controls (DMSO only). Assay interference
was calculated by dividing fluorescence intensity of a
compound-containing assay (Fc) by the average fluorescence intensity of the negative controls (F(")). Similar
results were obtained with both assay formats as long as
DMSO concentrations remained below 5%.
Compounds in the HCV helicase inhibitor library were
either purchased (Sigma, St. Louis, MO) or synthesized as
described (21) and screened at 20 mM. Samples in Sigma’s
Compounds
Homogeneous time resolved fluorescence (HTRF! ) assay
Assays were performed in 20.2 ml in 384-well, flat-bottom,
small volume, white microplates (Greiner Bio-One,
catalog #784075). The procedure was the same as that
for the FP assay except that 15 ml of the reaction
mixtures (7.5 nM Cy5-dT15 DNA, 50 nM NS3h, 25 mM
Tris, pH 7.5, 1.25 mM MgCl2, 0.05 mg/ml BSA, 0.1%
(v/v) Tween20, and 0.5 mM DTT) was first dispensed in
each well before the addition of 0.2 ml of dT20 or H2O.
After addition, 5 ml of a (1:50 dilution) of Lumi4! -Tb
Cryptate-conjugated anti-6 Histidine mouse monoclonal
antibody (catalog #61HISTLA, CISBIO US) was added
and the plate was incubated for 60 min at 4! C. TR-FRET
was monitored with a Fluostar Omega multimodal plate
reader (BMG Labtech, Inc.) by excitation of the donor
fluorophore at 340 nm. TR-FRET ratio was calculated
as emission of acceptor fluorophore at 665 nm over the
emission of donor fluorophore at 620 nm (gain 2300, integration time 400 ms, integration start time 60 ms, positioning delay 0.2 s, measurement start time 0 s, number of
flashes per well 200).
AlphaScreen! assay
All assays were similar to the above assays except
that they contained a final concentration of 10 nM
biotinylated oligonucleotide (Bio-d18, 50 -Bio-GCC TCG
CTG CCG TCG CCA-30 ), instead of Cy5-dT15, and
they used reagents from the AlphaScreen! Histidine
(Nickel Chelate) Detection Kit (catalog #6760619C,
Perkin Elmer). To 384-well, flat-bottom, low volume,
white microplates (Greiner Bio-One, catalog #784075),
12 ml of the reaction mixtures containing 10 nM Bio-d18,
20 nM NS3h, 25 mM HEPES, pH 7.5, 100 mM NaCl and
1.0 mg/ml BSA was dispensed, followed by 0.2 ml of dT20
or H2O and 4 ml Anti-His alpha screen donor beads. After
incubation for 30 min at 23! C, 4 ml of streptavidinacceptor beads was added, and the assays incubated
another 60 min. All work with the alpha reagents was performed under green filtered light conditions (<100 Lux).
Alpha counts were measured at 520–620 nm (Ex. 680 nm,
20 nm bandwidth) in a Fluostar Omega multimodal plate
reader (BMG Labtech, Inc.).
NS3h MBHAs
The ability of compounds to inhibit helicase action was
monitored using molecular beacons as described previously
(19,22). Assays contained 25 mM MOPS, 1.25 mM MgCl2,
5% DMSO, 5 mg/ml BSA, 0.01% (v/v) Tween20, 0.05 mM
DTT, 5 nM substrate, 12.5 nM NS3h and 1 mM ATP. The
partially duplex DNA substrates used in MBHAs consisted
of a 45-mer bottom strand 50 -GCT CCC CGT TCA TCG
ATT GGG GAG C(T)20-30 and the 25-mer HCV top
strand 5-/5Cy5/GCT CCC CAA TCG ATG AAC GGG
GAG C/3IAbRQSp/-3. The 3-stranded RNA substrate
used was made of two RNA strands, a 60 nucleotide
long bottom strand 50 -rGrGrA rGrCrU rGrGrU rGrGrC
rGrUrA rGrGrC rArArG rArGrU rGrCrC rUrUrG
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Binding assays containing 50 mM Tris, pH 7.4, 10%
glycerol, 100 nM DNA substrate (50 -Cy5-CC TAC GCC
ACC AGC TCC GTA GG–30 annealed to 50 -GGA GCT
GGT GGC GTA GG (T)20-30 ) and 650 nM NS3h were
incubated 20 min on ice. Following addition of indicated
concentrations of thioflavine S, the binding reactions were
incubated another 20 min on ice. A BioRad precast 15%
polyacrylamide Tris/Borate/EDTA gel was pre-run at 4! C
for 30 min at 120 V. Four microliters of each sample was
loaded onto the gel. The gel was run 1 min at 200 V to
allow samples to enter gel, then 40 min at 120 V. The gel
was scanned on a Molecular Dynamics Storm 860
Phosphorimager.
Library of Pharmaceutically Active
(LOPAC) were screened at 100 mM.
8610 Nucleic Acids Research, 2012, Vol. 40, No. 17
rArCrG rArUrA rCrArG rCrUrU rUrUrU rUrUrU
rUrUrU rUrUrU rUrUrU rUrUrU-30 , a 24 nucleotide
long top strand 50 -rArGrU rGrCrG rCrUrG rUrArU
rCrGrU rCrArA rGrGrC rArCrU-Cy5, and a
third DNA top strand with the sequence/5IAbRQ/CCT
ACG CCA CCA GCT CCG TAG G-3. In the screen
in Figure 5, percent inhibition was calculated with
equation (1) and interference with equation (2).
Inhibition ð%Þ ¼ ððFc0 =Fc30 Þ " ðFð"Þ0 =Fð"Þ30 Þ=
ð1 " ðFð"Þ0 =Fð"Þ30 ÞÞÞ & 100
ð1Þ
Interference ðratioÞ ¼ ðFc0 =Fð"Þ0 Þ
ð2Þ
HCV replicon assays
The ability of compounds to inhibit HCV replication was
judged using an HCV Renilla luciferase (HCV RLuc)
reporter construct that was a generous gift from
Seng-Lai Tan (35). Plasmid DNA expressing the
replicon was transcribed, and the subsequently purified
RNA was used to prepare stably transfected Huh7.5
cells by prolonged selection with G418. To test compounds, HCV RLuc replicon containing cells were
seeded at a density of 10 000 cells per well in 96-well
plates and incubated for 4–5 h to allow the cells to
attach to the plate in 100 ml of DMEM supplemented
with 10% fetal bovine serum (HyClone), 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 1x
non-essential amino-acids (Invitrogen). To each well,
0.5 ml of compounds dissolved in DMSO were added
such that the DMSO final concentration was 0.5%,
and the cells were incubated for 72 h at 37! C under 5%
CO2 atmosphere. The effect of compounds on HCV
replication was estimated by measuring the Renilla
luciferase activity using the Renilla luciferase assay
Cell viability assay
To assess compound toxicity towards Huh-7.5 cells, cells
were plated and treated as above and viability was
assessed using the Cell Titer-Glo luminescent cell viability
kit (Promega) following the manufacturer’s instructions.
Briefly, at the end of a 72 h incubation period, the medium
was removed and the cells were washed with growth
medium, then an equal volume of growth medium
and Cell Titer-Glo reagent was added and the lysis was
initiated by mixing on an orbital shaker. The plate
was incubated at 23! C for 30 min and luciferase activity
was measured for 1 s using a FLUOstar Omega microplate
reader (BMG Labtech, Inc.) in black 96-well microplates
(Thermo Scientific, catalog #9502867). Relative viability
was calculated by normalizing the values to those obtained
with cells treated with DMSO only.
Escherichia coli SSB assay
The procedure for screening with this assay was the same
as that for the FP-based DNA binding assay carried out in
384-well plates except that E. coli SSB (Promega) was used
at 20 nM instead of the HCV helicase. For IC50 determination, assays were performed with 60 ml total volume in
black flat bottomed 384-well microplates (Corning catalog
#3573). First, 3.0 ml of DMSO or compound dissolved in
DMSO was added, such that the final concentration of
DMSO was 5% (v/v) in each assay. Then 57 ml of a
FP-assay solution (5 nM Cy5-dT15, 20 nM SSB, 25 mM
MOPS, pH 7.5, 1.25 mM MgCl2, 0.0025 mg/ml BSA,
0.005% (v/v) Tween20 and 0.025 mM DTT) was dispensed
in each well. Polarization was monitored as described
above.
RESULTS
Belon and Frick (36) previously reported that thioflavine S
was an HCV helicase inhibitor, and thioflavine S was used
as a positive control for the screen of the NIH Molecular
Libraries Small Molecule Repository (PubChem BioAssay
AID #1800) and in other studies (37). While studying
the mechanism of action of thioflavine S (Direct Yellow
7, Sigma Cat. #T1892) and the related yellow dyes
primuline (Direct Yellow 59, MP Biomedicals Cat.
#195454) and titan yellow (Direct Yellow 9/Thiazole
Yellow G, Sigma Cat. #88390) (38), we observed that
they prevent NS3h from binding its nucleic acid substrate
(Figure 1A). In the absence of one of these dyes, NS3h
binds its substrate tightly enough that the complex will
migrate more slowly through a non-denaturing polyacrylamide gel. When thioflavine S was present the gel-shift
of the substrate decreased in a concentration-dependent
fashion (Figure 1B).
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In equations (1) and (2), Fc0 is the fluorescence of the
reactions containing the test compound before adding
ATP, Fc30 is the fluorescence of the test compound
reaction 30 min after adding ATP. F(")0 is the average
of three DMSO-only negative control reactions before
adding ATP and F(")30 is the average of three
DMSO-only reactions 30 min after adding ATP.
To monitor helicase reaction kinetics and to calculate
IC50 values, assays were performed in a volume of 60 ml in
white half-area 96-well plates (Corning Lifesciences,
catalog #3693) and measured in a Fluostar Omega multimodal plate reader (BMG Labtech, Inc.) using the 640 nm
excitation wavelength and 680 emission wavelength filters.
Reactions were performed by first incubating all components except for ATP for 2 min, then initiated by injecting
in 1/10 volume of ATP such that the final concentration of
all components was as noted above. Initial reaction
velocities were calculated by fitting a first order decay
equation to data obtained after ATP addition and
calculating an initial velocity from the resulting amplitude
and rate constant. The concentration at which a
compound causes a 50% reduction in reaction velocity
(IC50) was calculated using GraphPad Prism (v. 5).
system (Promega, Madison, WI) in 96-well black
microplates (Thermo Scientific, catalog #9502867) read
on a FLUOstar Omega multi-mode microplate reader
(BMG Labtech, Inc.). Relative percent inhibition was
calculated by normalizing values to those obtained with
cells treated with DMSO only.
Nucleic Acids Research, 2012, Vol. 40, No. 17 8611
A
B
Figure 1. Thioflavine S inhibits the ability of NS3h to bind to its DNA substrate. (A) Partially duplex DNA helicase substrate used for gel-shift
analysis. (B) Electrophoretic mobility shift assay (EMSA). Samples containing the MBHA substrate (100 nM), NS3h (650 nM) and indicated concentrations of thioflavine S were examined on a 15% native polyacrylamide gel using a phosphorimager to locate labeled DNA. Control lane with no
NS3h shows migration position of free DNA.
To unwind a substrate, HCV helicase binds a single
stranded nucleic acid tail then translocates in a 30 –50 direction (39) until it reaches a duplex region that it can
separate. The ability of NS3h to bind its substrate can
also be measured by monitoring changes in polarization
(or anisotropy) of a fluorescent helicase substrate as has
been done with related helicases (40,41). This loading step
can be monitored using a truncated substrate lacking
duplex regions, such as a 15-nucleotide long deoxythymidine polymer (dT15) (Figure 2A). Previous work has
shown that NS3h binds such single stranded DNA with
a high affinity and that 2-3 protomers bind such an oligonucleotide. A homopolymer was chosen to minimize the
possibility that the DNA would form a hairpin or other
secondary structures, and Ts were chosen because NS3h
prefers this sequence to others (10,30–32).
As seen before (30,31,42), the binding of NS3 to DNA
in this FP-assay was stoichiometric (Kd < 1 nM) and
about two to three molecules of NS3 were needed to
saturate Cy5-dT15. When 5 nM Cy5-dT15 was present,
increasing amounts of NS3h increased the FP of
Cy5-dT15 in a concentration dependent manner such
that the amount of NS3h needed to bind half of the
Cy5-dT15 (K0.5) was 9 ± 1.5 nM. Under these conditions,
the signal plateaued when about three times as much
NS3h (15 nM) was added as the amount of Cy5-dT15
(Figure 2B). The polarization of a Cy5-dT15–NS3h
complex decreased in the presence of either unlabeled
ligand (dT20) or a yellow dye in a concentration dependent manner (Figure 2C). The IC50 values measured for
dT20 and primuline were 7 ± 2 nM and 24 ± 3 mM, respectively. Thioflavine S decreased polarization less effectively than primuline with an IC50 of 35 ± 3.5 mM, and
titan yellow was 10 times more potent than either with
an IC50 value of 2.8 ± 0.2 mM (Table 1).
A Cy5-labeled oligonucleotide was chosen mainly
because its fluorescence intensity did not change upon
protein binding, and because it absorbs and emits light
in the far-red visible range, where it would be less likely
to interact with compounds in large chemical libraries.
One possible problem with using Cy5 as a tracer in such
a study is that it is coupled to the oligonucleotide with an
aliphatic linker so that the fluorophore could, in theory, be
still relatively free to rotate even when DNA is bound to
the enzyme, a phenomenon commonly referred to as ‘the
propeller effect’. We therefore also tested oligonucleotides
labeled with fluorescent moieties not bound to aliphatic
linkers such as with 6-carboxyfluorescein, hexachlorofluorescein and boron-dipyrromethene (BODIPY).
Polarization studies with each of these alternatives were
confounded by the fact that the fluorescence intensity of
each changed upon protein binding. The fact that fluorescence intensity of oligonucleotides changes when they bind
the unusually acidic DNA binding site of NS3 has been
previously documented (9,19). While screening fluorescent
oligonucleotides, we found two other red-shifted tracers
that did not change intensity upon binding, TyeTM 665
and Alexa Fluor 647TM. Identical (dT15) oligonucleotides
labeled on the 50 -end with either Cy5, Tye665, or Alexa
Fluor 647, bound NS3h with similar K0.5 s (5 ± 1, 8 ± 2
and 6 ± 2, respectively), and titan yellow inhibited
complex formation of each with a similar IC50 value
(7 ± 3, 5 ± 1 and 9 ± 4 mM, respectively). Repeated
assays (n = 40) with the three different fluorescence
tracers in the presence and absence of titan yellow
(100 mM) revealed similar Z0 factors (43). Assays with
Alexa Fluor and Tye 665 labeled oligonucleotides had
the largest difference between the positive and negative
controls, but their assay-to-assay variability was higher,
particularly in assays done in the absence of inhibitor
(Figure 2D). Further experiments were therefore performed with the Cy5-labeled oligonucleotide.
After optimizing conditions, FP-based binding assays
were then performed in a high throughput format to
judge necessary precision and reproducibility. To judge
well-to-well variation, 48 negative controls (DMSO only)
and 48 positive controls (100 mM primuline) were performed. The coefficient of variation was 2.2% for the
negative controls and 5.8% for the positive controls, resulting in a Z0 factor of 0.81 (Figure 2E). Similar Z0
factors, and concentration response curves were
obtained when plates were compared (Figure 2F) or
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HCV helicase-DNA binding assays that are suitable for
high throughput screening
8612 Nucleic Acids Research, 2012, Vol. 40, No. 17
A
B
C
E
F
G
H
Figure 2. Fluorescence polarization (FP)-based assay to monitor the
interaction of HCV helicase and a deoxythymidine polymer. (A) FP
based assay to monitor NS3h binding to Cy5-dT15. (B) Fluorescence
polarization of Cy5-dT15 (5 nM) at different concentrations of NS3h.
Data (n = 4) were fitted to a concentration-response equation (four
Figure 2. Continued
parameter, variable slope) with dotted lines showing the 95% confidence intervals for the curve fit. (C) Concentration response of unlabeled dT20 (squares) or primuline (circles) on the fluorescence
polarization of a Cy5-dT15-NS3h complex. Data (n = 4) were fitted
to a four-parameter concentration response equation (variable slope)
constrained to values obtained in the absence of inhibitor (top) and the
absence of NS3h (bottom), with indicated IC50 values and Hill slopes.
(D) Comparison of results obtained with Cy5-dT15 with those obtained
with dT15 labeled with either Tye665 or AlexaFluor 647.
Oligonucleotides were present at 5 nM and NS3h at 15 nM. Positive
controls (N = 40) contained 100 mM titan yellow, negative control contained DMSO only. (E) Fluorescence polarization of 48 positive control
assays (100 mM primuline (+)) and 48 negative control assays (DMSO
only (")). Solid lines represent means and dotted lines 3 times the
standard deviations of the mean of all assays. (F) Normalized percent
inhibition of Cy5-dT15 complex formation by various concentrations of
dT20 observed in FP-assays performed on two different plates.
(G) Normalized percent inhibition of Cy5-dT15 complex formation
by various concentrations of dT20 observed in FP-assays performed
on two different days. (H) Correlation plot of fluorescence polarization
values observed in duplicate assays at 20 mM of samples in an HCV
helicase inhibitor library (Table S1). Data were fitted to a straight line
through zero (slope = 0.97, R2 = 0.99). The dotted lines show values
representing 0% and 100% inhibition, as determined from negative
controls (DMSO only) and positive control (100 mM primuline).
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D
assays were performed on different days (Figure 2G). To
judge reproducibility, duplicate assays were performed
with 143 different HCV helicase inhibitors at a concentration of 20 mM. All but two samples in this library
decreased polarization of the Cy5-dT15–NS3h complex
to some extent in a reproducible fashion. Two compounds
in the collection, the DNA binding dyes H33258 and
TO-PRO3 (Invitrogen), increased polarization, suggesting
that they bound the Cy5–DNA–NS3h complex but did
not displace the oligonucleotide (Figure 2H).
Two other methods that are frequently used to monitor
protein nucleic acid interactions were compared with the
above FP-based assay. The first was a modified homogeneous time resolved fluorescence (HTRF! ) assay (Cisbio
bioassays), in which Cy5-dT15 was used as an acceptor of
long-lived lanthanide fluorescence. While optimizing this
TR-FRET assay, we tested three different lanthanide
donors: a Lumi4! -Tb Cryptate-conjugated anti-6 Histidine
mouse monoclonal antibody (catalog #61HISTLA,
Cisbio), an Eu3+ Cryptate-conjugated mouse anti-6
Histidine monoclonal antibody (catalog #61HISKLA,
Cisbio) and a LANCE! Europium Anti-6X Histidine
antibody (catalog #AD0110, Perkin Elmer). The highest
Z0 factors were obtained with the Tb3+ cryptate (data not
shown). The optimized TR-FRET-based assay was performed in the same buffer as the FP-based assays, except
that diluted anti-6 His antibody was added to each assay.
In the HTRF! assay setup, the ratio of signals from the
donor and acceptor is multiplied by 10 000 to estimate
TR-FRET. In our assay, time resolved fluorescence
occurring after excitation at 340 nm was measured at
625 nm, and the signal resulting from binding was detected
at a wavelength of 665 nm due to energy transfer to the
Cy5 (Figure 3A). The maximal energy transfer resulted in a
3.5-fold increase of the signal ratio in the presence of NS3h
bound to the Tb3+-conjugated anti-hexahistidine antibody.
This signal change returned to baseline upon addition of
Nucleic Acids Research, 2012, Vol. 40, No. 17 8613
Table 1. Effects of yellow dyes and small molecules on the interaction of NS3h with DNA, its ability to unwind DNA and RNA, and HCV replication in cells
DNA binding assaysa
Compound
Thioflavine S
Primuline
Titan yellow
ATA
AG 538
NF 023
Suramin
NS3h helicase assaysb
Cellular assays
NS3h IC50
(mM) ± SD
SSB IC50
(mM) ± SD
DNA IC50
(mM) ± SD
RNA IC50
(mM) ± SD
HCV repliconc IC50
(mM) ± SD
ViabilitycCC50
(mM) ± SD
35 ± 3.5
24 ± 3
2.8 ± 0.2
1.4 ± 0.1
3.6 ± 0.2
6.3 ± 0.6
3.6 ± 0.3
12 ± 0.9
5.0 ± 1.4
2.9 ± 0.2
1.0 ± 0.1
1.2 ± 0.1
1.7 ± 0.2
0.3 ± 0.0
24 ± 1.3
12 ± 1.3
12 ± 3.6
0.6 ± 0.1
>100
2.6 ± 0.4
3.7 ± 0.7
22 ± 6.3
15 ± 2.3
30 ± 3.6
0.8 ± 0.2
>100
5.1 ± 1.2
8.9 ± 2.0
>100
>100
>100
98 ± 30
18 ± 3.2
>25d
38 ± 9.3
>100
>100
>100
>100
60 ± 13
>25d
>50e
e
A
B
C
D
Figure 3. HTRF! and AlphaScreen! assays that detect NS3h interactions with DNA. (A) TR-FRET assay using the Lumi4! -Tb
cryptate-conjugated anti-hexahistidine (Cisbio Bioassays) as a donor and Cy5-dT15 as an acceptor. (B) TR-FRET observed with 5 nM Cy5-dT15
alone, with 15 nM NS3h, and with 15 nM NS3h and 200 nM dT20. Error bars are standard deviations (n = 16). (C) Use of AlphaScreen! Histidine
(Nickel Chelate) Detection Kit (Perkin Elmer) reagents to monitor NS3h binding to a biotinylated oligonucleotide. (D) AlphaScreen! counts for
control assays containing 10 nM Bio-d18 alone, with 20 nM NS3h, and with NS3h and 200 nM dT20. Error bars are standard deviations (n = 16).
unlabeled oligonucleotide, dT20 (200 nM) (Figure 3B).
Compounds that disrupt the Cy5-dT15-NS3h complex
also decreased the TR-FRET signal in a concentrationdependent fashion with IC50 values similar to those
determined with the FP-based assay (data not shown),
but the Z0 -factor (0.59) for the TR-FRET assay was less
than what was observed with FP-based assay (Figure 3B).
The second assay compared to the FP-based assay was
based on the AlphaScreen! Histidine (Nickel Chelate)
Detection Kit (catalog #6760619C, Perkin Elmer). This
AlphaScreen! assay monitored the binding of NS3h to
DNA, using donor beads containing Ni2+ ions that
interact with the C-terminal His-tag of NS3h and
streptavidin-bound acceptor beads binding to biotinylated
oligonucleotide (Bio-d18) (Figure 3C). Formation of an
NS3h–DNA complex brings the donor and acceptor
close enough that singlet oxygen can be transferred from
the donor to acceptor beads. Compounds that disrupt the
Bio-d18–NS3h complex (e.g. dT20) decrease the
AlphaScreen! signal (Figure 3D). The signal/background
in this AlphaScreen! was better than was seen in other
assays, with the complex yielding 80 - to 100-fold
increased counts, but assay variability was higher than
with the FP assay, leading to a Z0 factor of 0.62, which
was again less than what was observed in the FP-based
assay.
Compound interference and advantage
over unwinding assays
Most of the compounds in the above helicase inhibitor
library were previously shown to inhibit NS3h when
screened using an MBHA (21). The MBHA (22)
monitors the ability of a helicase to remove a molecular
beacon (44) bound to a complementary strand upon ATP
addition (Figure 4A). In the absence of inhibitors, fluorescence decreases in the MBHA upon ATP addition, but
when an inhibitor (e.g. titan yellow) is present, fluorescence decreases at a slower rate (Figure 4B). The main
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a
Average (±SD) IC50 value from three sets of FP-based binding assay performed with a 16 point 1.5-fold dilution series of each compound starting
at 100 mM.
b
Average (±SD) IC50 value from 3 sets of molecular beacon based helicase assays performed with a 16 point 1.5-fold dilution series of each
compound starting at 100 mM.
c
Average (±SD) IC50 value from three sets of assays performed with a 8 point 2-fold dilution series starting at 100 mM.
d
Average (±SD) IC50 value from three sets of assays performed with a 8 point 2-fold dilution series starting at 25 mM.
e
Average (±SD) IC50 value from three sets of assays performed with a 8 point 2-fold dilution series starting at 50 mM.
8614 Nucleic Acids Research, 2012, Vol. 40, No. 17
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Figure 4. Comparison of HCV helicase DNA binding and unwinding assays. (A) The molecular beacon helicase assay (MBHA). (B) Effect of
indicated concentrations of titan yellow on MBHAs. (C) Effect of indicated concentrations of thiazole orange on MBHAs. (B) and (C) show both the
fluorescence traces for each reaction and the curve fits used to determine initial rates of DNA unwinding. (D) Mean percentage inhibition (equation
(1)) and compound interference (equation (2)) of duplicate FP-based binding assays (+) performed with samples from a library of HCV helicase
inhibitors. (E) Mean percentage inhibition (equation (1)) and compound interference (equation (2)) of duplicate MBHAs performed with samples
from a library of HCV helicase inhibitors. In (D) and (E), the dotted line denotes 0% inhibition and the solid vertical line denotes no interference.
(F) Percentage inhibition seen in the MBHA and FP-based assay for DNA binding compounds present in the library screened in panels (D) and
(E). Note that most DNA-binding compounds that inhibited the MBHA screen did not inhibit the FP-binding assay. (G) Percentage inhibition seen
in the MBHA and FP-based assay for the primuline derivatives present in the library screened in panels (D) and (E). Line shows the correlation of
the ability to inhibit both unwinding and binding. Full data for both screens are in Table S1.
Nucleic Acids Research, 2012, Vol. 40, No. 17 8615
library samples. To identify compounds that decreased
FP by quenching of the fluorescence signal or intrinsic
fluorescence, the fluorescence intensity of each compound
(I) was divided by the fluorescence intensity observed for
negative controls (I(")). A plot of this data (I/I(")) revealed
that four hits increased or decreased the fluorescence intensity by more than 20% in comparison with the negative
controls. Four of the compounds with similar fluorescence
intensity to that of the negative controls decreased FP by
more than 60% at a concentration of 100 mM (Figure 5B).
Each of these compounds [ATA (Sigma Cat. #A1895),
suramin sodium salt (Sigma Cat. #S2671), NF 023
hydrate (Sigma Cat. #N8652) and tyrphostin AG 538
(Sigma Cat. #T7822)] decreased polarization in a
concentration-dependent manner (Figure 5C–F). The
IC50 values with the four best inhibitors were similar to
those seen with titan yellow and lower than those seen
with primuline and thioflavine S (Table 1).
To test if compounds that decrease FP of the
Cy5-dT15–NS3h complex inhibit the ability of NS3h to
unwind DNA, various concentrations of each of the compounds above were added to MBHAs that monitor either
DNA or RNA unwinding. Three of the four compounds
identified in the LOPAC screen inhibited DNA-based
MBHAs (Figure 6B). Those that inhibited the ability of
NS3h to unwind DNA also inhibited its activity on an
RNA substrate (Table 1).
To determine if compounds that interact with the
NS3h–DNA complex might also function as antiviral
agents, their ability to inhibit the replication of an HCV
subgenomic replicon was tested (46). HCV RNA replication was measured using a reporter system in which a
Renilla luciferase gene was fused to the 50 -end of the
neomycin phosphotransferase gene needed for replicon selection (Figure 6C), so that the cellular levels of Renilla
luciferase correlated directly with the amount of HCV
RNA present in cells (35). After replicon transfection
and selection, cells were treated in parallel in two sets of
triplicates. One set of cells was used for Renilla luciferase
assays and the other set was used to determine cell viability using a firefly luciferase-based assay. Three of the four
compounds identified in the LOPAC screen inhibited
replicon luciferase (Figure 6D), and all four compounds
showed little sign of toxicity at a concentration of 25 mM
(Figure 6E). Selectivity was estimated by comparing the
potency with which the compound inhibits the replicon to
its toxicity. By this measure, AG 538 was the most selective because three times more of this compound was needed
to reduce viability than was needed to inhibit HCV replication (Table 1).
Identification of NS3h inhibitors
in Sigma’s LOPAC 1280TM
A similar FP-assay using the E. coli SSB
To test if a DNA binding assay could be used to identify
new HCV helicase inhibitors, the above FP-based assay
was used to screen Sigma’s 1280-compound LOPAC. All
LOPAC compounds were screened at 100 mM in 384-well
plates, each containing positive (primuline or dT20) and
negative (DMSO or buffer only) controls (Figure 5A). Out
of the 1280 samples screened (Table S2), 18 compounds
exhibited FP signals significantly different from other
The specificity of the new HCV helicase inhibitors was
examined using a counterscreen in which NS3h was
substituted with the unrelated E. coli SSB (Figure 7A).
Like NS3h, SSB increased the FP of Cy5-dT15, but
more ('20 nM) SSB was needed to saturate the oligonucleotide with a K0.5 of 9.6 ± 2 nM in the presence of
5 nM Cy5-dT15 (Figure 7B). All the compounds that inhibited the Cy5-dT15–NS3h interaction also inhibited the
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problem with using the MBHA, or similar unwinding
assays to screen for helicase inhibitors, is that it is difficult
to distinguish helicase inhibitors from compounds that
simply interfere with the assay by binding DNA, such as
thiazole orange (45) (Figure 4C). In screens, the ability of
a compound to inhibit the MBHA is measured by
comparing the fluorescence of the MBHA substrate
before and after ATP addition. If a compound simply
reduces fluorescence of the Cy5 substrate before ATP is
added, as seen with concentrations of thiazole orange
>12.5 mM (Figure 4C), it might falsely appear to inhibit
the helicase. A simple method to identify true helicase
inhibitors is to plot percent inhibition vs. compound interference, which is calculated by dividing assay fluorescence
before ATP addition (F0) by the fluorescence seen in
negative control reactions (F0(")), which lack any inhibitory compounds (21). To compare the results of a DNA
binding assay with the MBHA, the same library of known
HCV helicase inhibitors, which was screened with the
FP-based binding assay (Figure 4D), was re-screened
with the MBHA at the same compound concentration
(20 mM) (Figure 4E). When each screen was analyzed for
both percent inhibition and compound interference, it was
clear that fewer compounds interfered with the FP-based
binding assay (Figure 4D) than with the MBHA
(Figure 4E). For full results, see Table S1 (Supplementary
Data). The fact that fewer compounds interfered with
the FP-based assay suggests that many library samples
did not decrease fluorescence by simply quenching Cy5
fluorescence.
Another way that a compound might decrease the fluorescence of the MBHA substrate would be to distort the
duplex region such that the quenching moiety of the
beacon is more likely to interact with the Cy5 fluorophore.
If that were the case, then most DNA binding compounds
should appear to inhibit the MBHA but not the FP-based
binding assay, which lacks a duplex region. To test this
hypothesis, the average percent inhibition observed with
each compound in FP-based binding assays was compared
with the average percent inhibition seen in the MBHA
unwinding assay. Such a plot reveals that most of the
known DNA binding compounds in our HCV helicase
inhibitor library (e.g. berenil, proflavin, netropsin and
SYBR green I) inhibit the MBHA but not the FP-based
binding assay (Figure 4F). In contrast, compounds, which
act mainly by inhibiting the ability of NS3h to bind DNA,
such as those derived from primuline (21), inhibit both the
FP-binding assay and MBHA with a similar potency
(Figure 4G).
8616 Nucleic Acids Research, 2012, Vol. 40, No. 17
A
B
D
E
F
Figure 5. Identification of inhibitors of Cy5-dT15-NS3h complex formation in a screen of the Sigma LOPAC 1280TM. (A) Summary of
screening results of a fluorescence polarization assay to identify inhibitors of the Cy5-dT15-NS3h interaction among the 1280 samples in
Sigma’s LOPAC (+). Positive controls contained primuline (squares)
or dT20 (circles) and negative controls contained DMSO only (not
shown). The solid line represents the mean of all assays (except
positive controls) and the dotted lines three standard deviations.
(B) Normalized inhibition (%) for compounds that fall outside the
above three standard deviation limit are plotted against the
compound interference, defined as fluorescence intensity divided by
the average fluorescence intensity of the negative control samples.
The vertical dotted lines denote defined boundaries of tolerance for
either possible quenching (I = 0.8) or possible intrinsic fluorescence
(I = 1.2). The horizontal dotted line denotes arbitrary cut-off criterion
of 60% inhibition. Fluorescence polarization of a Cy5-dT15-NS3h
DISCUSSION
This study shows how DNA-binding assays can be used to
discover and characterize small molecules that inhibit
helicases. The assays are simpler than those used to
monitor helicase-catalyzed DNA unwinding or ATP hydrolysis, making them more amenable to high throughput
screening (HTS). Despite its simplicity, the FP-based
DNA binding assay developed here was able to find four
compounds that disrupt the HCV helicase-DNA interaction, three of which also inhibit the NS3h’s ability to
unwind DNA and RNA. Three compounds also inhibit
replication of subgenomic HCV replicons. Similar binding
assays using unrelated protein SSB were shown to be
useful for judging compound specificity, as was demonstrated both with the newly identified helicase inhibitors
and with a panel of compounds created in a prior SAR
study of the primuline scaffold (21).
Figure 5. Continued
complex in the presence of increasing concentrations of (C) ATA,
(D) AG 538, (E) NF 023, or (F) Suramin. Data (n = 3) were fitted to
4-parameter concentration response curves constrained to values
obtained in the absence of inhibitor (top) and the absence of NS3h
(bottom) with parameters in Table 1. In (C)–(F), concentration
response curve for titan yellow is shown for comparison (dotted
lines). Raw data for all LOPAC samples can be found in Table S2.
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C
interaction between Cy5-dT15 and SSB (Figure 7C and
Table 1). The specificity of each compound was judged
by comparing the IC50 values obtained with NS3h and
SSB. By this measure, none of the new HCV helicase inhibitors were more specific than the yellow dyes (Table 1).
The yellow dyes were therefore selected to probe if the
SSB based counter screen could be used to identify specific
HCV helicase inhibitors. Thioflavine S was discovered to
inhibit HCV helicase in an MBHA-based screen of the
NCI Mechanistic Set of compounds (Figure 8). Li et al.
(21) isolated eight compounds from thioflavine S and the
related dye primuline and used the core dimeric
benzothiazole scaffold found in all eight components to
synthesize a library of semi-synthetic primuline derivatives. Since we showed above that primuline inhibits
NS3h from binding DNA in a non-specific manner, we
screened the entire primuline derivative collection for
compounds that might be more specific. To this end, we
compared the ability of the compounds synthesized from
the main component of primuline to disrupt the
Cy5-dT15–SSB complex with their ability to inhibit the
HCV helicase in a standard HCV helicase MBHA
(Figure 8 and Table S3). This structure activity relationship reveals that small changes to this scaffold can affect
the affinity of a compound for HCV helicase relative to its
ability to inhibit SSB from binding DNA. The most potent
and specific compound in this family, CID50930730, is
over 30 times more specific (as judged by the ratio of
IC50 values for each compound in the MBHA to its IC50
value in SSB-DNA binding assays, for each compound)
than the least specific compound with similar potency in
the MBHA, CID49849276 (Figure 8 and Table S3).
Nucleic Acids Research, 2012, Vol. 40, No. 17 8617
A
B
C
D
E
A
B
C
Figure 7. Effect of compounds on the polarization of a Cy5-dT15-Escherichia coli single-stranded DNA binding protein (SSB) complex. (A) FP
based assay to monitor SSB binding to Cy5-dT15. (B) Binding between Cy5-dT15 and SSB determined by FP. (C) Cy-dT15-SSB complexes were
titrated with titan yellow (squares), ATA (diamonds), NF 023 (x), AG 538 (circles) or Suramin (inverted triangles). Assays were performed in
triplicate, points show means, and error bars standard deviations. Data are fit to a 4-parameter concentration equation constrained to values
obtained in the absence of inhibitor (top) and the absence of SSB (bottom).
The interaction of HCV NS3 helicase with ssDNA was
monitored here with four different assays. The first, gel
shift analysis, is probably the most common method
used to study protein nucleic acid interactions. The gel
shift assay is laborious, not readily amenable to automation, and requires relatively large amounts of DNA and
protein. Of the three HTS compatible methods evaluated,
the FP-based method was the most precise, reproducible
and cost effective. FP-based assays have been used before
to measure the binding of helicases to a labeled substrate
(40,41), but have not been previously reported as methods
to screen for HCV helicase inhibitors. DNA binding to
NS3h can also be monitored either by measuring intrinsic
protein fluorescence (30,31) or by monitoring changes in
the fluorescence intensity of a fluorescein-labeled oligonucleotide when it binds NS3h (9). Monitoring intrinsic
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Figure 6. Ability of compounds that disrupt the Cy5-dT15-NS3h complex to inhibit the HCV helicase and HCV replication. (A) Fluorescence
intensity of the MBHA substrate in assays containing increasing concentrations of suramin. (B) Effect of AG 538 (squares), ATA (circles), NF 023
(x), and suramin (triangles) on the initial rates of HCV helicase catalyzed DNA unwinding. Points are means of duplicate reactions, and error bars
are standard deviations. Data are fit to a normalized concentration response equation with parameters listed in Table 1. (C) The sub-genomic Renilla
luciferase reporter replicon used to monitor compound effects on HCV replication. (D) Relative HCV RNA levels in the presence of various
concentrations of AG 538 (squares), ATA (circles), NF 023 (x), and Suramin (triangles). Data are fit to a normalized concentration response
equation with parameters in Table 1. (E) Average (±SD) cell viability in assays where cells were exposed to 25 mM of indicated compounds.
8618 Nucleic Acids Research, 2012, Vol. 40, No. 17
protein fluorescence is usually difficult in the presence of
small molecules, especially if they absorb light in the ultraviolet wavelengths. Use of fluorescence intensity based
assays in screening is also difficult, because many library
samples fluoresce in the same range as fluorescein, and as
noted above, we have not yet found an alternate
red-shifted fluorophore that changes intensity when it
interacts with NS3h.
The LOPAC samples that most potently disrupted the
NS3h–DNA complex were two polysulfonated naphthylureas (suramin and NF 023), a triphenylmethane (ATA)
and a tyrphostin (AG 538). All but the tyrphostin inhibit
helicase catalyzed strand separations. Tyrphostin AG 538
mimics a tyrosine kinase substrate so that it acts as a competitive inhibitor of the IGF-1 receptor tyrosine kinase
(47). Preliminary mechanistic studies on each compound
suggest that only ATA inhibits the ability of NS3h to
cleave ATP in the absence of DNA or RNA, and all but
AG 538 prevent RNA from stimulating ATP hydrolysis.
Similarly, gel shift assays show all but AG 538 displace
NS3h from DNA, as was seen with thioflavine S
(Figure 1B). These data suggest that the AG 538
induced decrease in the polarization of both NS3h–
Cy5-dT15 and SSB–Cy5-dT15 complexes might be due
to a fluorescence artifact or something other than the
ability of the tyrphostin to prevent protein from binding
DNA. The ability of AG 538 to inhibit growth of the HCV
replicon is likewise probably not due to its effects on NS3.
AG 538 is an inhibitor of the insulin-like growth factor I
(IGF-1) tyrosine kinase, and it is possible IGF-1-mediated
signaling is needed for efficient HCV replication. Both
HCV infection and a reduction of IGF-1 levels are
linked to the development of liver cancer (48).
Compounds binding in place of ATP could also cause
the helicase to release its grip on DNA in the above
binding assay because ATP binding and hydrolysis
causes HCV helicase to cycle between low affinity and
high affinity DNA-binding states (28). When NS3h
releases DNA, it slides from the 30 to 50 end of a nucleic
acid as a Brownian motor (49). Thus, in light of the recent
demonstration that most nucleoside triphosphates can fuel
this motor action (6), it is not surprising that 2-methylATP, 2-(methylthio)ATP and 2-chloroATP were also hits
in the LOPAC screen. The fact that they inhibited binding
by only 30–40% of NS3h (which was less than our arbitrary 60% cutoff) is also not surprising since they were
tested at concentrations far less than the concentration of
ATP needed to fuel unwinding, or NS3 translocation, at
half-maximum rates (6). In contrast, a,b-methylene ATP
did not inhibit NS3h from binding dT15 (Table S1), confirming the earlier observations that most of the canonical
non-hydrolysable ATP analogs are poor inhibitors of
HCV helicase (22,50).
The fact that the polysulfonated naphthylureas and
triphenylmethanes affect HCV helicase is noteworthy
because the anti-microbial properties of these compounds
are well documented. Suramin has long been used to treat
sleeping sickness caused by trypanosomes, and it inhibits
protein tyrosine phosphatases (51) and G-proteins (52).
G-proteins and helicases share a similar Walker-type
nucleotide-binding site (52), and it is possible that suramin
inhibits HCV helicase and G-proteins through a similar
molecular mechanism. NF 023 is a suramin analog, P2X
receptor antagonist (53), and inhibitor of RNA editing in
trypanosomes (54). Suramin and NF 023 behave similarly
in all in vitro assays here, but it is noteworthy that (among
the two) only suramin was effective against the HCV
replicon in cells. A simple explanation would be that the
somewhat more aromatic suramin is more likely to enter
cells to exert an intracellular effect. Our results also
confirm the recently reported antiviral effect of ATA
against the HCV replicon, which had been reported after
ATA was found to inhibit the HCV NS5B RNAdependent RNA polymerase (55). ATA exerts a similar
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Figure 8. Synthesis of specific HCV helicase inhibitors from a scaffold isolated from the yellow dye primuline. The flow chart summaries the source
of compounds tested (see text for details). The plot shows the ability of various primuline derivatives to inhibit HCV helicase (x-axis) and decrease
polarization of a Cy5-dT15-SSB complex. The dotted line shows where hypothetical compounds that inhibit both assays with the same potency
would lie on the plot. Structures of the four compounds with the most extreme properties are shown on the right, along with the ratios of IC50 values
obtained in the two different assays. All data, structures and CID numbers can be found in Table S3.
Nucleic Acids Research, 2012, Vol. 40, No. 17 8619
potent, specific probes needed to study the role of HCV
helicase in cells. On the other hand, more specific analogs
of non-specific screening hits could be synthesized, as was
demonstrated above with the primuline derivatives.
Screens of the LOPAC and helicase inhibitor libraries
show that the binding assays presented here are easier to
interpret, and less prone to compound interference, than
assays monitoring helicase catalyzed DNA separation.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online:
Supplementary Tables 1–3.
ACKNOWLEDGEMENTS
We would like to thank Peter Hodder (Scripps Florida)
for valuable advice in assay development, and Seng-Lai
Tan for providing the HCV replicon.
FUNDING
National Institutes of Health [RO1 AI088001]; Research
Growth Initiative Award [101X219] from the University
of Wisconsin-Milwaukee Research Foundation; National
Institutes of Health Molecular Libraries Initiative [U54
HG005031]. Funding for open access charge: University
of Wisconsin-Milwaukee Research Foundation.
Conflict of interest statement. None declared.
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inhibitors of NS3 helicase, which efficiently and specifically inhibit
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nonstructural protein 3 protease/helicase requires an intact
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Downloaded from http://nar.oxfordjournals.org/ by guest on September 14, 2013
effect against a wide variety of enzymes that manipulate
nucleic acids, like human flap endonuclease 1 (FEN1) (56).
ATA also inhibits replication of influenzas A and B (57).
Triphenylmethanes that resemble ATA have been developed from the dye Soluble Blue HT as HCV helicase
inhibitors (14).
This study is focused on finding compounds that inhibit
nucleic acids from binding to the known, high
affinity-binding site on the NS3 helicase region. To best
target this site, we have used truncated NS3 lacking the
protease region (i.e. NS3h). Another advantage to using
NS3h instead of full length NS3 is that the truncated
protein expresses at higher levels in E. coli and is more
stable after purification. The same assays used here have
also been performed with full-length NS3 and NS3–NS4A
fusion peptides, with similar results, and it might be
possible to perform the screens here with both fulllength NS3 and NS3h to identify compounds that target
the still poorly defined nucleic acid binding sites on the
protease or in the cleft separating the protease from the
helicase. Such compounds might simultaneously inhibit
both NS3 protease and helicase activities. To date, no
small molecules that simultaneously inhibit both the
NS3 protease and helicase have been reported in the
academic literature, although a recent structure shows
that a protease inhibitor can interact with residues in
both the helicase and protease domains (58).
In any HTS campaign, it is important to have an
appropriate counter screen to identify non-specific compounds. We show here that unrelated DNA binding
proteins can be substituted for NS3h in the FP-assay to
identify such compounds. The results show that the four
LOPAC hits affect both NS3h and E. coli SSB nonspecifically to inhibit the binding of the two proteins to
nucleic acids. As further evidence for a lack of specificity,
both suramin and ATA were hits in a LOPAC screen that
used a similar assay to identify compounds that prevent
the RNA-induced silencing complex from binding to
RNA (59). We show here how this SSB-based
counterscreen can be used to identify more specific NS3
helicase inhibitors using a library of recently disclosed
semi-synthetic analogs of potent helicase inhibitors
found in primuline (21). To identify primuline analogs
that have a greater affinity for HCV helicase than they
do for DNA, Li et al. (21) used an assay that monitors
the ability of the primuline derivatives to displace SYBR
Green I from DNA. As in this study, the most specific
derivative in the study by Li et al. was CID50930730
(Figure 7D), and the structure activity relationship
observed here with the SSB assay (Table S3) essentially
mirrors the relationships previously seen with DNA
binding data. The one important difference was that less
of each compound was needed to inhibit SSB binding than
was needed to displace SYBR Green I (21).
In conclusion, we established a new set of tools that can
be used to discover and analyze HCV helicase inhibitors.
None of these assays are new, and they have been extensively reviewed elsewhere in the context of both DNA (60)
and RNA binding proteins (61). While the helicase inhibitors identified from Sigma’s LOPAC are not specific for
NS3h, screens of larger, more diverse libraries might yield
8620 Nucleic Acids Research, 2012, Vol. 40, No. 17
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SUPPLEMENTAL INFORMATION
Identification and Analysis of Hepatitis C virus NS3 Helicase
Inhibitors Using Nucleic Acid Binding Assays
Sourav Mukherjee,1 Alicia M. Hanson,1 William R. Shadrick,1 Jean Ndjomou,1 Noreena L.
Sweeney,1 John J. Hernandez,1 Diana Bartczak,1 Kelin Li,2 Kevin J. Frankowski,2 Julie A.
Heck,3,4 Leggy A. Arnold,1 Frank J. Schoenen,3 and David N. Frick1,*
1
Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee,
Milwaukee, WI, 53211, USA. 2University of Kansas Specialized Chemistry Center,
University of Kansas, 2034 Becker Dr., Lawrence, KS 66047. 3Department of
Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY, 10595.
Table S1: Compound effects on the fluorescent polarization (FP)-based NS3h-DNA
binding assay and the molecular beacon based helicase assay (MBHAs). Means ±
standard deviations (n=2) are reported for normalized inhibition (%) and assay
interference (ratio). Compounds colored blue are primuline derivatives, green
compounds are known DNA binding agents, and pink compounds are other disclosed
NS3 helicase inhibitors. [Data relate to Fig. 4]
Table S2: Results of a screen of Sigma’s LOPAC for inhibitors of Cy5-dT15-NS3h
complex formation. Pink compounds inhibited complex formation more than 60%, and
green compounds interfered with the assay more than 20%. [Data relate to Fig. 5]
Table S3: The Cy-15-dT15-SSB binding assay as a screen for specific HCV
helicase inhibitors. Means ± standard deviations of IC50 values obtained in titrations
(n=3) with each of the shown primuline derivatives. Compounds are ranked based on
their ability to inhibit the formation of the Cy-15-dT15-SSB complex, with the least potent
compounds on the top. [Data relate to Fig. 8]!
Table&S1
CID$No./Compound$Name$
TO*PRO*3
Hoechst&33258
Chromomycin&A3
4049406
53308659
SYBR&Green&I
53255474
44251438
486270
53239937
44251434
44251428
247520
3413238
49849282
Titan&Yellow
483665
4176595
4111820
7080057
49849300
Soluble&Blue&HT
52939982
44251431
Ellipticine
49849299
Netropsin
Thiazole&Orange
50930741
50930749
50930730
49849293
49849293
50930737
50930733
50930738
DAPI
Rebeccamycin
46913723
Proflavin
53308658
50930755
49849287
Quinacrine
49849302
16060752
5479184
50930751
53312458
49849298
50930740
Thioflavin&T
3084034
Eva&Green
53255447
52914816
50930745
Berenil
49849280
46202556
50930743
49849276
50930732
50930756
9585555
3832647
Trioxsalen
Carrier&Blank/DMSO
50930734
49849284
$Cy52dT152NS3h$Binding$
%$Inhibition
*63&±&0
*96&±&2
*4&±&8
62&±&5
17&±&2
24&±&2
46&±&65
74&±&17
30&±&4
9&±&2
47&±&10
54&±&4
*17&±&2
63&±&2
22&±&3
97&±&4
*21&±&2
*13&±&3
4&±&2
*17&±&0
73&±&0
78&±&0
*9&±&2
73&±&8
*6&±&0
*2&±&1
8&±&1
*4&±&1
56&±&13
*1&±&5
46&±&30
39&±&2
20&±&6
58&±&14
22&±&6
91&±&1
4&±&0
*10&±&2
*10&±&4
*4&±&1
30&±&14
18&±&3
19&±&5
*5&±&4
28&±&1
11&±&5
10&±&3
19&±&6
43&±&6
13&±&1
34&±&6
*8&±&0
30&±&4
*3&±&4
18&±&7
10&±&2
11&±&6
*46&±&0
41&±&0
*8&±&0
38&±&2
28&±&11
4&±&0
52&±&10
*4&±&2
*6&±&2
*6&±&2
*7&±&2
18&±&4
26&±&8
$Cy52dT152NS3h$Binding$
Interference$
1.4&±&0.0
0.1&±&0.0
1.0&±&0.0
0.7&±&0.0
1.1&±&0.1
0.8&±&0.0
0.3&±&0.2
1.0&±&0.0
0.6&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
0.8&±&0.0
0.7&±&0.0
1.0&±&0.0
1.1&±&0.0
0.9&±&0.0
0.8&±&0.0
0.7&±&0.0
0.9&±&0.0
1.0&±&0.0
1.0&±&0.0
0.9&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
0.9&±&0.0
0.9&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.1&±&0.0
0.9&±&0.0
1.0&±&0.0
1.0&±&0.0
0.9&±&0.0
0.9&±&0.2
1.0&±&0.0
1.0&±&0.0
0.9&±&0.0
1.0&±&0.0
0.9&±&0.0
0.9&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
0.9&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.1&±&0.0
1.0&±&0.0
DNA$Unwinding$(MBHA)$ DNA$Unwinding$(MBHA)$
%Inhibition
Interference
115&±&2
0.5&±&0.0
104&±&6
0.3&±&0.0
96&±&1
1.0&±&0.0
96&±&2
0.7&±&0.0
95&±&0
1.0&±&0.0
91&±&3
0.7&±&0.0
90&±&2
0.4&±&0.0
89&±&3
0.9&±&0.0
87&±&4
0.6&±&0.0
87&±&5
1.0&±&0.0
82&±&4
1.0&±&0.0
81&±&12
1.0&±&0.0
80&±&4
0.7&±&0.0
78&±&7
0.7&±&0.0
77&±&8
1.0&±&0.0
75&±&3
1.0&±&0.0
74&±&4
0.8&±&0.0
69&±&2
0.9&±&0.0
68&±&7
0.9&±&0.0
67&±&1
0.9&±&0.0
63&±&2
1.0&±&0.0
63&±&12
0.8&±&0.0
59&±&4
1.0&±&0.0
58&±&23
1.0&±&0.0
57&±&4
0.6&±&0.0
53&±&14
1.0&±&0.0
51&±&20
0.8&±&0.1
49&±&7
0.7&±&0.0
46&±&5
1.0&±&0.0
41&±&13
1.0&±&0.0
41&±&25
1.0&±&0.0
32&±&12
0.9&±&0.0
31&±&4
0.9&±&0.0
31&±&11
1.0&±&0.0
29&±&4
1.0&±&0.0
28&±&2
1.0&±&0.0
28&±&9
0.8&±&0.0
27&±&11
0.8&±&0.1
27&±&3
0.9&±&0.0
27&±&4
0.8&±&0.0
27&±&2
1.0&±&0.0
26&±&1
0.9&±&0.0
25&±&1
0.9&±&0.0
25&±&2
0.9&±&0.0
24&±&6
0.9&±&0.0
24&±&3
0.8&±&0.0
24&±&1
0.9&±&0.0
23&±&0
1.0&±&0.0
23&±&2
0.9&±&0.0
22&±&3
0.9&±&0.0
22&±&1
1.0&±&0.0
21&±&2
0.9&±&0.0
21&±&12
0.8&±&0.0
20&±&2
0.9&±&0.0
20&±&0
0.9&±&0.1
20&±&9
1.0&±&0.0
19&±&6
0.9&±&0.0
19&±&3
0.9&±&0.0
17&±&3
0.9&±&0.0
17&±&1
1.0&±&0.0
17&±&5
0.9&±&0.0
16&±&5
1.0&±&0.0
16&±&8
1.0&±&0.0
15&±&2
1.0&±&0.0
15&±&5
1.0&±&0.0
15&±&2
1.0&±&0.0
15&±&6
0.9&±&0.0
8&±&4
1.0&±&0.0
14&±&6
1.0&±&0.0
14&±&7
1.0&±&0.0
1
Table&S1
CID$No./Compound$Name$
53255452
5362502
Luciferin
9682118
3643004
Carrier&Blank/DMSO
46916208
5691857
4594423
3496625
2998553
6540194
Primulin
978069
5447034
45382099
6064372
53255449
49849286
1293387
6540199
7314627
5281571
45382098
49795072
4473963
44251428
16060753
Carrier&Blank/DMSO
46916209
49849289
5860596
4611776
16060655
50930748
46839370
5736971
1601813
2186405
6540163
5293297
45382102
50904396
2571817
4371917
2330223
46897855
49795071
16238633
1959044
4051948
2214934
4872167
49849294
45382104
49849279
1126404
53255450
49849295
45382101
53255448
24818137
44251427
44251433
2115891
45382097
44251429
46839371
49795073
45382103
$Cy52dT152NS3h$Binding$
%$Inhibition
13&±&5
*1&±&5
*4&±&1
*9&±&1
*5&±&1
*8&±&1
*9&±&2
3&±&0
*5&±&1
*5&±&0
*1&±&2
*9&±&0
31&±&3
1&±&1
*3&±&2
*4&±&1
*6&±&3
0&±&1
27&±&5
*7&±&1
*7&±&3
1&±&1
20&±&2
*4&±&3
*8&±&0
*11&±&1
24&±&6
*10&±&4
*8&±&0
*9&±&0
36&±&2
*4&±&0
*5&±&3
*8&±&2
31&±&2
29&±&5
*3&±&2
*5&±&3
*6&±&0
*10&±&1
*7&±&2
*4&±&2
*10&±&1
*8&±&2
*26&±&4
*47&±&0
37&±&2
2&±&2
*4&±&1
*1&±&2
*7&±&1
*4&±&3
*4&±&2
32&±&6
0&±&1
*5&±&6
*8&±&2
12&±&2
8&±&3
*5&±&2
9&±&1
*3&±&2
8&±&2
7&±&3
7&±&2
*7&±&2
18&±&0
*8&±&2
*9&±&1
0&±&2
$Cy52dT152NS3h$Binding$
Interference$
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
0.9&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
0.9&±&0.0
1.0&±&0.0
1.0&±&0.0
0.9&±&0.0
1.0&±&0.0
1.0&±&0.0
0.9&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
0.9&±&0.0
1.0&±&0.0
0.9&±&0.0
0.9&±&0.0
0.9&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
0.9&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
0.9&±&0.0
1.0&±&0.0
0.9&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
1.0&±&0.0
0.9&±&0.0
1.0&±&0.0
1.0&±&0.0
0.9&±&0.0
1.0&±&0.0
1.0&±&0.0
DNA$Unwinding$(MBHA)$ DNA$Unwinding$(MBHA)$
%Inhibition
Interference
14&±&0
0.9&±&0.0
13&±&3
0.9&±&0.0
13&±&2
0.9&±&0.0
13&±&4
1.0&±&0.0
12&±&5
1.0&±&0.0
12&±&4
1.0&±&0.0
11&±&4
1.0&±&0.0
11&±&4
1.0&±&0.0
11&±&4
1.0&±&0.0
11&±&3
1.0&±&0.0
11&±&9
0.9&±&0.0
10&±&4
1.0&±&0.0
10&±&4
1.0&±&0.0
10&±&8
1.0&±&0.0
10&±&14
0.9&±&0.1
10&±&2
1.0&±&0.0
10&±&2
1.0&±&0.0
10&±&6
1.0&±&0.0
10&±&3
1.0&±&0.0
10&±&7
1.0&±&0.0
9&±&5
1.0&±&0.0
9&±&6
1.0&±&0.0
9&±&5
1.0&±&0.0
9&±&3
1.0&±&0.0
9&±&9
1.0&±&0.0
8&±&2
1.0&±&0.0
8&±&2
1.0&±&0.0
8&±&3
1.0&±&0.0
14&±&6
0.9&±&0.0
8&±&5
1.0&±&0.0
8&±&6
1.0&±&0.0
8&±&6
1.0&±&0.0
8&±&2
1.0&±&0.0
7&±&4
1.0&±&0.0
7&±&1
1.0&±&0.0
7&±&3
1.0&±&0.0
7&±&2
1.1&±&0.0
7&±&3
1.0&±&0.0
7&±&11
1.0&±&0.0
7&±&2
1.0&±&0.0
7&±&0
1.0&±&0.0
7&±&3
1.0&±&0.0
6&±&13
1.0&±&0.0
6&±&4
1.0&±&0.0
6&±&6
1.0&±&0.0
6&±&5
1.0&±&0.0
5&±&2
1.0&±&0.0
5&±&11
1.0&±&0.0
5&±&4
1.0&±&0.0
4&±&1
1.0&±&0.0
4&±&11
1.0&±&0.0
4&±&3
1.0&±&0.0
4&±&5
1.0&±&0.0
4&±&5
1.0&±&0.0
4&±&3
1.0&±&0.0
4&±&9
1.0&±&0.0
3&±&6
1.0&±&0.0
3&±&4
1.0&±&0.0
3&±&1
1.0&±&0.0
2&±&2
1.0&±&0.0
2&±&6
1.0&±&0.0
1&±&7
1.0&±&0.0
0&±&16
1.1&±&0.0
0&±&11
1.1&±&0.0
*1&±&6
1.1&±&0.0
*1&±&9
1.0&±&0.0
*1&±&3
1.1&±&0.0
*1&±&8
1.0&±&0.0
*1&±&0
1.0&±&0.0
*2&±&5
1.1&±&0.0
2
Table&S1
CID$No./Compound$Name$
3923766
3291242
3138378
49849290
$Cy52dT152NS3h$Binding$
%$Inhibition
*9&±&2
*1&±&2
*9&±&0
1&±&0
$Cy52dT152NS3h$Binding$
Interference$
0.9&±&0.0
0.9&±&0.0
0.9&±&0.0
1.0&±&0.0
DNA$Unwinding$(MBHA)$ DNA$Unwinding$(MBHA)$
%Inhibition
Interference
*2&±&8
1.0&±&0.0
*4&±&14
1.1&±&0.0
*5&±&4
1.0&±&0.0
22&±&7
1.0&±&0.0
3
Table&S2
Compound(Name(
Sigma(Cat.(No.
Protoporphyrin IX disodium
Mitoxantrone dihydrochloride
Tyrphostin AG 538
Suramin sodium salt
Aurintricarboxylic acid
NF 023
Reactive Blue 2
PPNDS tetrasodium
MRS 2159
2-Methylthioadenosine triphosphate tetrasodium
I-OMe-Tyrphostin AG 538
Tyrphostin AG 537
2-Chloroadenosine triphosphate tetrasodium
Aurothioglucose
2-(Methylthio)adenosine 5'-diphosphate trisodium salt hydrate
Me-3,4-dephostatin
6-Hydroxy-DL-DOPA
Tyrphostin 51
Tetraethylammonium chloride
JFD00244
L-745,870 hydrochloride
Methoctramine tetrahydrochloride
SCH-202676 hydrobromide
Myricetin
SU 5416
Cefotaxime sodium
L-750,667 trihydrochloride
Ro 41-0960
Tyrphostin AG 879
U-73122
SKF 89626
3-Aminopropionitrile fumarate
P 8293
M 6545
T 7822
S 2671
A 1895
N 8652
R-115
P 2738
M 7684
A-023
T 7697
T 4693
C-145
A0606
M152
M 9440
H 2380
T 7665
T 2265
J4829
L-131
M-105
S 4063
M 6760
S 8442
C 7912
L-133
R-108
T 2067
U 6756
S 3066
A 3134
Cefsulodin sodium salt hydrate
C 8145
DL-erythro-Dihydrosphingosine
Pentolinium di[L(+)-tartrate]
Lercanidipine hydrochloride hemihydrate
Ebastine
GYKI 52466 hydrochloride
P1,P4-Di(adenosine-5')tetraphosphate triammonium
Hemicholinium-3
GW7647
R(-)-Isoproterenol (+)-bitartrate
Nicardipine hydrochloride
Pyrilamine maleate
MK-912
Tyrphostin 47
IPA-3
BIX 01294 trihydrochloride hydrate
(+)-Bromocriptine methanesulfonate
L-Canavanine sulfate
DPO-1
(2S,1'S,2'S)-2-(carboxycyclopropyl)glycine
Decamethonium dibromide
N-Acetyl-2,3-dehydro-2-deoxyneuraminic acid
(±)-8-Hydroxy-DPAT hydrobromide
Dequalinium chloride hydrate
1,10-Diaminodecane
Etoposide
7-Cyclopentyl-5-(4-phenoxy)phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine
Emetine dihydrochloride hydrate
Bisoprolol hemifumarate salt
BIO
Nimustine hydrochloride
(-)-Perillic acid
(±)-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride
Ketanserin tartrate
Rutaecarpine
L-Tryptophan
D 6908
P 3520
L 6668
E9531
G-119
D 1262
H-108
G 6793
I 2760
N 7510
P 5514
M 7065
T 7540
I 2285
B9311
B 2134
C 9758
D7443
C-237
D 1260
D 9050
H 8250
D 3768
D14204
E 1383
C 8863
E 2375
B 2185
B 1686
N 8659
218359
P 7340
S-006
R 3277
T 0254
(Cy53dT153NS3h(Binding(
%(Inhibition
140
119
100
96
96
91
86
58
43
41
37
37
37
36
33
27
22
19
14
13
12
11
8
8
6
6
6
5
5
5
5
4
4
4
4
3
3
3
3
3
3
3
3
3
3
3
3
3
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
(Cy53dT153NS3h(Binding(
Interference(
2.0
0.5
1.0
1.0
1.0
1.1
0.7
0.8
1.0
1.0
0.9
1.0
1.0
1.0
0.9
0.9
0.9
0.8
1.0
1.1
1.0
1.0
1.0
1.0
1.0
1.0
0.9
0.9
1.0
0.8
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
1.1
1.0
1.0
1.0
0.9
1.0
0.9
1.0
1.0
1.0
1.0
1
Table&S2
Compound(Name(
Sigma(Cat.(No.
Ziprasidone hydrochloride monohydrate
Tyrphostin AG 698
Vincristine sulfate
Quercetin dihydrate
Succinylcholine chloride
Salmeterol xinafoate
DL-Buthionine-[S,R]-sulfoximine
Amiloride hydrochloride
DL-p-Chlorophenylalanine methyl ester hydrochloride
Z2777
T 5193
V 8879
Q 0125
S 8251
S 5068
B 2640
A 7410
C 3635
63Methoxy31,2,3,43tetrahydro39Hpyrido[3,4b]&indole
291552
6-Aminohexanoic acid
CNS-1102
Benserazide hydrochloride
Cinnarizine
6-Fluoronorepinephrine hydrochloride
Calcimycin
Agmatine sulfate
Cortisone
SB 222200
CK2 Inhibitor 2
PD 169316
(±)-CPP
JX401
4-Hydroxy-3-methoxyphenylacetic acid
(-)-Eseroline fumarate
N,N-Dihexyl-2-(4-fluorophenyl)indole-3-acetamide
SP600125
L-3,4-Dihydroxyphenylalanine
Chloro-IB-MECA
Dihydrokainic acid
R(-)-2,10,11-Trihydroxy-N-propylnoraporphine hydrobromide
AC-93253 iodide
3,5-Dinitrocatechol
Hydroxylamine hydrochloride
5,5-Diphenylhydantoin
4-Hydroxybenzhydrazide
Glybenclamide
Guanfacine hydrochloride
2,4-Diamino-6-pyrimidinone
SCH-28080
Diacylglycerol Kinase Inhibitor II
Metolazone
IMID-4F hydrochloride
MRS 1845
Kenpaullone
NCS-356
Pancuronium bromide
(±)-Metoprolol (+)-tartrate
Cisplatin
LFM-A13
NNC 55-0396
NO-711 hydrochloride
MDL 28170
Pilocarpine nitrate
SIB 1757
Pyrazinecarboxamide
Primidone
13-cis-retinoic acid
PAC-1
Trimethoprim
REV 5901
Tranilast
Sobuzoxane
Trequinsin hydrochloride
Propentofylline
Sulfaphenazole
Pregnenolone sulfate sodium
Xylometazoline hydrochloride
A 7824
C 4238
B 7283
C 5270
B-012
C 7522
A 7127
C 2755
S 5192
C 7367
P 9248
C-104
J4774
H 1252
E-100
D 8555
S 5567
D 9628
C-277
D 1064
D-030
A9605
D-131
H 9876
D 4007
H 9882
G 0639
G 1043
D1920-6
S 4443
D 5794
M 1195
I 2279
M 1692
K 3888
N 4034
P 1918
M 5391
P 4394
L 8789
N 0287
N-142
M 6690
P 6628
S 9186
P 7136
P 7295
R 3255
P0115
T 7883
R 5523
T 0318
S 4692
T 2057
P 9689
S 0758
P-162
X 6000
(Cy53dT153NS3h(Binding(
%(Inhibition
2
2
2
2
2
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
(Cy53dT153NS3h(Binding(
Interference(
1.0
0.9
1.0
0.9
1.0
1.0
1.0
0.9
1.0
0.9
0.9
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.1
1.0
1.0
1.1
1.0
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
0.8
1.0
1.0
1.0
1.1
1.0
0.9
1.0
1.0
0.9
0.9
1.0
0.9
1.0
1.0
0.9
1.0
1.0
1.0
1.0
2
Table&S2
Compound(Name(
Sigma(Cat.(No.
Quinacrine dihydrochloride
Triprolidine hydrochloride
Sulindac
IMS2186
Zonisamide sodium
Zimelidine dihydrochloride
Q 3251
T 6764
S 8139
I 7160
Z 2001
Z-101
N3Phenylanthranilic&acid
(±)3Nipecotic&acid
144509
211672
N-Acetyl-L-Cysteine
Cyclosporin A
Captopril
A 7250
C 3662
C 4042
Acetamide
A&0500
L-Aspartic acid
Cimetidine
Carbetapentane citrate
Fulvestrant
2-Chloroadenosine
A 9256
C 4522
C 4662
I4409
C 5134
GABA
A&2129
(+)-Butaclamol hydrochloride
8-(p-Sulfophenyl)theophylline
Indirubin-3'-oxime
Cefaclor
Caffeic Acid C 0625
CGP-13501
1-(4-Chlorobenzyl)-5-methoxy-2-methylindole-3-acetic acid
S-(+)-PD 123177 trifluoroacetate salt hydrate
Z-L-Phe chloromethyl ketone
(S)-(+)-Camptothecin
8-Cyclopentyl-1,3-dipropylxanthine
GW9508
Guanabenz acetate
(±)-Epinephrine hydrochloride
Ethosuximide
Emodin
Disopyramide
7,7-Dimethyl-(5Z,8Z)-eicosadienoic acid
CGS-21680 hydrochloride
(+)-Cyclazocine
Capsazepine
N-Methyl-beta-carboline-3-carboxamide
Chlormezanone
Danazol
4-Imidazolemethanol hydrochloride
Hexamethonium dichloride
(R,R)-cis-Diethyl tetrahydro-2,8-chrysenediol
CGP 20712A methanesulfonate
UCL 2077
17alpha-hydroxyprogesterone
Dipropyldopamine hydrobromide
Fenofibrate
D-ribofuranosylbenzimidazole
L-Histidine hydrochloride
Forskolin
Hydroquinone
R(+)-Butylindazone
Fluspirilene
N,N-Dipropyl-5-carboxamidotryptamine maleate
N-(3,3-Diphenylpropyl)glycinamide
(-)-alpha-Methylnorepinephrine
Anisotropine methyl bromide
R-(+)-7-Hydroxy-DPAT hydrobromide
Vanillic acid diethylamide
CPNQ
Daphnetin
L-Canavanine
2',3'-dideoxycytidine
Cambinol
GBR-12935 dihydrochloride
D-033
A-013
I0404
C 6895
C 0987
C 1610
P5749
C 9511
C 9911
C-101
G 9797
G-110
E 4642
E 7138
E 7881
D 7644
D 8008
C-141
C-147
C-191
E-006
C-192
D 8399
H 1877
H 2138
D 8690
C-231
U6758
H 5752
D-031
F 6020
D 1916
H 8125
F 6886
H 9003
D-129
F-100
D-132
D 8816
D 5290
A5181
H-168
E 0137
C 3118
D 5564
C 1625
D 5782
C0494
G 9659
(Cy53dT153NS3h(Binding(
%(Inhibition
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
(Cy53dT153NS3h(Binding(
Interference(
1.0
1.0
1.0
1.0
1.0
1.0
0.9
0.9
0.9
0.9
1.0
0.9
0.9
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.1
1.0
1.1
1.1
1.1
1.0
1.0
1.1
1.1
1.0
1.1
1.1
1.0
1.0
1.1
1.1
1.0
1.0
1.0
3
Table&S2
Compound(Name(
Sigma(Cat.(No.
Enoximone
ET-18-OCH3
5'-N-Ethylcarboxamidoadenosine
E-64
Isoliquiritigenin
Mecamylamine hydrochloride
SD-169
ODQ
MK-886
MDL 105,519
K 185
Meclofenamic acid sodium
AS605240
LY-367,265
(-)-Nicotine hydrogen tartrate salt
alpha-Lobeline hydrochloride
Valproic acid sodium
Loratadine
T0070907
L-655,708
NG-Monomethyl-L-arginine acetate
(±)-3-(3,4-dihydroxyphenyl)-2-methyl-DL-alanine
SR 59230A oxalate
SIB 1893
Ritodrine hydrochloride
Granisetron hydrochloride
Ritanserin
Rauwolscine hydrochloride
3-Tropanyl-indole-3-carboxylate hydrochloride
3-Tropanylindole-3-carboxylate methiodide
5alpha-Pregnan-3alpha-ol-20-one
Propantheline bromide
5HPP-33
Tyrphostin A9
Rilmenidine hemifumarate
Trihexyphenidyl hydrochloride
Paromomycin sulfate
Procaine hydrochloride
Tyrphostin AG 490
Org 27569
TTNPB
Bicalutamide (CDX)
Tyrphostin AG 1478
(±)-gamma-Vinyl GABA
PPADS
Bay 11-7082
NSC348884 hydrate
Albuterol hemisulfate
(±)-alpha-Lipoic Acid
DL-Thiorphan
Trazodone hydrochloride
SMER28
WIN 62,577
SR 2640
AC-55649
SKF 96365
SCH 58261
Quazinone
LP 12 hydrochloride hydrate
Tyrphostin 23
Pifithrin-mu
Cortexolone
1-[2-(Trifluoromethyl)phenyl]imidazole
Yohimbine hydrochloride
CV-3988
YC-1
Caroverine hydrochloride
(-)-Scopolamine methyl bromide
E 1279
E 1779
E 2387
E 3132
I 3766
M 9020
S0572
O 3636
M 2692
M-216
K 1888
M 4531
A 0233
L 2411
N 5260
L 4376
P 4543
L 9664
T 8703
L 9787
M 7033
M 7277
S 8688
S 9311
R 0758
G 3796
R-103
R-104
T-104
T-113
P 8887
P 8891
H 9415
T-182
R-134
T 1516
P 9297
P 9879
T 3434
O8014
T 3757
B9061
T 4182
V 8261
P-178
B 5556
N 3414
S 5013
T 5625
T 6031
T 6154
S 8197
W-104
S 7690
A9480
S 7809
S4568
Q 3504
L 3169
T 7165
P0122
R 0500
T 7313
Y 3125
C 7238
Y-102
C 1119
S 8502
(Cy53dT153NS3h(Binding(
%(Inhibition
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
(Cy53dT153NS3h(Binding(
Interference(
1.1
1.0
1.0
1.0
0.9
0.9
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
0.9
1.0
0.9
1.0
1.0
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.1
1.0
1.0
1.0
1.0
4
Table&S2
Compound(Name(
Sigma(Cat.(No.
S(3)3p3Bromotetramisole&oxalate
190047
(±)-Brompheniramine maleate
B 2390
Azelaic&acid
Tryptamine&hydrochloride
246379
246557
Cephalosporin C zinc salt
C 3270
Atropine&sulfate
A&0257
BWB70C
Bepridil hydrochloride
(+)-Brompheniramine maleate
Amiodarone hydrochloride
3-aminobenzamide
Buspirone hydrochloride
Altretamine
Cephalothin sodium
N-(4-Amino-2-chlorophenyl)phthalimide
N-Acetyl-5-hydroxytryptamine
BRL 50481
Adenosine 3',5'-cyclic monophosphate
Clemizole hydrochloride
(±)-HA-966
Benoxathian hydrochloride
Roscovitine
1,3-Dipropyl-8-p-sulfophenylxanthine
B 4558
B 5016
B 5275
A 8423
A 0788
B 7148
A 8723
C 4520
A 9345
A 1824
B 0936
A 9501
C 5040
A 9699
B-016
R 7772
A-022
Gabaculine&hydrochloride
A&3539
DAPH
BU224 hydrochloride
N-(2-[4-(4-Chlorophenyl)piperazin-1-yl]ethyl)-3-methoxybenzamide
Psora-4
AS-252424
PK 11195
Acetylthiocholine chloride
Cantharidin
Chelidamic acid
CGP-7930
L-Cycloserine
Arecoline hydrobromide
A-315456
(+)-Catechin Hydrate
Cephradine
D 3943
B-154
C 7230
P 9872
A8981
C 0424
A 5626
C 7632
C 8011
C 0862
C 1159
A 6134
A 6351
C 1251
C 8395
Actinonin
A&6671
Chlorprothixene hydrochloride
Corticosterone
Centrophenoxine hydrochloride
p-Benzoquinone
TBBz
CPCCOEt
Bromoenol lactone
Benzamide
ML-7
Mifepristone
(±)-Ibotenic acid
L-alpha-Methyl-p-tyrosine
SB-215505
Isotharine mesylate
Methapyrilene hydrochloride
Naltriben methanesulfonate
3-Isobutyl-1-methylxanthine
1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride
(+)-MK-801 hydrogen maleate
N-Oleoylethanolamine
Oxolinic acid
Stevioside
Ethopropazine hydrochloride
Iofetamine hydrochloride
Mevastatin
Oxotremorine methiodide
Niflumic acid
3-(1H-Imidazol-4-yl)propyl di(p-fluorophenyl)methyl ether hydrochloride
C 1671
C 2505
C 8773
B 1266
T6951
C 9611
B 1552
B 2009
I 2764
M 8046
I 2765
M 8131
S 1068
I 3639
M 9125
N-156
I 5879
I 7016
M-107
O 0383
O 0877
S3572
E5406
I-120
M 2537
O-100
N 0630
I-160
(Cy53dT153NS3h(Binding(
%(Inhibition
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
(Cy53dT153NS3h(Binding(
Interference(
0.9
0.9
0.9
1.0
0.9
0.9
1.0
1.0
1.0
0.9
1.0
1.0
0.9
1.0
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.1
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
0.9
0.9
0.9
0.9
0.9
1.0
1.0
0.9
0.9
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
0.9
1.0
5
Table&S2
Compound(Name(
Sigma(Cat.(No.
(±)-Octoclothepin maleate
Pentamidine isethionate
O-Phospho-L-serine
Gossypol
L-162,313
Papaverine hydrochloride
LY-310,762 hydrochloride
Lomefloxacin hydrochloride
6-Methyl-2-(phenylethynyl)pyridine hydrochloride
(±)-Methoxyverapamil hydrochloride
Nifedipine
7-Nitroindazole
CI-976
GW405833 hydrochloride
2-methoxyestradiol
alpha,beta-Methylene adenosine 5'-triphosphate dilithium
Propafenone hydrochloride
5alpha-Pregnan-3alpha-ol-11,20-dione
PNU-282987
(-)-Tetramisole hydrochloride
Piroxicam
3-n-Propylxanthine
Phenylephrine hydrochloride
Perphenazine
Cyclothiazide
N6-Cyclohexyladenosine
Efaroxan hydrochloride
OXA-22
8-Cyclopentyl-1,3-dimethylxanthine
Disopyramide phosphate
Diclofenac sodium
Glipizide
Staurosporine aglycone
EGTA
Cetirizine dihydrochloride
Cilnidipine
Y-27632 dihydrochloride
Dephostatin
MHPG piperazine
2-Chloro-2-deoxy-D-glucose
Felbamate
CNQX disodium
CX 546
Diazoxide
DL-threo-beta-hydroxyaspartic acid
3,4-Dihydroxyphenylacetic acid
Lithium Chloride
Hydrochlorothiazide
SB 218795
Hispidin
WB-4101 hydrochloride
Fluvoxamine maleate
Dihydroouabain
Dobutamine hydrochloride
p-Fluoro-L-phenylalanine
Doxycycline hydrochloride
6,7-ADTN hydrobromide
1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole
Harmane
Debrisoquin sulfate
5-Fluorouracil
R(+)-SCH-23390 hydrochloride
Diltiazem hydrochloride
Dextromethorphan hydrobromide monohydrate
Flunarizine dihydrochloride
Diphenhydramine hydrochloride
5-fluoro-5'-deoxyuridine
Flutamide
O-111
P 0547
P 0878
G8761
L 1415
P 3510
L 2536
L 2906
M 5435
M 5644
N 7634
N 7778
C3743
G1421
M 6383
M 6517
P 4670
P 5052
P6499
L 9756
P 5654
P 5679
P 6126
P 6402
C 9847
C 9901
E 3263
C-011
C-102
D 6035
D 6899
G-117
S 3939
E 4378
C3618
C1493
Y 0503
D 8065
H 1377
C-203
F 0778
C-239
C-271
D 9035
H 2775
D 9128
L 4408
H 4759
S 8817
H 5257
D 0411
F 2802
D 0670
D 0676
F 4646
D 9891
D-002
H 6036
H 7258
D 1306
F 6627
D-054
D 2521
D 2531
F 8257
D 3630
F 8791
F 9397
(Cy53dT153NS3h(Binding(
%(Inhibition
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
(Cy53dT153NS3h(Binding(
Interference(
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.1
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.1
1.0
1.0
1.1
1.1
1.0
1.0
1.0
1.0
6
Table&S2
Compound(Name(
Sigma(Cat.(No.
L-Hyoscyamine
BU99006
Propofol
MNS
Serotonin hydrochloride
L-165,041
(S)-3,5-Dihydroxyphenylglycine
Fexofenadine hydrochloride
Formoterol
Felodipine
Desipramine hydrochloride
cis-(Z)-Flupenthixol dihydrochloride
Furafylline
FPL 64176
D(-)-2-Amino-5-phosphonopentanoic acid
3,7-Dimethyl-1-propargylxanthine
5,7-Dichlorokynurenic acid
Histamine, R(-)-alpha-methyl-, dihydrochloride
GW2974
L-Glutamic acid hydrochloride
2,3-Dimethoxy-1,4-naphthoquinone
Ibudilast
Venlafaxine hydrochloride
Imidazole-4-acetic acid hydrochloride
Imazodan
DM 235
GW5074
Genistein
1,1-Dimethyl-4-phenyl-piperazinium iodide
Iodoacetamide
Idarubicin
GR 55562 dihydrobromide
Phenelzine sulfate
Pheniramine maleate
SKF 89976A hydrochloride
1-(1-Naphthyl)piperazine hydrochloride
Taxol
Retinoic acid
Pirenzepine dihydrochloride
Prazosin hydrochloride
L-Beta-threo-benzyl-aspartate
SQ 22536
Rotenone
ARP 101
Tranylcypromine hydrochloride
Ro 8-4304
U-75302
Piribedil maleate
Prilocaine hydrochloride
Pyridostigmine bromide
Tolazamide
Uridine 5'-diphosphate sodium
U-69593
(-)-Scopolamine methyl nitrate
Phaclofen
IC 261
U0126
Spermine tetrahydrochloride
A-134974 dihydrochloride hydrate
SC 19220
PD 98,059
Tyrphostin AG 835
AMG 9810
Wortmannin from Penicillium funiculosum
Quinidine sulfate
BAY 61-3606 hydrochloride hydrate
N,N,N-trimethyl-1-(4-trans-stilbenoxy)-2-propylammonium iodide
(-)-Sulpiride
H 9002
B 8433
D126608
M 7445
H 9523
L 2167
D 3689
F 9427
F 9552
F 9677
D 3900
F-114
F-124
F-131
A 8054
D-134
D-138
H-128
G 0668
G 2128
D 5439
I 0157
V7264
I 0375
I 0782
D 5689
G 6416
G 6649
D 5891
I 1149
I 1656
G 0419
P 6777
P 6902
S 9066
S-003
T 7402
R 2625
P 7412
P 7791
B6436
S-153
R 8875
A8356
P 8511
R 8900
U 1508
P 9233
P 9547
P 9797
T 2408
U 4125
U-103
S 2250
P-118
I 0658
U-120
S 2876
A2846
S 3065
P-215
T 5568
A 2731
W 1628
Q 0875
B 9685
T 6692
S 7771
(Cy53dT153NS3h(Binding(
%(Inhibition
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
(Cy53dT153NS3h(Binding(
Interference(
1.1
0.9
1.0
1.0
1.0
1.0
1.1
1.1
1.1
1.0
1.0
1.0
1.0
1.0
1.1
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.1
1.0
1.0
1.1
1.0
1.0
1.0
1.1
0.8
1.0
0.9
0.9
0.9
1.0
0.9
0.9
1.0
0.9
1.0
1.0
1.0
0.9
1.0
0.9
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
7
Table&S2
Compound(Name(
Sigma(Cat.(No.
(-)-Scopolamine,n-Butyl-, bromide
Quipazine, 6-nitro-, maleate
Quinelorane dihydrochloride
(±)-Quinpirole dihydrochloride
Aprindine hydrochloride
(±)-Sulpiride
YS-035 hydrochloride
Zaprinast
Olprinone hydrochloride
S 7882
Q-109
Q-110
Q-111
A 7606
S 8010
Y-101
Z 0878
O 7389
DL3alpha3Methyl3p3tyrosine
120693
3-Bromo-7-nitroindazole
B 2050
53Aminovaleric&acid&hydrochloride
194336
N-Bromoacetamide
Benzamil hydrochloride
L-2-aminoadipic acid
(±)-Atenolol
Cyproterone acetate
5-Bromo-2'-deoxyuridine
GR 113808
Carbamazepine
(±)-Baclofen
Betamethasone
4-Amino-1,8-naphthalimide
Carbachol
Adenosine
Astaxanthin
B 2377
B 2417
A 7275
A 7655
C 3412
B 5002
G 5918
C 4024
B 5399
B 7005
A 0966
C 4382
A 9251
A 9335
Amantadine&hydrochloride
A&1260
N-arachidonylglycine
BP 897
A 1977
B 9308
3'3Azido33'3deoxythymidine
A&2169
(E)-5-(2-Bromovinyl)-2'-deoxyuridine
5-azacytidine
Bromoacetyl alprenolol menthane
Apigenin
Phenoxybenzamine hydrochloride
Paroxetine hydrochloride hemihydrate (MW = 374.83)
SB 204741
Bupropion hydrochloride
1-Aminobenzotriazole
3-Amino-1-propanesulfonic acid sodium
Cyproheptadine hydrochloride
Arecaidine propargyl ester hydrobromide
B 9647
A 2385
B-015
A 3145
B-019
P 9623
S 0693
B-102
A 3940
A 4147
C 6022
A-140
O3(Carboxymethyl)hydroxylamine&hemiHCl
A&4508
Sandoz 58-035
3-Morpholinosydnonimine hydrochloride
Diacylglycerol kinase inhibitor I
S 9318
M5793
D 5919
(±)323Amino373phosphonoheptanoic&acid
A&5157
Ciprofibrate
6-Chloromelatonin
Cilostazol
N6-2-(4-Aminophenyl)ethyladenosine
Cilostamide
gamma-Acetylinic GABA
Alloxazine
Acetazolamide
GR 46611
Chlorpropamide
cis-Azetidine-2,4-dicarboxylic acid
trans-Azetidine-2,4-dicarboxylic acid
AIDA
Imipenem monohydrate
ATPA
ARL 67156 trisodium salt
Carisoprodol
C 0330
C 0331
C 0737
A-202
C 7971
A-230
A-242
A 6011
G 8543
C 1290
A-243
A-244
A-254
I 0160
A-263
A-265
C 8759
Methotrexate&hydrate
A&6770
Amperozide hydrochloride
Aminoguanidine hemisulfate
3-Aminopropylphosphonic acid
A 6976
A 7009
A 7162
(Cy53dT153NS3h(Binding(
%(Inhibition
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
31
(Cy53dT153NS3h(Binding(
Interference(
1.0
1.0
1.0
1.0
1.0
1.1
1.0
1.0
1.0
0.9
0.9
0.9
0.9
1.0
0.9
0.9
0.9
1.0
1.0
1.0
0.9
1.0
1.0
0.9
0.9
1.0
0.9
1.0
1.0
0.9
1.0
0.9
1.0
1.0
1.0
0.9
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.1
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
8
Table&S2
Compound(Name(
Sigma(Cat.(No.
2,3-Butanedione monoxime
Pyrocatechol
Colchicine
1-Methylimidazole
(±)-Ibuprofen
Memantine hydrochloride
Nitrendipine
BW 723C86
SB 242084 dihydrochloride hydrate
Naftopidil dihydrochloride
Fenobam
(±)-Muscarine chloride
DFB
Ivermectin
Methysergide maleate
Olomoucine
Oxiracetam
Indatraline hydrochloride
S15535
Orphenadrine hydrochloride
Mexiletene hydrochloride
IB-MECA
Isonipecotic acid
Nomifensine maleate
N-Methyl-D-aspartic acid
JL-18
nor-Binaltorphimine dihydrochloride
Niclosamide
Metergoline
L-701,324
Nordihydroguaiaretic acid from Larrea divaricata (creosote bush)
3-alpha,21-Dihydroxy-5-alpha-pregnan-20-one
(±)-alpha-Methyl-4-carboxyphenylglycine
PRE-084
NG-Nitro-L-arginine
NG-Nitro-L-arginine methyl ester hydrochloride
Mibefradil dihydrochloride
N6-Methyladenosine
NU2058
Leflunomide
Lidocaine N-ethyl bromide quaternary salt
4-Amidinophenylmethanesulfonyl fluoride hydrochloride
NS 521 oxalate
LE 300
Lansoprazole
6-Nitroso-1,2-benzopyrone
Nilutamide
Promethazine hydrochloride
Cysteamine hydrochloride
Methoxamine hydrochloride
O-Methylserotonin hydrochloride
Phosphomycin disodium
Norcantharidin
Noscapine hydrchloride
Pentylenetetrazole
(+)-Pilocarpine hydrochloride
2,6-Difluoro-4-[2-(phenylsulfonylamino)ethylthio]phenoxyacetamide
SB 415286
S-Ethylisothiourea hydrobromide
Epinastine hydrochloride
Ebselen
Diethylenetriaminepentaacetic acid
R-(-)-Desmethyldeprenyl hydrochloride
(±)-Vanillylmandelic acid
6-Hydroxymelatonin
Hexamethonium bromide
(-)-Physostigmine
NBI 27914
B 0753
C 9510
C 9754
M 8878
I 4883
M 9292
N-144
B175
S 8061
N-158
F 0430
M-104
D 0943
I 8898
M-137
O 0886
O 3011
I-119
S 5321
O 3752
M 2727
I-146
I18008
N 1530
M 3262
J-102
N 1771
N 3510
M 3668
L 0258
N 5023
P 2016
M 4796
P 2607
N 5501
N 5751
M 5441
M 5501
N 4286
L 5025
L 5783
A 6664
N 7904
L 8401
L 8533
N 8403
N 8534
P 4651
M 6500
M 6524
M 6628
P 5396
N 8784
N 9007
P 6500
P 6503
D 8941
S 3567
E 3149
E 5156
E 3520
D 6518
D 6940
H 0131
H 0627
H 0879
E 8375
N 3911
(Cy53dT153NS3h(Binding(
%(Inhibition
31
31
31
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
(Cy53dT153NS3h(Binding(
Interference(
1.1
1.0
1.0
0.9
1.0
1.0
0.9
0.9
0.9
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
0.9
1.0
0.9
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
9
Table&S2
Compound(Name(
Sigma(Cat.(No.
8-(3-Chlorostyryl)caffeine
Imperatorin
CGS-15943
Fusidic acid sodium
S-(+)-Fluoxetine hydrochloride
DCEBIO
1-Deoxynojirimycin hydrochloride
Dipyridamole
Furegrelate sodium
Furosemide
Fluphenazine dihydrochloride
R(-)-2,10,11-Trihydroxyaporphine hybrobromide
2',3'-didehydro-3'-deoxythymidine
Droperidol
L-3,4-Dihydroxyphenylalanine methyl ester hydrochloride
GBR-12909 dihydrochloride
MHPG sulfate potassium
(±)-2,3-Dichloro-alpha-methylbenzylamine hydrochloride
5-Hydroxyindolacetic acid
Carvedilol
4-DAMP methiodide
Doxylamine succinate
N^G,N^G-Dimethylarginine hydrochloride
Clodronic acid
N-Desmethylclozapine
Hexahydro-sila-difenidol hydrochloride, p-fluoro analog
AL-8810
Phenytoin sodium
S(-)-Pindolol
Ganaxolone
Ganciclovir
Sematilide monohydrochloride monohydrate
Epibestatin hydrochloride
Bezafibrate
5,5-Dimethyl-1-pyrroline-N-oxide
N-Methyldopamine hydrochloride
Gallamine triethiodide
Ipratropium bromide
Etazolate hydrochloride
SU 4312
BRL 52537 hydrochloride
Tetracaine hydrochloride
Phorbol 12-myristate 13-acetate
NU6027
D-609 potassium
Phosphoramidon disodium
Picotamide
Ammonium pyrrolidinedithiocarbamate
Tamoxifen citrate
Ibandronate sodium
(±)-cis-Piperidine-2,3-dicarboxylic acid
Tetramisole hydrochloride
S(+)-Raclopride L-tartrate
Procainamide hydrochloride
Tetradecylthioacetic acid
(±)-Sotalol hydrochloride
SB-366791
(±)-Synephrine
SKF 95282 dimaleate
4-Imidazoleacrylic acid
N2-Ethyl-2'-deoxyguanosine
Terbutaline hemisulfate
Triamterene
Auranofin
DL-Stearoylcarnitine chloride
BF-170 hydrochloride
Tyrphostin AG 494
A3 hydrochloride
C-197
I6659
C-199
F 0881
F 1553
D 9190
D 9305
D 9766
F 3764
F 4381
F 4765
D-029
D 1413
D 1414
D 1507
D-052
H 8759
D-103
H 8876
C3993
D-104
D 3775
D 4268
D 4434
D5676
H-127
A 3846
D 4505
P-152
G 7795
G 2536
S0323
E 0381
B 7273
D 5766
D 5886
G 8134
I 1637
E 1896
S 8567
B 5559
T 7508
P 8139
N 4411
T 8543
R 7385
P 8477
P 8765
T 9262
I5784
P 8782
T 1512
R-121
P 9391
T 1698
S 0278
S 0441
S 0752
S 5317
U 7500
N 3289
T 2528
T 4143
A 6733
S 2381
B 4311
T 4318
A 1980
(Cy53dT153NS3h(Binding(
%(Inhibition
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
(Cy53dT153NS3h(Binding(
Interference(
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.1
1.1
1.0
1.0
1.0
1.0
1.0
1.1
1.0
1.0
1.0
1.1
1.0
1.1
1.0
1.0
1.0
1.0
1.0
0.9
0.9
0.9
1.0
0.9
1.0
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
10
Table&S2
Compound(Name(
Sigma(Cat.(No.
Spermidine trihydrochloride
U-99194A maleate
Vinblastine sulfate salt
Vinpocetine
Phenamil methanesulfonate
Tyrphostin AG 555
(±)-PD 128,907 hydrochloride
Stattic
ICI 63,137
Sanguinarine chloride
LY2183240
N-Succinyl-L-proline
SU 6656
Quinolinic acid
Triamcinolone
Quinine sulfate
S(-)-Timolol maleate
(+)-Quisqualic acid
IRAK-1/4 Inhibitor I
Ara-G hydrate
Xylazine hydrochloride
Xanthine amine congener
Tyrphostin AG 112
(-)-Quinpirole hydrochloride
Tyrphostin 1
O6-benzylguanine
L-Buthionine-sulfoximine
S 2501
U-116
V 1377
V 6383
P-203
T 4818
P-216
S7947
I 8283
S 5890
L 9044
S 6633
S 9692
P63204
T 6376
Q 1250
T 6394
Q 2128
I 5409
A 4233
X 1251
X-103
T 6943
Q-102
T 7040
B 2292
B 2515
53Fluoroindole323carboxylic&acid
265128
Chlormethiazole hydrochloride
CGP-74514A hydrochloride
C1240
C 3353
S3(43Nitrobenzyl)363thioguanosine
861669
1-Aminocyclopropanecarboxylic acid hydrochloride
D-Cycloserine
4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride
A 0430
C 3909
A 8456
Org&24598&lithium&salt
O7639
Benzamidine hydrochloride
Cyclobenzaprine hydrochloride
B 6506
C 4542
Aminophylline&ethylenediamine
A&1755
Ro 20-1724
Chlorothiazide
Bethanechol chloride
Acetyl-beta-methylcholine chloride
5-(N-Ethyl-N-isopropyl)amiloride
(±)-AMT hydrochloride
Ceftriaxone sodium
HEMADO
(-)-Cotinine
Clotrimazole
B 8279
C 4911
C 5259
A 2251
A 3085
A 9834
C 5793
H3288
C 5923
C 6019
YM&976
Y&4877
AA-861
9-Amino-1,2,3,4-tetrahydroacridine hydrochloride
Bromoacetylcholine bromide
R(+)-6-Bromo-APB hydrobromide
S(-)-Atenolol
A 3711
A 3773
B-121
B-135
A-143
53(N,N3Dimethyl)amiloride&hydrochloride
A&4562
Azathioprine
Acyclovir
BRL 54443 maleate B-173
(±)-2-Amino-3-phosphonopropionic acid
Supercinnamaldehyde
DL-Cycloserine
1-Amino-1-cyclohexanecarboxylic acid hydrochloride
McN-A-343
Cystamine dihydrochloride
SB 200646 hydrochloride
Caffeine
Acetohexamide
AB-MECA
A 4638
A 4669
A 4910
S3322
C 7005
A-162
C 7041
C 7255
S 0568
C 0750
A-178
A-236
(Cy53dT153NS3h(Binding(
%(Inhibition
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
32
(Cy53dT153NS3h(Binding(
Interference(
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.1
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
1.0
1.0
0.9
0.9
1.0
0.9
0.9
0.9
1.0
1.0
0.9
1.0
0.9
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
11
Table&S2
Compound(Name(
Sigma(Cat.(No.
ML-9 C 1172
2-Hydroxysaclofen
Chlorpromazine hydrochloride
CBIQ
Caffeic acid phenethyl ester
DSP-4 hydrochloride
CB 1954
4-Aminobenzamidine dihydrochloride
1-benzoyl-5-methoxy-2-methylindole-3-acetic acid
8-Bromo-cGMP sodium
(±)-Isoproterenol hydrochloride
R(-)-Me5
Iproniazid phosphate
(-)-MK-801 hydrogen maleate
Cibenzoline succinate
L-alpha-Methyl DOPA
4-Methylpyrazole hydrochloride
Oxybutynin Chloride
MRS 1523
ZM 39923 hydrochloride
SB-525334
Mianserin hydrochloride
SB 216763
Imetit dihydrobromide
1,5-Isoquinolinediol
Nimesulide
Molsidomine
BBMP
Mizoribine
MG 624
alpha-Methyl-DL-tyrosine methyl ester hydrochloride
Kainic acid
Ketorolac tris salt
Ketotifen fumarate
NAN-190 hydrobromide
Pimozide
U-73343
S-Nitrosoglutathione
Milrinone
1-Methylhistamine dihydrochloride
1,3-Dimethyl-8-phenylxanthine
S-Methyl-L-thiocitrulline acetate
Melatonin
L-Methionine sulfoximine
Naphazoline hydrochloride
3-Nitropropionic acid
(±)-Normetanephrine hydrochloride
1-Phenyl-3-(2-thiazolyl)-2-thiourea
Podophyllotoxin
VER-3323 hemifumarate salt
Lidocaine hydrochloride
L-687,384 hydrochloride
Praziquantel
LY-294,002 hydrochloride
Loxapine succinate
GR 127935 hydrochloride hydrate
Edrophonium chloride
Ellipticine
Isoguvacine hydrochloride
Guvacine hydrochloride
Demeclocycline hydrochloride
Muscimol hydrobromide
2,2'-Bipyridyl
CGS-12066A maleate
N-Ethylmaleimide
2-Cyclooctyl-2-hydroxyethylamine hydrochloride
5-Carboxamidotryptamine maleate
(±)-CGP-12177A hydrochloride
A 6566
C 8138
C 6617
C 8221
C 8417
C 2235
A 7148
B 1183
B 1381
I 5627
M 0814
I 7627
M-108
C1618
M-129
M 1387
O 2881
M 1809
Z 4626
S 8822
M 2525
S 3442
I-135
I-138
N 1016
M 2901
B7936
M 3047
M 3184
M 3281
K 0250
K 1136
K 2628
N 3529
P 1793
U 6881
N 4148
M 4659
M 4910
P 2278
M 5171
M 5250
M 5379
N 5504
N 5636
N 7127
P 4015
P 4405
V 1889
L 5647
L 8539
P 4668
L 9908
L-106
G 5793
E 3256
E 3380
G-002
G-007
D 6140
G-019
D 7505
C-106
E 3876
C-108
C-117
C-125
(Cy53dT153NS3h(Binding(
%(Inhibition
32
32
32
32
32
32
32
32
32
32
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
(Cy53dT153NS3h(Binding(
Interference(
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
0.9
0.9
1.0
0.9
0.9
1.0
0.9
1.0
0.9
1.0
1.0
0.9
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
12
Table&S2
Compound(Name(
Sigma(Cat.(No.
DL-Homatropine hydrobromide
3,4-Dichloroisocoumarin
beta-Estradiol
Phenserine
Hypotaurine
Haloperidol
Cytidine 5'-diphosphocholine sodium salt hydrate
Hydralazine hydrochloride
S-(-)-Eticlopride hydrochloride
Fenoterol hydrobromide
Ciproxifan hydrochloride
Dantrolene sodium
Hydrocortisone
R(-)-Propylnorapomorphine hydrochloride
Histamine dihydrochloride
JS-K
Retinoic acid p-hydroxyanilide
Fenspiride hydrochloride
Genipin
1,4-Dideoxy-1,4-imino-D-arabinitol
Fusaric acid
Esomeprazole magnesium dihydrate
Fenoldopam bromide
R(-)-N-Allylnorapomorphine hydrobromide
(±)-SKF-38393 hydrochloride
Hydroxyurea
(+)-Hydrastine
(±)-DOI hydrochloride
Famotidine
FSCPX
Dihydroergotamine methanesulfonate
NS8593 hydrochloride
2,3-Butanedione
Flupirtine maleate
1,3-Dipropyl-7-methylxanthine
Domperidone
Eliprodil
5-Hydroxy-L-tryptophan
N-Methyl-1-deoxynojirimycin
Fluoxetine hydrochloride
Dihydro-beta-erythroidine hydrobromide
Dilazep hydrochloride
1,7-Dimethylxanthine
L-Glutamine
DL-alpha-Difluoromethylornithine hydrochloride
ATPO
NSC 95397
Etodolac
alpha-Guanidinoglutaric acid
HA-100
Phosphonoacetic acid
Ropinirole hydrochloride
Resveratrol
Putrescine dihydrochloride
PAPP
SR-95531
Pargyline hydrochloride
Rottlerin
Ro 25-6981 hydrochloride
Thioridazine hydrochloride
Thapsigargin
Sepiapterin
Amisulpride
SB 206553 hydrochloride
(S)-Propranolol hydrochloride
RX 821002 hydrochloride
Terfenadine
Ranitidine hydrochloride
H 0126
D 7910
E 8875
P0111
H 1384
H 1512
C0256
H 1753
E-101
F 1016
H 3132
D 9175
H 4001
D-027
H 7250
J 4137
H 7779
F 6145
G4796
D 1542
F 6513
E 7906
F 6800
D-042
D-047
H 8627
H 8645
D-101
F 6889
F 7927
D 2763
N 2538
D 3634
F 8927
D-108
D-122
E 2031
H 9772
M 1777
F-132
D-149
D 5294
D 5385
G 3126
D-193
A 7845
N 1786
E 0516
G 7788
I 1392
P 6909
R 2530
R 5010
P 7505
S-009
S-106
P 8013
R 5648
R 7150
T 9025
T 9033
S-154
A 2729
S-180
P 8688
R 9525
T 9652
R-101
(Cy53dT153NS3h(Binding(
%(Inhibition
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
(Cy53dT153NS3h(Binding(
Interference(
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.1
1.0
1.0
1.0
1.0
1.1
1.0
0.9
1.1
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.1
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
1.0
0.9
0.9
0.9
1.0
0.9
1.0
0.9
1.0
0.9
0.9
0.9
1.0
0.9
0.9
0.9
13
Table&S2
Compound(Name(
Sigma(Cat.(No.
Trifluperidol hydrochloride
Ro 41-1049 hydrochloride
Tiapride hydrochloride
Protriptyline hydrochloride
6(5H)-Phenanthridinone
Tetraethylthiuram disulfide
TCPOBOP
K114
Prochlorperazine dimaleate
Telenzepine dihydrochloride
Thioperamide maleate
Riluzole
Steviol
A-68930 hydrochloride
Tizanidine hydrochloride
Theophylline
1,10-Phenanthroline monohydrate
Ro 04-6790 dihydrochloride
4-Hydroxyphenethylamine hydrochloride
R(+)-3PPP hydrochloride
S(-)-3PPP hydrochloride
L-765,314
UK 14,304
Tetrabenazine
(+)-Norfenfluramine hydrochloride
Semicarbazide hydrochloride
U-101958 maleate
Tetrahydrozoline hydrochloride
1-Phenylbiguanide
SID7969543
Pirenperone
cDPCP
(±)-Taxifolin
SNC80
(±)-Verapamil hydrochloride
Terazosin hydrochloride
N-Oleoyldopamine
L-Mimosine from Koa hoale seeds
D-Serine
PD 168,077 maleate
SB 269970 hydrochloride
Spiperone hydrochloride
WAY-100635 maleate
SB 205384
Na-p-Tosyl-L-lysine chloromethyl ketone hydrochloride
Chelerythrine chloride
N-Acetyltryptamine
Cortisone 21-acetate
L-allylglycine
Bumetanide
Benazoline oxalate
T-103
R-107
T 0410
P 8813
P 8852
T 1132
T 1443
K 1015
P 9178
T-122
T-123
R-116
H8664
A 8852
T 6950
T 1633
P 9375
R-140
T 2879
P-102
P-103
L 3040
U-104
T2952
N3288
S 2201
U-115
T 4264
P-120
S1323
P-126
C 0996
T 4512
S 2812
V 4629
T 4680
O 2139
M0253
S 4250
P-233
S 7389
S 7395
W-108
S 7936
T 7254
C 2932
A 7342
C 3130
A 7762
B 3023
B 4555
Atropine&methyl&nitrate
Arcaine&sulfate
A&0382
A&0384
ATPO
Calmidazolium chloride
SB 202190
(±)-Norepinephrine (+)bitartrate
Brefeldin A from Penicillium brefeldianum
(±)-2-Amino-4-phosphonobutyric acid
Alfuzosin hydrochloride
Cefazolin sodium
(±)-p-Aminoglutethimide
Aniracetam
CL 316,243
7-Chloro-4-hydroxy-2-phenyl-1,8-naphthyridine
(-)-Bicuculline methbromide, 1(S), 9(R)
10058-F4
BTCP hydrochloride
A 7845
C 3930
S 7067
A 0937
B 7651
A 1910
A 0232
C 5020
A 9657
A 9950
C 5976
C 5982
B-103
F3680
B-138
(Cy53dT153NS3h(Binding(
%(Inhibition
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
(Cy53dT153NS3h(Binding(
Interference(
1.0
1.0
0.9
0.9
1.0
1.0
1.0
1.0
1.0
1.0
0.9
0.9
1.0
0.9
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
0.9
0.9
1.0
0.9
0.9
1.0
1.0
0.9
0.9
0.9
0.9
0.9
1.0
1.0
0.9
1.0
0.9
1.0
1.0
1.0
1.0
1.0
14
Table&S2
Compound(Name(
Sigma(Cat.(No.
5'-Amino-5'-deoxyadenosine p-toluenesulfonate salt
GR 79236X
Clozapine
Aminobenztropine
(±)-p-Chlorophenylalanine
Chloroquine diphosphate
B-HT 933 dihydrochloride
Biperiden hydrochloride
Chlorambucil
L-Arginine
1-Allyl-3,7-dimethyl-8-p-sulfophenylxanthine
A283
G5794
C 6305
A-138
C 6506
C 6628
B-161
B 5311
C 0253
A 5006
A-145
(±)323Amino353phosphonopentanoic&acid
A&5282
L-732,138
Acetylsalicylic acid
4-Androsten-4-ol-3,17-dione
2-(2-Aminoethyl)isothiourea dihydrobromide
Cyclophosphamide monohydrate
SKF-89145 hydrobromide
cis-4-Aminocrotonic acid
A 5330
A 5376
A 5791
A 5879
C 0768
S 3316
A-201
N3Acetylprocainamide&HCl
A&5909
Amifostine
A-77636 hydrochloride
Beclomethasone
Choline bromide
Carboplatin
Clemastine fumarate
beta-Chloro-L-alanine hydrochloride
Ifenprodil tartrate
TMPH hydrochloride
Nimodipine
cis(+/-)-8-OH-PBZI hydrobromide
Levallorphan tartrate
AFMK
Minocycline hydrochloride
Proglumide
Metoclopramide hydrochloride
NS 2028
(±)-Octopamine hydrochloride
L-N6-(1-Iminoethyl)lysine hydrochloride
3-Iodo-L-tyrosine
Imiloxan hydrochloride
Oleic Acid
Moxonidine hydrochloride
Ouabain
ICI 204,448 hydrochloride
p-MPPI hydrochloride
Metaproterenol hemisulfate
TG003
Oxotremorine sesquifumarate salt
8-Methoxymethyl-3-isobutyl-1-methylxanthine
Trandolapril
Oxaprozin
Molindone hydrochloride
p-MPPF dihydrochloride
Levetiracetam
Nialamide
JWH-015
Progesterone
S-Methylisothiourea hemisulfate
S-(4-Nitrobenzyl)-6-thioinosine
S-Nitroso-N-acetylpenicillamine
(±)-Propranolol hydrochloride
MRS 2179
Meloxicam sodium
Morin
Pentoxifylline
Minoxidil
L-Glutamic acid, N-phthaloyl-
A 5922
A-255
B 0385
C 1754
C 2538
C 8903
C 9033
I 2892
T5576
N-149
P 0618
L-121
A 2355
M 9511
M-001
M 0763
N-211
O 0250
I 8021
I 8250
I 9531
O 1008
M 1559
O 3125
I-122
M-204
M 2398
T 5575
O 9126
M 2547
T4827
O 9637
M 1818
M-226
L8668
N 1392
J 4252
P 0130
M 3127
N 2255
N 3398
P 0884
M 3808
M 3935
M 4008
P 1784
M 4145
P 1801
(Cy53dT153NS3h(Binding(
%(Inhibition
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
33
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
(Cy53dT153NS3h(Binding(
Interference(
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
0.9
0.9
0.9
0.9
1.0
1.0
0.9
0.9
1.0
1.0
1.0
0.9
0.9
1.0
0.9
0.9
1.0
1.0
0.9
0.9
0.9
0.9
0.9
1.0
1.0
0.9
0.9
1.0
1.0
1.0
0.9
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
15
Table&S2
Compound(Name(
Sigma(Cat.(No.
NCS-382
loxoprofen
Labetalol hydrochloride
5-Nitro-2-(3-phenylpropylamino)benzoic acid
AMN082
Pirfenidone
Olvanil
Loperamide hydrochloride
Lonidamine
NADPH tetrasodium
Thiolactomycin
(S)-MAP4 hydrochloride
Palmitoyl-DL-Carnitine chloride
Naloxone hydrochloride
Piracetam
Naltrindole hydrochloride
Sertraline hydrochloride
(±)-AMPA hydrobromide
S-(-)-Carbidopa
(±)-Chloro-APB hydrobromide
Paliperidone
Daidzein
GR-89696 fumarate
Dicyclomine hydrochloride
(±) trans-U-50488 methanesulfonate
1-(m-Chlorophenyl)-biguanide hydrochloride
Estrone
3-deazaadenosine
Bendamustine hydrochloride
erythro-9-(2-Hydroxy-3-nonyl)adenine hydrochloride
Cirazoline hydrochloride
Alinidine
Hydroxytacrine maleate
Doxazosin mesylate
DNQX
R(-)-Apocodeine hydrochloride
1-(4-Hydroxybenzyl)imidazole-2-thiol
Flumazenil
SANT-1
Mephetyl tetrazole
Icilin
Dopamine hydrochloride
(±)-7-Hydroxy-DPAT hydrobromide
N,N,N',N'-Tetramethylazodicarboxamide
BNTX maleate salt hydrate
N-Methylhistaprodifen dioxalate salt
5-hydroxydecanoic acid sodium
1-(2-Methoxyphenyl)piperazine hydrochloride
Propionylpromazine hydrochloride
(±)-6-Chloro-PB hydrobromide
Phloretin
BIA 2-093
(±)-SKF 38393, N-allyl-, hydrobromide
Phenylbutazone
Taurine
CCT007093
Sodium nitroprusside dihydrate
Urapidil hydrochloride
Sulindac sulfone
2-Phenylaminoadenosine
Trimipramine maleate
(±)-PPHT hydrochloride
SKF 86466
(-)-Scopolamine hydrobromide
S(-)-UH-301 hydrochloride
(-)-trans-(1S,2S)-U-50488 hydrochloride
N-p-Tosyl-L-phenylalanine chloromethyl ketone
(6R)-5,6,7,8-Tetrahydro-L-biopterin hydrochloride
N 4159
L 0664
L 1011
N 4779
A6605
P 2116
O 0257
L 4762
L 4900
N 7505
T 9567
M 5560
P 4509
N 7758
P 5295
N-115
S 6319
G-017
C1335
C-130
P0099
D 7802
G-133
D 7909
D 8040
C-144
E 9750
D 8296
B 5437
E-114
C-223
A7230
H 3146
D 9815
D 0540
D-003
H 6892
F 6300
S4572
M 7945
I 9532
H 8502
H 8653
D 3648
B 8312
M 7320
H-135
S-008
P 7780
S-143
P 7912
B 5435
S-168
P 8386
T 0625
C 9369
S 0501
U-100
S 1438
P-101
T 3146
P-105
S 1563
S 1875
U-108
U-111
T 4376
T 4425
(Cy53dT153NS3h(Binding(
%(Inhibition
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
(Cy53dT153NS3h(Binding(
Interference(
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.1
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
0.9
0.6
1.0
1.0
0.9
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
16
Table&S2
Compound(Name(
Sigma(Cat.(No.
SKF 83959 hydrobromide
S(+)-PD 128,907 hydrochloride
Spironolactone
(±)-Vesamicol hydrochloride
Tulobuterol hydrochloride
1-(2-Chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane
S 2816
P-183
S 3378
V-100
T 6050
C 3010
TMB38&hydrochloride
43aminopyridine
861804
A&0152
Allopurinol
Alprenolol hydrochloride
Bay 11-7085
p-Aminoclonidine hydrochloride
Reserpine
BW 284c51
Aminopterin
Bestatin hydrochloride
Cephalexin hydrate
L(-)-Norepinephrine bitartrate
BRL 15572
Chloroethylclonidine dihydrochloride
Antozoline hydrochloride
PNU-37883A
Amoxapine
Cytosine-1-beta-D-arabinofuranoside hydrochloride
BRL 37344 sodium
Amiprilose hydrochloride
Alaproclate hydrochloride
Clomipramine hydrochloride
Carmustine
Aconitine
Citalopram hydrobromide
Clonidine hydrochloride
CP55940
Cinoxacin
Atropine methyl bromide
BTO-1
Lorglumide sodium
Nylidrin hydrochloride
(-)-Isoproterenol hydrochloride
Indomethacin
Isoxanthopterin
Dihydrocapsaicin
S(+)-Isoproterenol (+)-bitartrate
Nocodazole
Melphalan
m-Iodobenzylguanidine hemisulfate
Methiothepin mesylate
Nemadipine-A
3-Morpholinosydnonimine hydrochloride
3-Methoxy-morphanin hydrochloride
Methylergonovine maleate
Sivelestat sodium salt hydrate
ML 10302
Pindolol
Neostigmine bromide
Ketoconazole
Ketoprofen
(-)-cis-(1S,2R)-U-50488 tartrate
Picrotoxin
LP44
Rufinamide
Nalidixic acid sodium
CyPPA
beta-Lapachone
Lamotrigine
Nortriptyline hydrochloride
Metrazoline oxalate
GW9662
A 8003
A 8676
B 5681
A 0779
R 0875
A 9013
A 1784
B 8385
C 4895
A 9512
B 9929
B-003
A 9899
P 0248
A-129
C 6645
B-169
A 4687
A-164
C 7291
C 0400
A 8001
C 7861
C 7897
C 1112
C 8645
A 6883
B 6311
L-109
N-153
I 6504
I 7378
I 7388
M 1022
I 8005
M 1404
M 2011
I 9890
M-149
N4163
M-184
M-187
M 2776
S 7198
M 7319
P 0778
N2001
K 1003
K 1751
U-106
P 1675
L9793
R 8404
N 4382
C5493
L 2037
L 3791
N 7261
M 5685
M 6191
(Cy53dT153NS3h(Binding(
%(Inhibition
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
34
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
(Cy53dT153NS3h(Binding(
Interference(
1.0
1.0
1.0
1.0
1.0
0.9
0.9
0.9
0.9
1.0
0.9
0.9
1.0
0.9
0.9
1.0
0.9
0.9
0.9
1.0
0.9
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
0.9
0.9
1.0
1.0
1.0
0.9
0.9
1.0
0.9
0.9
0.9
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
17
Table&S2
Compound(Name(
Sigma(Cat.(No.
Se-(methyl)selenocysteine hydrochloride
Ro 90-7501
(-)-Epinephrine bitartrate
DBO-83
Tamoxifen
Tocainide hydrochloride
AS 604850
Fiduxosin hydrochloride
Diphenyleneiodonium chloride
Nefiracetam
R-(+)-8-Hydroxy-DPAT hydrobromide
Doxepin hydrochloride
Tomoxetine
1,4-PBIT dihydrobromide
THIP hydrochloride
XCT790
Tolbutamide
Tetraisopropyl pyrophosphoramide
TPMPA
(E)-4-amino-2-butenoic acid
U-74389G maleate
Urapidil, 5-MethylTriflupromazine hydrochloride
Enalaprilat dihydrate
U-62066
CGP 57380
Theobromine
Pinacidil
SKF 75670 hydrobromide
Vancomycin hydrochloride from Streptomyces orientalis
Phenylbenzene-omega-phosphono-alpha-amino acid
Thio-NADP sodium
Betaine hydrochloride
Betaine aldehyde chloride
Amitriptyline hydrochloride
Ancitabine hydrochloride
L-azetidine-2-carboxylic acid
Chlorzoxazone
Aminoguanidine hydrochloride
L-Cysteinesulfinic Acid
Budesonide
S-(p-Azidophenacyl)glutathione
8-Bromo-cAMP sodium
Benztropine mesylate
5-(N,N-hexamethylene)amiloride
4-Chloromercuribenzoic acid
(±)-Bay K 8644
BMY 7378 dihydrochloride
Tracazolate
Cefmetazole sodium
Clofibrate
(±)-Butaclamol hydrochloride
trans-(±)-ACPD
Opipramol dihydrochloride
N6-Cyclopentyladenosine
Cantharidic Acid
M 6680
R 0529
E 4375
D 7938
T 5648
T0202
A 0231
F 4303
D 2926
N 2288
H-140
D 4526
T 7947
P 8352
T-101
X4753
T 0891
T 1505
T-200
T 1694
U 5882
U-101
T 2896
E9658
U-105
C0993
T 4500
P-154
S 2941
V 8138
P-204
T 5515
B 3501
B 3650
A 8404
A 8598
A 0760
C 4397
A 8835
C 4418
B 7777
A 1782
B 7880
B 8262
A 9561
C 5913
B-112
B-134
T-112
C 6048
C 6643
B-168
A-155
O 5889
C 8031
C 8088
Sodium&Taurocholate&hydrate
T&9034
Nisoxetine hydrochloride
Maprotiline hydrochloride
1-(5-Isoquinolinylsulfonyl)-3-methylpiperazine dihydrochloride
H-8 dihydrochloride
Naloxone benzoylhydrazone
NS-1619
Naloxonazine dihydrochloride
NBQX disodium
(-)-Naproxen sodium
2-Methyl-5-hydroxytryptamine maleate
Metolazone
N-151
M 9651
I 6391
M 9656
N-165
N-170
N-176
N-183
M 1275
M-109
M-116
(Cy53dT153NS3h(Binding(
%(Inhibition
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
35
36
36
36
36
36
36
36
36
36
36
36
(Cy53dT153NS3h(Binding(
Interference(
1.0
0.6
1.0
1.0
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
0.9
0.9
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
0.9
0.9
1.0
0.9
0.9
0.9
0.9
0.9
0.9
0.9
1.0
0.9
1.0
0.9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.9
0.9
0.9
0.9
0.9
0.9
0.9
1.0
1.0
0.9
1.0
18
Table&S2
Compound(Name(
Sigma(Cat.(No.
N-omega-Methyl-5-hydroxytryptamine oxalate salt
Oxymetazoline hydrochloride
S(+)-Ibuprofen
p-Iodoclonidine hydrochloride
Moclobemide
R(+)-IAA-94
Ofloxacin
Piceatannol
TBB
Parthenolide
CR 2249
Naltrexone hydrochloride
Kynurenic acid
Clorgyline hydrochloride
SKF-525A hydrochloride
Moxisylyte hydrochloride
Gabapentin
ABT-702 dihydrochloride
Guanidinyl-naltrindole di-trifluoroacetate
Raloxifene hydrochloride
Phentolamine mesylate
Spiroxatrine
Trifluoperazine dihydrochloride
SDZ-205,557 hydrochloride
Ribavirin
Tropicamide
Pergolide methanesulfonate
FAUC 213
(±)-Thalidomide
SR 57227A
(±)-Chlorpheniramine maleate
ABT-418 hydrochloride
Betaxolol hydrochloride
9-cyclopentyladenine
(+)-Chlorpheniramine maleate
Apomorphine hydrochloride hemihydrate
CHM-1 hydrate
Idazoxan hydrochloride
Imipramine hydrochloride
Linopirdine
alpha-Methyl-5-hydroxytryptamine maleate
Sodium Oxamate
MDL 26,630 trihydrochloride
Palmitoylethanolamide
Ranolazine dihydrochloride
3-Tropanyl-3,5-dichlorobenzoate
Rolipram
L-741,626
L-703,606 oxalate salt hydrate
R(-)-N6-(2-Phenylisopropyl)adenosine
( R)-(+)-WIN 55,212-2 mesylate
8-(4-Chlorophenylthio)-cAMP sodium
Amsacrine hydrochloride
Promazine hydrochloride
AGK2
Ruthenium red
M 1514
O 2378
I-106
I-114
M3071
I-117
O 8757
P 0453
T 0826
P 0667
N 2034
N 3136
K 3375
M 3778
P 1061
M 5154
G-154
A2721
G 3416
R 1402
P 7561
S-103
T 8516
S-174
R 9644
T 9778
P 8828
F 4429
T-144
S 1688
C 3025
A 6476
B 5683
C 4479
C 4915
A 4393
C1244
I 6138
I 7379
L-134
M-110
O 2751
M-166
P 0359
R 6152
T 8160
R 6520
L-135
L119
P 4532
W-102
C 3912
A 9809
P 6656
A 8231
R 2751
(Cy53dT153NS3h(Binding(
%(Inhibition
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
36
37
37
37
37
37
37
37
37
37
37
37
38
38
38
38
38
38
39
312
398
(Cy53dT153NS3h(Binding(
Interference(
0.9
0.9
0.9
0.9
0.9
1.0
0.9
0.9
1.0
1.0
1.0
1.0
1.0
1.0
0.9
1.0
1.0
0.9
0.9
0.9
0.9
0.9
0.9
1.0
0.9
0.9
1.0
0.9
1.0
1.0
0.9
1.0
0.9
0.9
0.9
1.0
0.9
0.9
1.0
0.9
0.9
0.9
0.9
1.0
0.9
0.9
1.0
0.9
0.9
1.0
1.0
0.9
0.9
0.9
0.8
0.0
19
Table&S3
Structure
CID*No.
DNA*Unwinding* *Cy5;dT15;SSB*Binding*
IC50,*µM
IC50,*µM
50930730
3&±&1
201&±&119
46839370
5&±&1
179&±&239
50930756
22&±&2
173&±&354
50930737
4&±&1
128&±&57
50930751
9&±&3
71&±&13
53377551
81&±&22
57&±&13
53239937
4&±&0
57&±&16
50930734
52&±&20
55&±&20
49849302
3&±&0
46&±&52
1
Table&S3
Structure
CID*No.
DNA*Unwinding* *Cy5;dT15;SSB*Binding*
IC50,*µM
IC50,*µM
50930749
22&±&4
36&±&7
49849299
8&±&1
32&±&7
49849293
4&±&0
28&±&9
50930740
5&±&4
27&±&6
53377547
12&±&4
27&±&5
53377546
29&±&7
23&±&5
53308658
10&±&2
23&±&6
49849293
3&±&1
22&±&4
53377548
44&±&12
20&±&4
2
Table&S3
Structure
CID*No.
DNA*Unwinding* *Cy5;dT15;SSB*Binding*
IC50,*µM
IC50,*µM
44251433
122&±&5
19&±&8
53377553
29&±&4
17&±&5
53255450
227&±&107
17&±&4
53255447
5&±&3
15&±&3
50930733
19&±&15
14&±&2
49849294
5&±&1
14&±&4
50930745
17&±&6
13&±&1
50930748
14&±&1
13&±&2
44251427
70&±&31
13&±&4
3
Table&S3
Structure
CID*No.
DNA*Unwinding* *Cy5;dT15;SSB*Binding*
IC50,*µM
IC50,*µM
50930741
6&±&2
12&±&8
53356770
8&±&2
11&±&2
49849298
5&±&1
10&±&3
50930743
17&±&17
10&±&1
49849295
24&±&2
10&±&2
53308659
5&±&0
9&±&2
52914816
14&±&0
9&±&2
44251428
95&±&14
9&±&1
53356771
9&±&4
7&±&1
4
Table&S3
Structure
CID*No.
DNA*Unwinding* *Cy5;dT15;SSB*Binding*
IC50,*µM
IC50,*µM
53377543
3&±&0
7&±&3
44251429
33&±&24
7&±&2
53356772
4&±&1
6&±&1
49849287
4&±&2
5&±&1
53312458
11&±&2
5&±&1
415713
12&±&1
5&±&1
53356653
4&±&1
5&±&1
49849286
3&±&0
5&±&2
49849284
11&±&7
4&±&1
5
Table&S3
Structure
CID*No.
DNA*Unwinding* *Cy5;dT15;SSB*Binding*
IC50,*µM
IC50,*µM
Primulin
10&±&5
4&±&1
49849280
11&±&2
4&±&1
53356656
5&±&2
4&±&1
49849276
2&±&0
4&±&2
49849289
6&±&2
3&±&0
49849300
10&±&2
3&±&1
49849282
10&±&3
2&±&1
44251428
6&±&2
2&±&1
44251431
5&±&2
2&±&0
6
Table&S3
Structure
CID*No.
DNA*Unwinding* *Cy5;dT15;SSB*Binding*
IC50,*µM
IC50,*µM
49849290
10&±&5
2&±&0
44251434
7&±&4
1&±&0
7