Identification and Analysis of Hepatitis C Virus NS3 Helicase Inhibitors Using Nucleic Acid Binding Assays Sourav Mukherjee1, Alicia M

Home , Ciproxifan, Dihydroxyphenylglycine, Fenobam, MPPF, Propylnorapomorphine, Quazinone

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,*

1Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, 2University 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

Received March 26, 2012; Revised May 30, 2012; Accepted June 4, 2012 Downloaded from

ABSTRACT INTRODUCTION Typical assays used to discover and analyze small All cells and viruses need helicases to read, replicate and molecules that inhibit the hepatitis C virus (HCV) repair their genomes. Cellular organisms encode

NS3 helicase yield few hits and are often con- numerous specialized helicases that unwind DNA, RNA http://nar.oxfordjournals.org/ founded by compound interference. Oligonucleotide or displace nucleic acid binding proteins in reactions binding assays are examined here as an alternative. fuelled by ATP hydrolysis. Small molecules that inhibit After comparing fluorescence polarization (FP), helicases would therefore be valuable as molecular homogeneous time-resolved fluorescence (HTRFÕ; probes to understand the biological role of a particular Cisbio) and AlphaScreenÕ (Perkin Elmer) assays, helicase, or as antibiotic or antiviral drugs (1,2). For an FP-based assay was chosen to screen Sigma’s example, several compounds that inhibit a helicase Library of Pharmacologically Active Compounds encoded by herpes simplex virus (HSV) are potent drugs in animal models (3,4). Despite this clear need, relatively (LOPAC) for compounds that inhibit NS3-DNA by guest on September 14, 2013 few speciﬁc helicase inhibitors have been reported, and the complex formation. Four LOPAC compounds in- mechanisms through which the most potent compounds hibited the FP-based assay: aurintricarboxylic acid exert their action are still not clear. Although HSV (ATA) (IC50 = 1.4 kM), suramin sodium salt (IC50 = helicase inhibitors have progressed furthest in pre-clinical 3.6 kM), NF 023 hydrate (IC50 = 6.2 kM) and tyrphostin trials (5), the viral helicase that has been most widely AG 538 (IC50 = 3.6 kM). All but AG 538 inhibited studied as a drug target is the one encoded by the hepatitis helicase-catalyzed strand separation, and all but C virus (HCV). The uniquely promiscuous HCV helicase NF 023 inhibited replication of subgenomic HCV rep- unwinds duplex DNA and RNA in a reaction fuelled by licons. A counterscreen using Escherichia coli virtually any nucleoside triphosphate (6). The ability of single-stranded DNA binding protein (SSB) revealed HCV helicase to act on DNA is particularly intriguing that none of the new HCV helicase inhibitors were because the HCV genome and replication cycle are specific for NS3h. However, when the SSB-based entirely RNA-based. There is no convincing evidence that HCV helicase ever encounters DNA in host cells. assay was used to analyze derivatives of another Compounds that disrupt the interaction of the helicase non-specific helicase inhibitor, the main component and DNA, therefore, would be useful to understand why of the dye primuline, it revealed that some primuline an RNA virus encodes a helicase that acts on DNA. They derivatives (e.g. PubChem CID50930730) are up to also might be useful antivirals because HCV needs a func- 30-fold more specific for HCV NS3h than similarly tional helicase to replicate in cells (7) and helicase inhibi- potent HCV helicase inhibitors. tors 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.

ß 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. 8608 Nucleic Acids Research, 2012, Vol. 40, No. 17

The HCV helicase resides in the C-terminal two-thirds of both DNA and RNA is intriguing because other similar the viral multifunctional non-structural protein 3 (NS3), enzymes typically prefer DNA or RNA and because NS3 which is also a protease. The NS3 protease and helicase likely never encounters DNA. The RNA virus replicates in are covalently associated during HCV replication for the cytoplasm and has no DNA stage in its replication unknown reasons. HCV and related viruses encode the cycle. Numerous NS3 crystal structures show how the only proteins known that are both proteases and helicases. protein binds DNA (26–28) or RNA (29), both in the Recombinant DNA technology can be used to separate the absence or presence of ATP analogs (28,29). These struc- two NS3 functional domains, and express the proteins sep- tural studies reveal that amino acid side chains in the arately in Escherichia coli or other model organisms. Both NS3h nucleic acid binding cleft do not directly contact mono-functional, recombinant, truncated NS3 proteins the 20-hydroxyl of RNA, explaining the enzyme’s (called NS3p and NS3h) retain their activities in vitro. unusual promiscuity, and justifying the use of DNA oligo- Although NS3h retains a helicase function, its ability to nucleotides as surrogates for RNA to probe the enzyme’s unwind RNA is somewhat diminished (9). The NS3 functions. HCV helicase binds nucleic acids with low helicase was one of the first HCV enzymes to be charac- nanomolar affinity (30,31) and NS3h preferentially inter- terized, and crystal structures of NS3h were first solved in acts with polypyrimidine tracts like those found in the 30 the mid 1990s (10,11). However, helicase inhibitor develop- untranslated region of the virus genome (10,32). While it is ment has been far slower than it has been for other HCV clear that one strand of DNA (or RNA) binds in a cleft

drug targets (2,12). To date, only a few classes of helicase separating the two conserved helicase motor domains Downloaded from inhibitors have been reported to slow HCV RNA replica- from a third helical domain, it is not clear where else on tion in cells. HCV helicase inhibitors reported to act as anti- the protein nucleic acids might bind. Based on modeling virals include nucleoside mimics (13), triphenylmethanes studies, some groups have suggested that RNA might bind (14), acridones (8,15), amidinoanthracyclines (16), tropo- in the positively charged cleft separating the protease from lones (17), symmetrical benzimidazoles (18–20) and pri- the helicase (12,33), and more recent evidence suggests the muline derivatives (21). protease region binds certain sequences in the internal http://nar.oxfordjournals.org/ One reason that so few molecular probes targeting HCV ribosome entry site of the HCV RNA genome (34). helicase are available is because high throughput screens We show here how DNA binding assays can be used for helicase inhibitors yield few hits. For example, a sen- to identify new helicase inhibitors and how DNA binding sitive molecular beacon-based helicase assay (MBHA) (22) assays with unrelated proteins can be used to screen a has been used to screen 290 735 compounds in the library of helicase inhibitors for speciﬁc compounds. A NIH Molecular Libraries Small Molecule Repository truncated NS3 lacking the protease domain (i.e. NS3h) by the Scripps Research Institute Molecular Screening is used because it is still unclear exactly how the

Center (PubChem Project: http://pubchem.ncbi.nlm.nih. protease region affects NS3–RNA interactions, and by guest on September 14, 2013 gov/assay/assay.cgi?aid=1800, 14 June 2011, date last DNA is used here instead of more costly RNA, because accessed), and only 500 compounds (0.2%) were con- the nucleic acid-binding site on NS3h does not differenti- firmed as hits upon retesting (PubChem Project: http:// ate between the DNA and RNA. First, we compare pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=1943, 14 various DNA binding assays for their screening utility. June 2011, date last accessed). The most potent hits in Next, we use a fluorescence-polarization (FP)-based the NIH screen did not, however, directly inhibit binding assay to identify three new HCV helicase helicase action, but instead they interfered with the assay inhibitors. Binding assays with the unrelated E. coli (PubChem Project: http://pubchem.ncbi.nlm.nih.gov/ single-stranded DNA binding protein (SSB) are then assay/assay.cgi?aid=485301, 14 June 2011, date last used to reveal that the new compounds, like helicase in- accessed). They also did not inhibit HCV replication in hibitors discovered in a prior screen of the NCI cells (PubChem Project: http://pubchem.ncbi.nlm.nih. Mechanistic Set (21), are not specific for HCV helicase. gov/assay/assay.cgi?aid=463235, 14 June 2011, date last In the final part of this study, we use a library of com- accessed) (23). pounds derived from a scaffold identified in the prior Screens for helicase inhibitors typically rely on assays screen (21) to show that binding assays can be used to that monitor either helicase catalyzed strand separation or differentiate specific inhibitors from non-specific HCV ATP hydrolysis. Both assays are relatively complex, and helicase inhibitors. 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 MATERIALS AND METHODS that the protein conformational changes that take place in Materials these assays, or some other unknown factor, obfuscates inhibitor identification in large screens. Here, we test DNA oligonucleotides were obtained from Integrated whether simpler DNA-binding assays might be more DNA Technologies (Coralville, IA). HCV NS3h was useful for HCV helicase inhibitor discovery. expressed and purified as described (6). Helicase sub- Hepatitis C virus helicase binds single stranded DNA strates were prepared by combining DNA oligonucleo- and RNA with similar high affinities in the absence of tides (Integrated DNA Technologies, Coralville, IA) at a ATP. When ATP is present, it fuels helicase movements 1:1 molar ratio to a concentration of 20 mM in 10 mM and the subsequent separation of both DNA and RNA Tris–HCl pH 8.5, placing in 95C water, and allowing duplexes (25). The ability of HCV helicase to separate them to cool to room temperature for 1 h. The partially Nucleic Acids Research, 2012, Vol. 40, No. 17 8609

duplex helicase substrates possessing a 30 ssDNA tail Library of Pharmaceutically Active Compounds were then purified of free oligonucleotides by mixing (LOPAC) were screened at 100 mM. DNA 6:1 with 6X loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 40% sucrose) and sepa- Homogeneous time resolved fluorescence (HTRFÕ) assay rating with 20% non-denaturing PAGE at a constant Assays were performed in 20.2 ml in 384-well, flat-bottom, 200 V for 1 h. small volume, white microplates (Greiner Bio-One, catalog #784075). The procedure was the same as that Electrophoretic mobility shift assay for the FP assay except that 15 ml of the reaction Binding assays containing 50 mM Tris, pH 7.4, 10% mixtures (7.5 nM Cy5-dT15 DNA, 50 nM NS3h, 25 mM glycerol, 100 nM DNA substrate (50-Cy5-CC TAC GCC Tris, pH 7.5, 1.25 mM MgCl2, 0.05 mg/ml BSA, 0.1% ACC AGC TCC GTA GG–30 annealed to 50-GGA GCT (v/v) Tween20, and 0.5 mM DTT) was first dispensed in GGT GGC GTA GG (T)20-30) and 650 nM NS3h were each well before the addition of 0.2 ml of dT20 or H2O. incubated 20 min on ice. Following addition of indicated After addition, 5 ml of a (1:50 dilution) of Lumi4Õ-Tb concentrations of thioflavine S, the binding reactions were Cryptate-conjugated anti-6 Histidine mouse monoclonal incubated another 20 min on ice. A BioRad precast 15% antibody (catalog #61HISTLA, CISBIO US) was added polyacrylamide Tris/Borate/EDTA gel was pre-run at 4C and the plate was incubated for 60 min at 4C. TR-FRET for 30 min at 120 V. Four microliters of each sample was was monitored with a Fluostar Omega multimodal plate loaded onto the gel. The gel was run 1 min at 200 V to reader (BMG Labtech, Inc.) by excitation of the donor Downloaded from allow samples to enter gel, then 40 min at 120 V. The gel fluorophore at 340 nm. TR-FRET ratio was calculated was scanned on a Molecular Dynamics Storm 860 as emission of acceptor fluorophore at 665 nm over the Phosphorimager. emission of donor fluorophore at 620 nm (gain 2300, integration time 400 ms, integration start time 60 ms, position- ing delay 0.2 s, measurement start time 0 s, number of FP-based DNA-binding assay http://nar.oxfordjournals.org/ flashes per well 200). For screening, assays were performed in a total volume of 20.2 ml in 384-well, flat-bottom, low volume, black micro- AlphaScreenÕ assay plates (Greiner Bio-One, catalog #784076). First, 20 ml of a All assays were similar to the above assays except FP-assay solution (5 nM Cy5-TTTTTTTTTTTTTTT-30 (Cy5-dT15), 15 nM NS3h, 25 mM MOPS, pH 7.5, that they contained a final concentration of 10 nM biotinylated oligonucleotide (Bio-d18, 5 -Bio-GCC TCG 1.25 mM MgCl2, 0.0025 mg/ml BSA, 0.005% (v/v) 0 Tween20 and 0.025 mM DTT) was dispensed in each CTG CCG TCG CCA-30), instead of Cy5-dT15, and they used reagents from the AlphaScreenÕ Histidine well, then 0.2 ml of dimethylsulfoxide (DMSO) or by guest on September 14, 2013 compound dissolved in DMSO was added by pin (Nickel Chelate) Detection Kit (catalog #6760619C, transfer, such that the final concentration of DMSO was Perkin Elmer). To 384-well, flat-bottom, low volume, 1% (v/v) in each assay. white microplates (Greiner Bio-One, catalog #784075), 12 ml of the reaction mixtures containing 10 nM Bio-d18, For confirmation and IC50 value determination, assays were performed in half area 96-well microplates (Corning 20 nM NS3h, 25 mM HEPES, pH 7.5, 100 mM NaCl and Life Sciences, catalog #3694). First, 47.5 ml of a FP-assay 1.0 mg/ml BSA was dispensed, followed by 0.2 ml of dT20 solution was dispensed in each well, then 2.5 ml of DMSO or H2O and 4 ml Anti-His alpha screen donor beads. After or compound dissolved in DMSO was added, such that incubation for 30 min at 23C, 4 ml of streptavidin- the final concentrations in each assay were 5 nM Cy5- acceptor beads was added, and the assays incubated dT15, 15 nM NS3h, 25 mM MOPS, pH 7.5, 1.25 mM another 60 min. All work with the alpha reagents was performed under green filtered light conditions (<100 Lux). MgCl2, 0.0025 mg/ml BSA, 0.005% (v/v) Tween20, 0.025 mM DTT and 5% DMSO (v/v). Alpha counts were measured at 520–620 nm (Ex. 680 nm, Polarization was monitored with a TECAN Infinite 20 nm bandwidth) in a Fluostar Omega multimodal plate M1000 PRO multi-mode microplate reader by exciting reader (BMG Labtech, Inc.). at 635 nm (5 nm bandwidth) and measuring total fluorescence intensity, parallel and perpendicular polarized light NS3h MBHAs at 667 nm (20 nm bandwidth). G-factors were calculated The ability of compounds to inhibit helicase action was from wells with Cy5-dT15 alone. Inhibition (%) was monitored using molecular beacons as described previously calculated by normalizing data to values obtained with (19,22). Assays contained 25 mM MOPS, 1.25 mM MgCl2, positive controls (200 nM dT20 or 100 mM primuline) 5% DMSO, 5 mg/ml BSA, 0.01% (v/v) Tween20, 0.05 mM and negative controls (DMSO only). Assay interference DTT, 5 nM substrate, 12.5 nM NS3h and 1 mM ATP. The was calculated by dividing fluorescence intensity of a partially duplex DNA substrates used in MBHAs consisted compound-containing assay (Fc) by the average fluores- of a 45-mer bottom strand 50-GCT CCC CGT TCA TCG cence intensity of the negative controls (F( )). Similar ATT GGG GAG C(T)20-30 and the 25-mer HCV top results were obtained with both assay formatsÀ as long as strand 5-/5Cy5/GCT CCC CAA TCG ATG AAC GGG DMSO concentrations remained below 5%. GAG C/3IAbRQSp/-3. The 3-stranded RNA substrate Compounds in the HCV helicase inhibitor library were used was made of two RNA strands, a 60 nucleotide either purchased (Sigma, St. Louis, MO) or synthesized as long bottom strand 50-rGrGrA rGrCrU rGrGrU rGrGrC described (21) and screened at 20 mM. Samples in Sigma’s rGrUrA rGrGrC rArArG rArGrU rGrCrC rUrUrG 8610 Nucleic Acids Research, 2012, Vol. 40, No. 17 rArCrG rArUrA rCrArG rCrUrU rUrUrU rUrUrU system (Promega, Madison, WI) in 96-well black rUrUrU rUrUrU rUrUrU rUrUrU-30, a 24 nucleotide microplates (Thermo Scientific, catalog #9502867) read long top strand 50-rArGrU rGrCrG rCrUrG rUrArU on a FLUOstar Omega multi-mode microplate reader rCrGrU rCrArA rGrGrC rArCrU-Cy5, and a (BMG Labtech, Inc.). Relative percent inhibition was third DNA top strand with the sequence/5IAbRQ/CCT calculated by normalizing values to those obtained with ACG CCA CCA GCT CCG TAG G-3. In the screen cells treated with DMSO only. in Figure 5, percent inhibition was calculated with equation (1) and interference with equation (2). Cell viability assay To assess compound toxicity towards Huh-7.5 cells, cells Inhibition % Fc0=Fc30 F =F = ð Þ ¼ ðð ÞÀð ðÀÞ0 ðÀÞ30Þ 1 were plated and treated as above and viability was 1 F 0=F 30 100 ð Þ assessed using the Cell Titer-Glo luminescent cell viability ð Àð ðÞÀ ðÞÀ ÞÞÞ Â kit (Promega) following the manufacturer’s instructions. Briefly, at the end of a 72 h incubation period, the medium Interference ratio Fc0=F 0 2 ðÞ¼ð ðÀÞ ÞðÞ was removed and the cells were washed with growth medium, then an equal volume of growth medium In equations (1) and (2), Fc0 is the fluorescence of the and Cell Titer-Glo reagent was added and the lysis was reactions containing the test compound before adding initiated by mixing on an orbital shaker. The plate Downloaded from ATP, Fc30 is the fluorescence of the test compound was incubated at 23C for 30 min and luciferase activity reaction 30 min after adding ATP. F( )0 is the average was measured for 1 s using a FLUOstar Omega microplate À of three DMSO-only negative control reactions before reader (BMG Labtech, Inc.) in black 96-well microplates adding ATP and F( )30 is the average of three (Thermo Scientific, catalog #9502867). Relative viability À DMSO-only reactions 30 min after adding ATP. was calculated by normalizing the values to those obtained

To monitor helicase reaction kinetics and to calculate with cells treated with DMSO only. http://nar.oxfordjournals.org/ IC50 values, assays were performed in a volume of 60 ml in white half-area 96-well plates (Corning Lifesciences, Escherichia coli SSB assay catalog #3693) and measured in a Fluostar Omega multimodal plate reader (BMG Labtech, Inc.) using the 640 nm The procedure for screening with this assay was the same excitation wavelength and 680 emission wavelength filters. as that for the FP-based DNA binding assay carried out in Reactions were performed by first incubating all compo- 384-well plates except that E. coli SSB (Promega) was used nents except for ATP for 2 min, then initiated by injecting at 20 nM instead of the HCV helicase. For IC50 determin- in 1/10 volume of ATP such that the final concentration of ation, assays were performed with 60 ml total volume in all components was as noted above. Initial reaction black flat bottomed 384-well microplates (Corning catalog by guest on September 14, 2013 velocities were calculated by fitting a first order decay #3573). First, 3.0 ml of DMSO or compound dissolved in equation to data obtained after ATP addition and DMSO was added, such that the final concentration of calculating an initial velocity from the resulting amplitude DMSO was 5% (v/v) in each assay. Then 57 ml of a and rate constant. The concentration at which a FP-assay solution (5 nM Cy5-dT15, 20 nM SSB, 25 mM compound causes a 50% reduction in reaction velocity MOPS, pH 7.5, 1.25 mM MgCl2, 0.0025 mg/ml BSA, (IC50) was calculated using GraphPad Prism (v. 5). 0.005% (v/v) Tween20 and 0.025 mM DTT) was dispensed in each well. Polarization was monitored as described HCV replicon assays above. The ability of compounds to inhibit HCV replication was judged using an HCV Renilla luciferase (HCV RLuc) RESULTS reporter construct that was a generous gift from Seng-Lai Tan (35). Plasmid DNA expressing the Belon and Frick (36) previously reported that thioflavine S replicon was transcribed, and the subsequently purified was an HCV helicase inhibitor, and thioflavine S was used RNA was used to prepare stably transfected Huh7.5 as a positive control for the screen of the NIH Molecular cells by prolonged selection with G418. To test com- Libraries Small Molecule Repository (PubChem BioAssay pounds, HCV RLuc replicon containing cells were AID #1800) and in other studies (37). While studying seeded at a density of 10 000 cells per well in 96-well the mechanism of action of thioflavine S (Direct Yellow plates and incubated for 4–5 h to allow the cells to 7, Sigma Cat. #T1892) and the related yellow dyes attach to the plate in 100 ml of DMEM supplemented primuline (Direct Yellow 59, MP Biomedicals Cat. with 10% fetal bovine serum (HyClone), 2 mM L-glutam- #195454) and titan yellow (Direct Yellow 9/Thiazole ine, 100 U/ml penicillin, 100 mg/ml streptomycin and 1x Yellow G, Sigma Cat. #88390) (38), we observed that non-essential amino-acids (Invitrogen). To each well, they prevent NS3h from binding its nucleic acid substrate 0.5 ml of compounds dissolved in DMSO were added (Figure 1A). In the absence of one of these dyes, NS3h such that the DMSO final concentration was 0.5%, binds its substrate tightly enough that the complex will and the cells were incubated for 72 h at 37C under 5% migrate more slowly through a non-denaturing polyacryl- CO2 atmosphere. The effect of compounds on HCV amide gel. When thioflavine S was present the gel-shift replication was estimated by measuring the Renilla of the substrate decreased in a concentration-dependent luciferase activity using the Renilla luciferase assay fashion (Figure 1B). Nucleic Acids Research, 2012, Vol. 40, No. 17 8611

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. Downloaded from HCV helicase-DNA binding assays that are suitable for One possible problem with using Cy5 as a tracer in such high throughput screening a study is that it is coupled to the oligonucleotide with an To unwind a substrate, HCV helicase binds a single aliphatic linker so that the fluorophore could, in theory, be still relatively free to rotate even when DNA is bound to stranded nucleic acid tail then translocates in a 30–50 dir- ection (39) until it reaches a duplex region that it can the enzyme, a phenomenon commonly referred to as ‘the http://nar.oxfordjournals.org/ separate. The ability of NS3h to bind its substrate can propeller effect’. We therefore also tested oligonucleotides also be measured by monitoring changes in polarization labeled with fluorescent moieties not bound to aliphatic (or anisotropy) of a fluorescent helicase substrate as has linkers such as with 6-carboxyfluorescein, hexachloro- been done with related helicases (40,41). This loading step fluorescein and boron-dipyrromethene (BODIPY). can be monitored using a truncated substrate lacking Polarization studies with each of these alternatives were duplex regions, such as a 15-nucleotide long deoxythy- confounded by the fact that the fluorescence intensity of midine polymer (dT15) (Figure 2A). Previous work has each changed upon protein binding. The fact that fluorescence intensity of oligonucleotides changes when they bind

shown that NS3h binds such single stranded DNA with by guest on September 14, 2013 a high affinity and that 2-3 protomers bind such an oligo- the unusually acidic DNA binding site of NS3 has been nucleotide. A homopolymer was chosen to minimize the previously documented (9,19). While screening fluorescent possibility that the DNA would form a hairpin or other oligonucleotides, we found two other red-shifted tracers that did not change intensity upon binding, TyeTM 665 secondary structures, and Ts were chosen because NS3h TM prefers this sequence to others (10,30–32). and Alexa Fluor 647 . Identical (dT15) oligonucleotides As seen before (30,31,42), the binding of NS3 to DNA labeled on the 50-end with either Cy5, Tye665, or Alexa Fluor 647, bound NS3h with similar K s (5 ± 1, 8 ± 2 in this FP-assay was stoichiometric (Kd < 1 nM) and 0.5 about two to three molecules of NS3 were needed to and 6 ± 2, respectively), and titan yellow inhibited saturate Cy5-dT15. When 5 nM Cy5-dT15 was present, complex formation of each with a similar IC50 value increasing amounts of NS3h increased the FP of (7 ± 3, 5 ± 1 and 9 ± 4 mM, respectively). Repeated Cy5-dT15 in a concentration dependent manner such assays (n = 40) with the three different fluorescence that the amount of NS3h needed to bind half of the tracers in the presence and absence of titan yellow Cy5-dT15 (K0.5) was 9 ± 1.5 nM. Under these conditions, (100 mM) revealed similar Z0 factors (43). Assays with the signal plateaued when about three times as much Alexa Fluor and Tye 665 labeled oligonucleotides had NS3h (15 nM) was added as the amount of Cy5-dT15 the largest difference between the positive and negative (Figure 2B). The polarization of a Cy5-dT15–NS3h controls, but their assay-to-assay variability was higher, complex decreased in the presence of either unlabeled particularly in assays done in the absence of inhibitor ligand (dT20) or a yellow dye in a concentration depend- (Figure 2D). Further experiments were therefore per- ent manner (Figure 2C). The IC50 values measured for formed with the Cy5-labeled oligonucleotide. dT20 and primuline were 7 ± 2 nM and 24 ± 3 mM, re- After optimizing conditions, FP-based binding assays spectively. Thioflavine S decreased polarization less effect- were then performed in a high throughput format to ively than primuline with an IC50 of 35 ± 3.5 mM, and judge necessary precision and reproducibility. To judge titan yellow was 10 times more potent than either with well-to-well variation, 48 negative controls (DMSO only) an IC50 value of 2.8 ± 0.2 mM (Table 1). and 48 positive controls (100 mM primuline) were per- A Cy5-labeled oligonucleotide was chosen mainly formed. The coefficient of variation was 2.2% for the because its fluorescence intensity did not change upon negative controls and 5.8% for the positive controls, re- protein binding, and because it absorbs and emits light sulting in a Z0 factor of 0.81 (Figure 2E). Similar Z0 in the far-red visible range, where it would be less likely factors, and concentration response curves were to interact with compounds in large chemical libraries. obtained when plates were compared (Figure 2F) or 8612 Nucleic Acids Research, 2012, Vol. 40, No. 17

A 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 BC 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 (HTRFÕ) 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 Lumi4Õ-Tb Cryptate-conjugated anti-6 Histidine Downloaded from D mouse monoclonal antibody (catalog #61HISTLA, Cisbio), an Eu3+ Cryptate-conjugated mouse anti-6 Histidine monoclonal antibody (catalog #61HISKLA, Cisbio) and a LANCEÕ Europium Anti-6X Histidine antibody (catalog #AD0110, Perkin Elmer). The highest 3+ http://nar.oxfordjournals.org/ Z0 factors were obtained with the Tb 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 HTRFÕ assay setup, the ratio of signals from the donor and acceptor is multiplied by 10 000 to estimate E 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 by guest on September 14, 2013 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 F G Figure 2. Continued parameter, variable slope) with dotted lines showing the 95% confi- dence 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. H 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 standardÀ 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 Figure 2. Fluorescence polarization (FP)-based assay to monitor the values observed in duplicate assays at 20 mM of samples in an HCV interaction of HCV helicase and a deoxythymidine polymer. (A) FP helicase inhibitor library (Table S1). Data were fitted to a straight line based assay to monitor NS3h binding to Cy5-dT15. (B) Fluorescence through zero (slope = 0.97, R2 = 0.99). The dotted lines show values polarization of Cy5-dT15 (5 nM) at different concentrations of NS3h. representing 0% and 100% inhibition, as determined from negative Data (n = 4) were fitted to a concentration-response equation (four controls (DMSO only) and positive control (100 mM primuline). 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

Compound DNA binding assaysa NS3h helicase assaysb Cellular assays

c c NS3h IC50 SSB IC50 DNA IC50 RNA IC50 HCV replicon IC50 Viability CC50 (mM) ± SD (mM) ± SD (mM) ± SD (mM) ± SD (mM) ± SD (mM) ± SD

Thioﬂavine S 35 ± 3.5 12 ± 0.9 24 ± 1.3 22 ± 6.3 >100 >100 Primuline 24 ± 3 5.0 ± 1.4 12 ± 1.3 15 ± 2.3 >100 >100 Titan yellow 2.8 ± 0.2 2.9 ± 0.2 12 ± 3.6 30 ± 3.6 >100 >100 ATA 1.4 ± 0.1 1.0 ± 0.1 0.6 ± 0.1 0.8 ± 0.2 98 ± 30 >100 AG 538 3.6 ± 0.2 1.2 ± 0.1 >100 >100 18 ± 3.2 60 ± 13 NF 023 6.3 ± 0.6 1.7 ± 0.2 2.6 ± 0.4 5.1 ± 1.2 >25d >25d Suramin 3.6 ± 0.3 0.3 ± 0.0 3.7 ± 0.7 8.9 ± 2.0 38 ± 9.3 e >50e 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. Downloaded from e Average (±SD) IC50 value from three sets of assays performed with a 8 point 2-fold dilution series starting at 50 mM.

A B C D http://nar.oxfordjournals.org/ by guest on September 14, 2013

Figure 3. HTRFÕ and AlphaScreenÕ assays that detect NS3h interactions with DNA. (A) TR-FRET assay using the Lumi4Õ-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 AlphaScreenÕ Histidine (Nickel Chelate) Detection Kit (Perkin Elmer) reagents to monitor NS3h binding to a biotinylated oligonucleotide. (D) AlphaScreenÕ 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).

Õ unlabeled oligonucleotide, dT20 (200 nM) (Figure 3B). AlphaScreen signal (Figure 3D). The signal/background Compounds that disrupt the Cy5-dT15-NS3h complex in this AlphaScreenÕ was better than was seen in other also decreased the TR-FRET signal in a concentration- assays, with the complex yielding 80 - to 100-fold dependent fashion with IC50 values similar to those increased counts, but assay variability was higher than determined with the FP-based assay (data not shown), with the FP assay, leading to a Z0 factor of 0.62, which but the Z0-factor (0.59) for the TR-FRET assay was less was again less than what was observed in the FP-based than what was observed with FP-based assay (Figure 3B). assay. The second assay compared to the FP-based assay was based on the AlphaScreenÕ Histidine (Nickel Chelate) Compound interference and advantage Detection Kit (catalog #6760619C, Perkin Elmer). This over unwinding assays AlphaScreenÕ assay monitored the binding of NS3h to Most of the compounds in the above helicase inhibitor DNA, using donor beads containing Ni2+ ions that library were previously shown to inhibit NS3h when interact with the C-terminal His-tag of NS3h and screened using an MBHA (21). The MBHA (22) streptavidin-bound acceptor beads binding to biotinylated monitors the ability of a helicase to remove a molecular oligonucleotide (Bio-d18) (Figure 3C). Formation of an beacon (44) bound to a complementary strand upon ATP NS3h–DNA complex brings the donor and acceptor addition (Figure 4A). In the absence of inhibitors, ﬂuor- close enough that singlet oxygen can be transferred from escence decreases in the MBHA upon ATP addition, but the donor to acceptor beads. Compounds that disrupt the when an inhibitor (e.g. titan yellow) is present, ﬂuores- Bio-d18–NS3h complex (e.g. dT20) decrease the cence decreases at a slower rate (Figure 4B). The main 8614 Nucleic Acids Research, 2012, Vol. 40, No. 17 Downloaded from http://nar.oxfordjournals.org/ by guest on September 14, 2013

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 problem with using the MBHA, or similar unwinding library samples. To identify compounds that decreased assays to screen for helicase inhibitors, is that it is difficult FP by quenching of the fluorescence signal or intrinsic to distinguish helicase inhibitors from compounds that fluorescence, the fluorescence intensity of each compound simply interfere with the assay by binding DNA, such as (I) was divided by the fluorescence intensity observed for thiazole orange (45) (Figure 4C). In screens, the ability of negative controls (I( )). A plot of this data (I/I( )) revealed a compound to inhibit the MBHA is measured by that four hits increasedÀ or decreased the fluorescenceÀ in- comparing the fluorescence of the MBHA substrate tensity by more than 20% in comparison with the negative before and after ATP addition. If a compound simply controls. Four of the compounds with similar fluorescence reduces fluorescence of the Cy5 substrate before ATP is intensity to that of the negative controls decreased FP by added, as seen with concentrations of thiazole orange more than 60% at a concentration of 100 mM(Figure5B). >12.5 mM(Figure4C),itmightfalselyappeartoinhibit Each of these compounds [ATA (Sigma Cat. #A1895), the helicase. A simple method to identify true helicase suramin sodium salt (Sigma Cat. #S2671), NF 023 inhibitors is to plot percent inhibition vs. compound inter- hydrate (Sigma Cat. #N8652) and tyrphostin AG 538 ference, which is calculated by dividing assay fluorescence (Sigma Cat. #T7822)] decreased polarization in a before ATP addition (F0) by the fluorescence seen in concentration-dependent manner (Figure 5C–F). The negative control reactions (F0( )), which lack any inhibi- IC50 values with the four best inhibitors were similar to tory compounds (21). To compareÀ the results of a DNA those seen with titan yellow and lower than those seen binding assay with the MBHA, the same library of known with primuline and thioflavine S (Table 1). Downloaded from HCV helicase inhibitors, which was screened with the To test if compounds that decrease FP of the FP-based binding assay (Figure 4D), was re-screened Cy5-dT15–NS3h complex inhibit the ability of NS3h to with the MBHA at the same compound concentration unwind DNA, various concentrations of each of the com- (20 mM) (Figure 4E). When each screen was analyzed for pounds above were added to MBHAs that monitor either both percent inhibition and compound interference, it was DNA or RNA unwinding. Three of the four compounds clear that fewer compounds interfered with the FP-based identified in the LOPAC screen inhibited DNA-based http://nar.oxfordjournals.org/ binding assay (Figure 4D) than with the MBHA MBHAs (Figure 6B). Those that inhibited the ability of (Figure 4E). For full results, see Table S1 (Supplementary NS3h to unwind DNA also inhibited its activity on an Data). The fact that fewer compounds interfered with RNA substrate (Table 1). the FP-based assay suggests that many library samples To determine if compounds that interact with the did not decrease fluorescence by simply quenching Cy5 NS3h–DNA complex might also function as antiviral fluorescence. agents, their ability to inhibit the replication of an HCV Another way that a compound might decrease the fluor- subgenomic replicon was tested (46). HCV RNA replica- escence of the MBHA substrate would be to distort the tion was measured using a reporter system in which a by guest on September 14, 2013 duplex region such that the quenching moiety of the Renilla luciferase gene was fused to the 50-end of the beacon is more likely to interact with the Cy5 fluorophore. neomycin phosphotransferase gene needed for replicon se- If that were the case, then most DNA binding compounds lection (Figure 6C), so that the cellular levels of Renilla should appear to inhibit the MBHA but not the FP-based luciferase correlated directly with the amount of HCV binding assay, which lacks a duplex region. To test this RNA present in cells (35). After replicon transfection hypothesis, the average percent inhibition observed with and selection, cells were treated in parallel in two sets of each compound in FP-based binding assays was compared triplicates. One set of cells was used for Renilla luciferase with the average percent inhibition seen in the MBHA assays and the other set was used to determine cell viabil- unwinding assay. Such a plot reveals that most of the ity using a firefly luciferase-based assay. Three of the four known DNA binding compounds in our HCV helicase compounds identified in the LOPAC screen inhibited inhibitor library (e.g. berenil, proflavin, netropsin and replicon luciferase (Figure 6D), and all four compounds SYBR green I) inhibit the MBHA but not the FP-based showed little sign of toxicity at a concentration of 25 mM binding assay (Figure 4F). In contrast, compounds, which (Figure 6E). Selectivity was estimated by comparing the act mainly by inhibiting the ability of NS3h to bind DNA, potency with which the compound inhibits the replicon to such as those derived from primuline (21), inhibit both the its toxicity. By this measure, AG 538 was the most select- FP-binding assay and MBHA with a similar potency ive because three times more of this compound was needed (Figure 4G). 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 The specificity of the new HCV helicase inhibitors was new HCV helicase inhibitors, the above FP-based assay examined using a counterscreen in which NS3h was was used to screen Sigma’s 1280-compound LOPAC. All substituted with the unrelated E. coli SSB (Figure 7A). LOPAC compounds were screened at 100 mM in 384-well Like NS3h, SSB increased the FP of Cy5-dT15, but plates, each containing positive (primuline or dT ) and more ( 20 nM) SSB was needed to saturate the oligo- 20 negative (DMSO or buffer only) controls (Figure 5A). Out nucleotide with a K0.5 of 9.6 ± 2 nM in the presence of of the 1280 samples screened (Table S2), 18 compounds 5 nM Cy5-dT15 (Figure 7B). All the compounds that in- exhibited FP signals significantly different from other hibited the Cy5-dT15–NS3h interaction also inhibited the 8616 Nucleic Acids Research, 2012, Vol. 40, No. 17

A 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 B 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 Downloaded from 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 relation- http://nar.oxfordjournals.org/ C D ship 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). by guest on September 14, 2013

DISCUSSION E F 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 Figure 5. Identification of inhibitors of Cy5-dT15-NS3h complex for- assays using unrelated protein SSB were shown to be mation in a screen of the Sigma LOPAC 1280TM.(A) Summary of useful for judging compound specificity, as was demon- screening results of a fluorescence polarization assay to identify inhibi- strated both with the newly identified helicase inhibitors tors of the Cy5-dT15-NS3h interaction among the 1280 samples in Sigma’s LOPAC (+). Positive controls contained primuline (squares) and with a panel of compounds created in a prior SAR or dT20 (circles) and negative controls contained DMSO only (not study of the primuline scaffold (21). 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 Figure 5. Continued above three standard deviation limit are plotted against the complex in the presence of increasing concentrations of (C) ATA, compound interference, defined as fluorescence intensity divided by (D) AG 538, (E) NF 023, or (F) Suramin. Data (n = 3) were fitted to the average fluorescence intensity of the negative control samples. 4-parameter concentration response curves constrained to values The vertical dotted lines denote defined boundaries of tolerance for obtained in the absence of inhibitor (top) and the absence of NS3h either possible quenching (I = 0.8) or possible intrinsic fluorescence (bottom) with parameters in Table 1. In (C)–(F), concentration (I = 1.2). The horizontal dotted line denotes arbitrary cut-off criterion response curve for titan yellow is shown for comparison (dotted of 60% inhibition. Fluorescence polarization of a Cy5-dT15-NS3h lines). Raw data for all LOPAC samples can be found in Table S2. Nucleic Acids Research, 2012, Vol. 40, No. 17 8617

A B

DE Downloaded from http://nar.oxfordjournals.org/

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. by guest on September 14, 2013

ABC

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 and cost effective. FP-based assays have been used before monitored here with four different assays. The first, gel to measure the binding of helicases to a labeled substrate shift analysis, is probably the most common method (40,41), but have not been previously reported as methods used to study protein nucleic acid interactions. The gel to screen for HCV helicase inhibitors. DNA binding to shift assay is laborious, not readily amenable to automa- NS3h can also be monitored either by measuring intrinsic tion, and requires relatively large amounts of DNA and protein fluorescence (30,31) or by monitoring changes in protein. Of the three HTS compatible methods evaluated, the fluorescence intensity of a fluorescein-labeled oligo- the FP-based method was the most precise, reproducible nucleotide when it binds NS3h (9). Monitoring intrinsic 8618 Nucleic Acids Research, 2012, Vol. 40, No. 17

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 Downloaded from 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. http://nar.oxfordjournals.org/ protein fluorescence is usually difficult in the presence of high affinity DNA-binding states (28). When NS3h small molecules, especially if they absorb light in the ultra- releases DNA, it slides from the 30 to 50 end of a nucleic violet wavelengths. Use of fluorescence intensity based acid as a Brownian motor (49). Thus, in light of the recent assays in screening is also difficult, because many library demonstration that most nucleoside triphosphates can fuel samples fluoresce in the same range as fluorescein, and as this motor action (6), it is not surprising that 2-methyl- noted above, we have not yet found an alternate ATP, 2-(methylthio)ATP and 2-chloroATP were also hits red-shifted fluorophore that changes intensity when it in the LOPAC screen. The fact that they inhibited binding interacts with NS3h. by only 30–40% of NS3h (which was less than our arbi- The LOPAC samples that most potently disrupted the trary 60% cutoff) is also not surprising since they were by guest on September 14, 2013 NS3h–DNA complex were two polysulfonated naphthy- tested at concentrations far less than the concentration of lureas (suramin and NF 023), a triphenylmethane (ATA) ATP needed to fuel unwinding, or NS3 translocation, at and a tyrphostin (AG 538). All but the tyrphostin inhibit half-maximum rates (6). In contrast, a,b-methylene ATP helicase catalyzed strand separations. Tyrphostin AG 538 did not inhibit NS3h from binding dT15 (Table S1), con- mimics a tyrosine kinase substrate so that it acts as a com- firming the earlier observations that most of the canonical petitive inhibitor of the IGF-1 receptor tyrosine kinase non-hydrolysable ATP analogs are poor inhibitors of (47). Preliminary mechanistic studies on each compound HCV helicase (22,50). suggest that only ATA inhibits the ability of NS3h to The fact that the polysulfonated naphthylureas and cleave ATP in the absence of DNA or RNA, and all but triphenylmethanes affect HCV helicase is noteworthy AG 538 prevent RNA from stimulating ATP hydrolysis. because the anti-microbial properties of these compounds Similarly, gel shift assays show all but AG 538 displace are well documented. Suramin has long been used to treat NS3h from DNA, as was seen with thioflavine S sleeping sickness caused by trypanosomes, and it inhibits (Figure 1B). These data suggest that the AG 538 protein tyrosine phosphatases (51) and G-proteins (52). induced decrease in the polarization of both NS3h– G-proteins and helicases share a similar Walker-type Cy5-dT15 and SSB–Cy5-dT15 complexes might be due nucleotide-binding site (52), and it is possible that suramin to a fluorescence artifact or something other than the inhibits HCV helicase and G-proteins through a similar ability of the tyrphostin to prevent protein from binding molecular mechanism. NF 023 is a suramin analog, P2X DNA. The ability of AG 538 to inhibit growth of the HCV receptor antagonist (53), and inhibitor of RNA editing in replicon is likewise probably not due to its effects on NS3. trypanosomes (54). Suramin and NF 023 behave similarly AG 538 is an inhibitor of the insulin-like growth factor I in all in vitro assays here, but it is noteworthy that (among (IGF-1) tyrosine kinase, and it is possible IGF-1-mediated the two) only suramin was effective against the HCV signaling is needed for efficient HCV replication. Both replicon in cells. A simple explanation would be that the HCV infection and a reduction of IGF-1 levels are somewhat more aromatic suramin is more likely to enter linked to the development of liver cancer (48). cells to exert an intracellular effect. Our results also Compounds binding in place of ATP could also cause confirm the recently reported antiviral effect of ATA the helicase to release its grip on DNA in the above against the HCV replicon, which had been reported after binding assay because ATP binding and hydrolysis ATA was found to inhibit the HCV NS5B RNA- causes HCV helicase to cycle between low affinity and dependent RNA polymerase (55). ATA exerts a similar Nucleic Acids Research, 2012, Vol. 40, No. 17 8619 effect against a wide variety of enzymes that manipulate potent, specific probes needed to study the role of HCV nucleic acids, like human flap endonuclease 1 (FEN1) (56). helicase in cells. On the other hand, more specific analogs ATA also inhibits replication of influenzas A and B (57). of non-specific screening hits could be synthesized, as was Triphenylmethanes that resemble ATA have been de- demonstrated above with the primuline derivatives. veloped from the dye Soluble Blue HT as HCV helicase Screens of the LOPAC and helicase inhibitor libraries inhibitors (14). show that the binding assays presented here are easier to This study is focused on finding compounds that inhibit interpret, and less prone to compound interference, than nucleic acids from binding to the known, high assays monitoring helicase catalyzed DNA separation. 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 SUPPLEMENTARY DATA NS3h instead of full length NS3 is that the truncated Supplementary Data are available at NAR Online: protein expresses at higher levels in E. coli and is more Supplementary Tables 1–3. 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 ACKNOWLEDGEMENTS possible to perform the screens here with both full- We would like to thank Peter Hodder (Scripps Florida) length NS3 and NS3h to identify compounds that target Downloaded from for valuable advice in assay development, and Seng-Lai the still poorly defined nucleic acid binding sites on the Tan for providing the HCV replicon. 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 FUNDING small molecules that simultaneously inhibit both the NS3 protease and helicase have been reported in the National Institutes of Health [RO1 AI088001]; Research http://nar.oxfordjournals.org/ academic literature, although a recent structure shows Growth Initiative Award [101X219] from the University that a protease inhibitor can interact with residues in of Wisconsin-Milwaukee Research Foundation; National both the helicase and protease domains (58). Institutes of Health Molecular Libraries Initiative [U54 In any HTS campaign, it is important to have an HG005031]. Funding for open access charge: University appropriate counter screen to identify non-specific com- of Wisconsin-Milwaukee Research Foundation. pounds. We show here that unrelated DNA binding Conflict of interest statement. None declared. proteins can be substituted for NS3h in the FP-assay to

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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,*

1Department 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

$Cy52dT152NS3h$Binding$ $Cy52dT152NS3h$Binding$ DNA$Unwinding$(MBHA)$ DNA$Unwinding$(MBHA)$ CID$No./Compound$Name$ %$Inhibition Interference$ %Inhibition Interference TO*PRO*3 *63&±&0 1.4&±&0.0 115&±&2 0.5&±&0.0 Hoechst&33258 *96&±&2 0.1&±&0.0 104&±&6 0.3&±&0.0 Chromomycin&A3 *4&±&8 1.0&±&0.0 96&±&1 1.0&±&0.0 4049406 62&±&5 0.7&±&0.0 96&±&2 0.7&±&0.0 53308659 17&±&2 1.1&±&0.1 95&±&0 1.0&±&0.0 SYBR&Green&I 24&±&2 0.8&±&0.0 91&±&3 0.7&±&0.0 53255474 46&±&65 0.3&±&0.2 90&±&2 0.4&±&0.0 44251438 74&±&17 1.0&±&0.0 89&±&3 0.9&±&0.0 486270 30&±&4 0.6&±&0.0 87&±&4 0.6&±&0.0 53239937 9&±&2 1.0&±&0.0 87&±&5 1.0&±&0.0 44251434 47&±&10 1.0&±&0.0 82&±&4 1.0&±&0.0 44251428 54&±&4 1.0&±&0.0 81&±&12 1.0&±&0.0 247520 *17&±&2 0.8&±&0.0 80&±&4 0.7&±&0.0 3413238 63&±&2 0.7&±&0.0 78&±&7 0.7&±&0.0 49849282 22&±&3 1.0&±&0.0 77&±&8 1.0&±&0.0 Titan&Yellow 97&±&4 1.1&±&0.0 75&±&3 1.0&±&0.0 483665 *21&±&2 0.9&±&0.0 74&±&4 0.8&±&0.0 4176595 *13&±&3 0.8&±&0.0 69&±&2 0.9&±&0.0 4111820 4&±&2 0.7&±&0.0 68&±&7 0.9&±&0.0 7080057 *17&±&0 0.9&±&0.0 67&±&1 0.9&±&0.0 49849300 73&±&0 1.0&±&0.0 63&±&2 1.0&±&0.0 Soluble&Blue&HT 78&±&0 1.0&±&0.0 63&±&12 0.8&±&0.0 52939982 *9&±&2 0.9&±&0.0 59&±&4 1.0&±&0.0 44251431 73&±&8 1.0&±&0.0 58&±&23 1.0&±&0.0 Ellipticine *6&±&0 1.0&±&0.0 57&±&4 0.6&±&0.0 49849299 *2&±&1 1.0&±&0.0 53&±&14 1.0&±&0.0 Netropsin 8&±&1 0.9&±&0.0 51&±&20 0.8&±&0.1 Thiazole&Orange *4&±&1 0.9&±&0.0 49&±&7 0.7&±&0.0 50930741 56&±&13 1.0&±&0.0 46&±&5 1.0&±&0.0 50930749 *1&±&5 1.0&±&0.0 41&±&13 1.0&±&0.0 50930730 46&±&30 1.0&±&0.0 41&±&25 1.0&±&0.0 49849293 39&±&2 1.0&±&0.0 32&±&12 0.9&±&0.0 49849293 20&±&6 1.0&±&0.0 31&±&4 0.9&±&0.0 50930737 58&±&14 1.0&±&0.0 31&±&11 1.0&±&0.0 50930733 22&±&6 1.0&±&0.0 29&±&4 1.0&±&0.0 50930738 91&±&1 1.1&±&0.0 28&±&2 1.0&±&0.0 DAPI 4&±&0 0.9&±&0.0 28&±&9 0.8&±&0.0 Rebeccamycin *10&±&2 1.0&±&0.0 27&±&11 0.8&±&0.1 46913723 *10&±&4 1.0&±&0.0 27&±&3 0.9&±&0.0 Proflavin *4&±&1 0.9&±&0.0 27&±&4 0.8&±&0.0 53308658 30&±&14 0.9&±&0.2 27&±&2 1.0&±&0.0 50930755 18&±&3 1.0&±&0.0 26&±&1 0.9&±&0.0 49849287 19&±&5 1.0&±&0.0 25&±&1 0.9&±&0.0 Quinacrine *5&±&4 0.9&±&0.0 25&±&2 0.9&±&0.0 49849302 28&±&1 1.0&±&0.0 24&±&6 0.9&±&0.0 16060752 11&±&5 0.9&±&0.0 24&±&3 0.8&±&0.0 5479184 10&±&3 0.9&±&0.0 24&±&1 0.9&±&0.0 50930751 19&±&6 1.0&±&0.0 23&±&0 1.0&±&0.0 53312458 43&±&6 1.0&±&0.0 23&±&2 0.9&±&0.0 49849298 13&±&1 1.0&±&0.0 22&±&3 0.9&±&0.0 50930740 34&±&6 1.0&±&0.0 22&±&1 1.0&±&0.0 Thioflavin&T *8&±&0 1.0&±&0.0 21&±&2 0.9&±&0.0 3084034 30&±&4 1.0&±&0.0 21&±&12 0.8&±&0.0 Eva&Green *3&±&4 1.0&±&0.0 20&±&2 0.9&±&0.0 53255447 18&±&7 1.0&±&0.0 20&±&0 0.9&±&0.1 52914816 10&±&2 1.0&±&0.0 20&±&9 1.0&±&0.0 50930745 11&±&6 1.0&±&0.0 19&±&6 0.9&±&0.0 Berenil *46&±&0 1.0&±&0.0 19&±&3 0.9&±&0.0 49849280 41&±&0 1.0&±&0.0 17&±&3 0.9&±&0.0 46202556 *8&±&0 1.0&±&0.0 17&±&1 1.0&±&0.0 50930743 38&±&2 1.0&±&0.0 17&±&5 0.9&±&0.0 49849276 28&±&11 1.0&±&0.0 16&±&5 1.0&±&0.0 50930732 4&±&0 1.0&±&0.0 16&±&8 1.0&±&0.0 50930756 52&±&10 1.0&±&0.0 15&±&2 1.0&±&0.0 9585555 *4&±&2 0.9&±&0.0 15&±&5 1.0&±&0.0 3832647 *6&±&2 1.0&±&0.0 15&±&2 1.0&±&0.0 Trioxsalen *6&±&2 1.0&±&0.0 15&±&6 0.9&±&0.0 Carrier&Blank/DMSO *7&±&2 1.0&±&0.0 8&±&4 1.0&±&0.0 50930734 18&±&4 1.1&±&0.0 14&±&6 1.0&±&0.0 49849284 26&±&8 1.0&±&0.0 14&±&7 1.0&±&0.0

1 Table&S1

$Cy52dT152NS3h$Binding$ $Cy52dT152NS3h$Binding$ DNA$Unwinding$(MBHA)$ DNA$Unwinding$(MBHA)$ CID$No./Compound$Name$ %$Inhibition Interference$ %Inhibition Interference 53255452 13&±&5 1.0&±&0.0 14&±&0 0.9&±&0.0 5362502 *1&±&5 1.0&±&0.0 13&±&3 0.9&±&0.0 Luciferin *4&±&1 1.0&±&0.0 13&±&2 0.9&±&0.0 9682118 *9&±&1 1.0&±&0.0 13&±&4 1.0&±&0.0 3643004 *5&±&1 1.0&±&0.0 12&±&5 1.0&±&0.0 Carrier&Blank/DMSO *8&±&1 1.0&±&0.0 12&±&4 1.0&±&0.0 46916208 *9&±&2 1.0&±&0.0 11&±&4 1.0&±&0.0 5691857 3&±&0 0.9&±&0.0 11&±&4 1.0&±&0.0 4594423 *5&±&1 1.0&±&0.0 11&±&4 1.0&±&0.0 3496625 *5&±&0 1.0&±&0.0 11&±&3 1.0&±&0.0 2998553 *1&±&2 1.0&±&0.0 11&±&9 0.9&±&0.0 6540194 *9&±&0 1.0&±&0.0 10&±&4 1.0&±&0.0 Primulin 31&±&3 1.0&±&0.0 10&±&4 1.0&±&0.0 978069 1&±&1 0.9&±&0.0 10&±&8 1.0&±&0.0 5447034 *3&±&2 1.0&±&0.0 10&±&14 0.9&±&0.1 45382099 *4&±&1 1.0&±&0.0 10&±&2 1.0&±&0.0 6064372 *6&±&3 0.9&±&0.0 10&±&2 1.0&±&0.0 53255449 0&±&1 1.0&±&0.0 10&±&6 1.0&±&0.0 49849286 27&±&5 1.0&±&0.0 10&±&3 1.0&±&0.0 1293387 *7&±&1 0.9&±&0.0 10&±&7 1.0&±&0.0 6540199 *7&±&3 1.0&±&0.0 9&±&5 1.0&±&0.0 7314627 1&±&1 1.0&±&0.0 9&±&6 1.0&±&0.0 5281571 20&±&2 1.0&±&0.0 9&±&5 1.0&±&0.0 45382098 *4&±&3 1.0&±&0.0 9&±&3 1.0&±&0.0 49795072 *8&±&0 1.0&±&0.0 9&±&9 1.0&±&0.0 4473963 *11&±&1 1.0&±&0.0 8&±&2 1.0&±&0.0 44251428 24&±&6 1.0&±&0.0 8&±&2 1.0&±&0.0 16060753 *10&±&4 1.0&±&0.0 8&±&3 1.0&±&0.0 Carrier&Blank/DMSO *8&±&0 1.0&±&0.0 14&±&6 0.9&±&0.0 46916209 *9&±&0 1.0&±&0.0 8&±&5 1.0&±&0.0 49849289 36&±&2 1.0&±&0.0 8&±&6 1.0&±&0.0 5860596 *4&±&0 1.0&±&0.0 8&±&6 1.0&±&0.0 4611776 *5&±&3 1.0&±&0.0 8&±&2 1.0&±&0.0 16060655 *8&±&2 1.0&±&0.0 7&±&4 1.0&±&0.0 50930748 31&±&2 1.0&±&0.0 7&±&1 1.0&±&0.0 46839370 29&±&5 1.0&±&0.0 7&±&3 1.0&±&0.0 5736971 *3&±&2 0.9&±&0.0 7&±&2 1.1&±&0.0 1601813 *5&±&3 1.0&±&0.0 7&±&3 1.0&±&0.0 2186405 *6&±&0 0.9&±&0.0 7&±&11 1.0&±&0.0 6540163 *10&±&1 0.9&±&0.0 7&±&2 1.0&±&0.0 5293297 *7&±&2 0.9&±&0.0 7&±&0 1.0&±&0.0 45382102 *4&±&2 1.0&±&0.0 7&±&3 1.0&±&0.0 50904396 *10&±&1 1.0&±&0.0 6&±&13 1.0&±&0.0 2571817 *8&±&2 1.0&±&0.0 6&±&4 1.0&±&0.0 4371917 *26&±&4 1.0&±&0.0 6&±&6 1.0&±&0.0 2330223 *47&±&0 0.9&±&0.0 6&±&5 1.0&±&0.0 46897855 37&±&2 1.0&±&0.0 5&±&2 1.0&±&0.0 49795071 2&±&2 1.0&±&0.0 5&±&11 1.0&±&0.0 16238633 *4&±&1 1.0&±&0.0 5&±&4 1.0&±&0.0 1959044 *1&±&2 0.9&±&0.0 4&±&1 1.0&±&0.0 4051948 *7&±&1 1.0&±&0.0 4&±&11 1.0&±&0.0 2214934 *4&±&3 0.9&±&0.0 4&±&3 1.0&±&0.0 4872167 *4&±&2 1.0&±&0.0 4&±&5 1.0&±&0.0 49849294 32&±&6 1.0&±&0.0 4&±&5 1.0&±&0.0 45382104 0&±&1 1.0&±&0.0 4&±&3 1.0&±&0.0 49849279 *5&±&6 1.0&±&0.0 4&±&9 1.0&±&0.0 1126404 *8&±&2 1.0&±&0.0 3&±&6 1.0&±&0.0 53255450 12&±&2 1.0&±&0.0 3&±&4 1.0&±&0.0 49849295 8&±&3 1.0&±&0.0 3&±&1 1.0&±&0.0 45382101 *5&±&2 1.0&±&0.0 2&±&2 1.0&±&0.0 53255448 9&±&1 1.0&±&0.0 2&±&6 1.0&±&0.0 24818137 *3&±&2 1.0&±&0.0 1&±&7 1.0&±&0.0 44251427 8&±&2 1.0&±&0.0 0&±&16 1.1&±&0.0 44251433 7&±&3 1.0&±&0.0 0&±&11 1.1&±&0.0 2115891 7&±&2 0.9&±&0.0 *1&±&6 1.1&±&0.0 45382097 *7&±&2 1.0&±&0.0 *1&±&9 1.0&±&0.0 44251429 18&±&0 1.0&±&0.0 *1&±&3 1.1&±&0.0 46839371 *8&±&2 0.9&±&0.0 *1&±&8 1.0&±&0.0 49795073 *9&±&1 1.0&±&0.0 *1&±&0 1.0&±&0.0 45382103 0&±&2 1.0&±&0.0 *2&±&5 1.1&±&0.0

2 Table&S1

$Cy52dT152NS3h$Binding$ $Cy52dT152NS3h$Binding$ DNA$Unwinding$(MBHA)$ DNA$Unwinding$(MBHA)$ CID$No./Compound$Name$ %$Inhibition Interference$ %Inhibition Interference 3923766 *9&±&2 0.9&±&0.0 *2&±&8 1.0&±&0.0 3291242 *1&±&2 0.9&±&0.0 *4&±&14 1.1&±&0.0 3138378 *9&±&0 0.9&±&0.0 *5&±&4 1.0&±&0.0 49849290 1&±&0 1.0&±&0.0 22&±&7 1.0&±&0.0

3 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( Protoporphyrin IX disodium P 8293 140 2.0 Mitoxantrone dihydrochloride M 6545 119 0.5 Tyrphostin AG 538 T 7822 100 1.0 Suramin sodium salt S 2671 96 1.0 Aurintricarboxylic acid A 1895 96 1.0 NF 023 N 8652 91 1.1 Reactive Blue 2 R-115 86 0.7 PPNDS tetrasodium P 2738 58 0.8 MRS 2159 M 7684 43 1.0 2-Methylthioadenosine triphosphate tetrasodium A-023 41 1.0 I-OMe-Tyrphostin AG 538 T 7697 37 0.9 Tyrphostin AG 537 T 4693 37 1.0 2-Chloroadenosine triphosphate tetrasodium C-145 37 1.0 Aurothioglucose A0606 36 1.0 2-(Methylthio)adenosine 5'-diphosphate trisodium salt hydrate M152 33 0.9 Me-3,4-dephostatin M 9440 27 0.9 6-Hydroxy-DL-DOPA H 2380 22 0.9 Tyrphostin 51 T 7665 19 0.8 Tetraethylammonium chloride T 2265 14 1.0 JFD00244 J4829 13 1.1 L-745,870 hydrochloride L-131 12 1.0 Methoctramine tetrahydrochloride M-105 11 1.0 SCH-202676 hydrobromide S 4063 8 1.0 Myricetin M 6760 8 1.0 SU 5416 S 8442 6 1.0 Cefotaxime sodium C 7912 6 1.0 L-750,667 trihydrochloride L-133 6 0.9 Ro 41-0960 R-108 5 0.9 Tyrphostin AG 879 T 2067 5 1.0 U-73122 U 6756 5 0.8 SKF 89626 S 3066 5 1.0 3-Aminopropionitrile fumarate A 3134 4 1.0 Cefsulodin sodium salt hydrate C 8145 4 1.0 DL-erythro-Dihydrosphingosine D 6908 4 0.9 Pentolinium di[L(+)-tartrate] P 3520 4 1.0 Lercanidipine hydrochloride hemihydrate L 6668 3 1.0 Ebastine E9531 3 1.0 GYKI 52466 hydrochloride G-119 3 1.0 P1,P4-Di(adenosine-5')tetraphosphate triammonium D 1262 3 1.0 Hemicholinium-3 H-108 3 1.0 GW7647 G 6793 3 1.0 R(-)-Isoproterenol (+)-bitartrate I 2760 3 1.0 Nicardipine hydrochloride N 7510 3 1.0 Pyrilamine maleate P 5514 3 1.0 MK-912 M 7065 3 1.0 Tyrphostin 47 T 7540 3 0.9 IPA-3 I 2285 3 1.0 BIX 01294 trihydrochloride hydrate B9311 3 1.0 (+)-Bromocriptine methanesulfonate B 2134 2 0.9 L-Canavanine sulfate C 9758 2 1.0 DPO-1 D7443 2 1.0 (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine C-237 2 1.0 Decamethonium dibromide D 1260 2 1.0 N-Acetyl-2,3-dehydro-2-deoxyneuraminic acid D 9050 2 1.0 (±)-8-Hydroxy-DPAT hydrobromide H 8250 2 1.0 Dequalinium chloride hydrate D 3768 2 0.9 1,10-Diaminodecane D14204 2 1.0 Etoposide E 1383 2 1.1 7-Cyclopentyl-5-(4-phenoxy)phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamine C 8863 2 1.0 Emetine dihydrochloride hydrate E 2375 2 1.0 Bisoprolol hemifumarate salt B 2185 2 1.0 BIO B 1686 2 0.9 Nimustine hydrochloride N 8659 2 1.0 (-)-Perillic acid 218359 2 0.9 (±)-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride P 7340 2 1.0 Ketanserin tartrate S-006 2 1.0 Rutaecarpine R 3277 2 1.0 L-Tryptophan T 0254 2 1.0

1 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( Ziprasidone hydrochloride monohydrate Z2777 2 1.0 Tyrphostin AG 698 T 5193 2 0.9 Vincristine sulfate V 8879 2 1.0 Quercetin dihydrate Q 0125 2 0.9 Succinylcholine chloride S 8251 2 1.0 Salmeterol xinafoate S 5068 2 1.0 DL-Buthionine-[S,R]-sulfoximine B 2640 1 1.0 Amiloride hydrochloride A 7410 1 0.9 DL-p-Chlorophenylalanine methyl ester hydrochloride C 3635 1 1.0 63Methoxy31,2,3,43tetrahydro39Hpyrido[3,4b]&indole 291552 1 0.9 6-Aminohexanoic acid A 7824 1 0.9 CNS-1102 C 4238 1 1.0 Benserazide hydrochloride B 7283 1 1.0 Cinnarizine C 5270 1 1.0 6-Fluoronorepinephrine hydrochloride B-012 1 0.9 Calcimycin C 7522 1 1.0 Agmatine sulfate A 7127 1 1.0 Cortisone C 2755 1 1.0 SB 222200 S 5192 1 1.0 CK2 Inhibitor 2 C 7367 1 1.0 PD 169316 P 9248 1 1.0 (±)-CPP C-104 1 1.0 JX401 J4774 1 1.0 4-Hydroxy-3-methoxyphenylacetic acid H 1252 1 1.0 (-)-Eseroline fumarate E-100 1 1.0 N,N-Dihexyl-2-(4-fluorophenyl)indole-3-acetamide D 8555 1 1.0 SP600125 S 5567 1 1.0 L-3,4-Dihydroxyphenylalanine D 9628 1 1.0 Chloro-IB-MECA C-277 1 1.0 Dihydrokainic acid D 1064 1 1.0 R(-)-2,10,11-Trihydroxy-N-propylnoraporphine hydrobromide D-030 1 1.0 AC-93253 iodide A9605 1 0.9 3,5-Dinitrocatechol D-131 1 1.0 Hydroxylamine hydrochloride H 9876 1 1.0 5,5-Diphenylhydantoin D 4007 1 1.0 4-Hydroxybenzhydrazide H 9882 1 1.1 Glybenclamide G 0639 1 1.0 Guanfacine hydrochloride G 1043 1 1.0 2,4-Diamino-6-pyrimidinone D1920-6 1 1.1 SCH-28080 S 4443 1 1.0 Diacylglycerol Kinase Inhibitor II D 5794 1 1.0 Metolazone M 1195 1 1.0 IMID-4F hydrochloride I 2279 1 1.0 MRS 1845 M 1692 1 0.9 Kenpaullone K 3888 1 1.0 NCS-356 N 4034 1 1.0 Pancuronium bromide P 1918 1 1.0 (±)-Metoprolol (+)-tartrate M 5391 1 1.0 Cisplatin P 4394 1 0.8 LFM-A13 L 8789 1 1.0 NNC 55-0396 N 0287 1 1.0 NO-711 hydrochloride N-142 1 1.0 MDL 28170 M 6690 1 1.1 Pilocarpine nitrate P 6628 1 1.0 SIB 1757 S 9186 1 0.9 Pyrazinecarboxamide P 7136 1 1.0 Primidone P 7295 1 1.0 13-cis-retinoic acid R 3255 1 0.9 PAC-1 P0115 1 0.9 Trimethoprim T 7883 1 1.0 REV 5901 R 5523 1 0.9 Tranilast T 0318 1 1.0 Sobuzoxane S 4692 1 1.0 Trequinsin hydrochloride T 2057 1 0.9 Propentofylline P 9689 1 1.0 Sulfaphenazole S 0758 1 1.0 Pregnenolone sulfate sodium P-162 1 1.0 Xylometazoline hydrochloride X 6000 1 1.0

2 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( Quinacrine dihydrochloride Q 3251 1 1.0 Triprolidine hydrochloride T 6764 1 1.0 Sulindac S 8139 1 1.0 IMS2186 I 7160 1 1.0 Zonisamide sodium Z 2001 1 1.0 Zimelidine dihydrochloride Z-101 1 1.0 N3Phenylanthranilic&acid 144509 0 0.9 (±)3Nipecotic&acid 211672 0 0.9 N-Acetyl-L-Cysteine A 7250 0 0.9 Cyclosporin A C 3662 0 0.9 Captopril C 4042 0 1.0 Acetamide A&0500 0 0.9 L-Aspartic acid A 9256 0 0.9 Cimetidine C 4522 0 1.0 Carbetapentane citrate C 4662 0 1.0 Fulvestrant I4409 0 0.9 2-Chloroadenosine C 5134 0 1.0 GABA A&2129 0 1.0 (+)-Butaclamol hydrochloride D-033 0 1.0 8-(p-Sulfophenyl)theophylline A-013 0 1.0 Indirubin-3'-oxime I0404 0 1.0 Cefaclor C 6895 0 1.0 Caffeic Acid C 0625 0 1.0 CGP-13501 C 0987 0 1.0 1-(4-Chlorobenzyl)-5-methoxy-2-methylindole-3-acetic acid C 1610 0 1.0 S-(+)-PD 123177 trifluoroacetate salt hydrate P5749 0 1.0 Z-L-Phe chloromethyl ketone C 9511 0 1.0 (S)-(+)-Camptothecin C 9911 0 1.0 8-Cyclopentyl-1,3-dipropylxanthine C-101 0 1.0 GW9508 G 9797 0 1.0 Guanabenz acetate G-110 0 1.0 (±)-Epinephrine hydrochloride E 4642 0 1.0 Ethosuximide E 7138 0 1.0 Emodin E 7881 0 1.0 Disopyramide D 7644 0 1.0 7,7-Dimethyl-(5Z,8Z)-eicosadienoic acid D 8008 0 0.9 CGS-21680 hydrochloride C-141 0 1.0 (+)-Cyclazocine C-147 0 1.0 Capsazepine C-191 0 1.0 N-Methyl-beta-carboline-3-carboxamide E-006 0 1.0 Chlormezanone C-192 0 1.0 Danazol D 8399 0 1.0 4-Imidazolemethanol hydrochloride H 1877 0 1.0 Hexamethonium dichloride H 2138 0 1.0 (R,R)-cis-Diethyl tetrahydro-2,8-chrysenediol D 8690 0 1.0 CGP 20712A methanesulfonate C-231 0 1.0 UCL 2077 U6758 0 1.0 17alpha-hydroxyprogesterone H 5752 0 1.0 Dipropyldopamine hydrobromide D-031 0 1.0 Fenofibrate F 6020 0 1.1 D-ribofuranosylbenzimidazole D 1916 0 1.0 L-Histidine hydrochloride H 8125 0 1.1 Forskolin F 6886 0 1.1 Hydroquinone H 9003 0 1.1 R(+)-Butylindazone D-129 0 1.0 Fluspirilene F-100 0 1.0 N,N-Dipropyl-5-carboxamidotryptamine maleate D-132 0 1.1 N-(3,3-Diphenylpropyl)glycinamide D 8816 0 1.1 (-)-alpha-Methylnorepinephrine D 5290 0 1.0 Anisotropine methyl bromide A5181 0 1.1 R-(+)-7-Hydroxy-DPAT hydrobromide H-168 0 1.1 Vanillic acid diethylamide E 0137 0 1.0 CPNQ C 3118 0 1.0 Daphnetin D 5564 0 1.1 L-Canavanine C 1625 0 1.1 2',3'-dideoxycytidine D 5782 0 1.0 Cambinol C0494 0 1.0 GBR-12935 dihydrochloride G 9659 0 1.0

3 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( Enoximone E 1279 0 1.1 ET-18-OCH3 E 1779 0 1.0 5'-N-Ethylcarboxamidoadenosine E 2387 0 1.0 E-64 E 3132 0 1.0 Isoliquiritigenin I 3766 0 0.9 Mecamylamine hydrochloride M 9020 0 0.9 SD-169 S0572 0 1.0 ODQ O 3636 0 1.0 MK-886 M 2692 0 0.9 MDL 105,519 M-216 0 1.0 K 185 K 1888 0 1.0 Meclofenamic acid sodium M 4531 0 1.0 AS605240 A 0233 0 1.0 LY-367,265 L 2411 0 1.0 (-)-Nicotine hydrogen tartrate salt N 5260 0 1.0 alpha-Lobeline hydrochloride L 4376 0 1.0 Valproic acid sodium P 4543 0 1.0 Loratadine L 9664 0 1.0 T0070907 T 8703 0 1.0 L-655,708 L 9787 0 1.0 NG-Monomethyl-L-arginine acetate M 7033 0 1.0 (±)-3-(3,4-dihydroxyphenyl)-2-methyl-DL-alanine M 7277 0 1.0 SR 59230A oxalate S 8688 0 0.9 SIB 1893 S 9311 0 1.0 Ritodrine hydrochloride R 0758 0 0.9 Granisetron hydrochloride G 3796 0 1.0 Ritanserin R-103 0 0.9 Rauwolscine hydrochloride R-104 0 1.0 3-Tropanyl-indole-3-carboxylate hydrochloride T-104 0 1.0 3-Tropanylindole-3-carboxylate methiodide T-113 0 1.0 5alpha-Pregnan-3alpha-ol-20-one P 8887 0 1.0 Propantheline bromide P 8891 0 1.0 5HPP-33 H 9415 0 0.9 Tyrphostin A9 T-182 0 1.0 Rilmenidine hemifumarate R-134 0 1.0 Trihexyphenidyl hydrochloride T 1516 0 1.0 Paromomycin sulfate P 9297 0 1.0 Procaine hydrochloride P 9879 0 1.0 Tyrphostin AG 490 T 3434 0 1.0 Org 27569 O8014 0 1.0 TTNPB T 3757 0 1.0 Bicalutamide (CDX) B9061 0 1.0 Tyrphostin AG 1478 T 4182 0 1.0 (±)-gamma-Vinyl GABA V 8261 0 1.0 PPADS P-178 0 1.0 Bay 11-7082 B 5556 0 1.0 NSC348884 hydrate N 3414 0 1.0 Albuterol hemisulfate S 5013 0 1.0 (±)-alpha-Lipoic Acid T 5625 0 1.0 DL-Thiorphan T 6031 0 1.0 Trazodone hydrochloride T 6154 0 1.0 SMER28 S 8197 0 1.0 WIN 62,577 W-104 0 1.0 SR 2640 S 7690 0 1.0 AC-55649 A9480 0 1.0 SKF 96365 S 7809 0 1.0 SCH 58261 S4568 0 1.0 Quazinone Q 3504 0 1.0 LP 12 hydrochloride hydrate L 3169 0 1.0 Tyrphostin 23 T 7165 0 1.0 Pifithrin-mu P0122 0 1.0 Cortexolone R 0500 0 1.0 1-[2-(Trifluoromethyl)phenyl]imidazole T 7313 0 1.0 Yohimbine hydrochloride Y 3125 0 1.1 CV-3988 C 7238 0 1.0 YC-1 Y-102 0 1.0 Caroverine hydrochloride C 1119 0 1.0 (-)-Scopolamine methyl bromide S 8502 0 1.0

4 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( S(3)3p3Bromotetramisole&oxalate 190047 0 0.9 (±)-Brompheniramine maleate B 2390 0 0.9 Azelaic&acid 246379 0 0.9 Tryptamine&hydrochloride 246557 0 1.0 Cephalosporin C zinc salt C 3270 0 0.9 Atropine&sulfate A&0257 0 0.9 BWB70C B 4558 0 1.0 Bepridil hydrochloride B 5016 0 1.0 (+)-Brompheniramine maleate B 5275 0 1.0 Amiodarone hydrochloride A 8423 0 0.9 3-aminobenzamide A 0788 0 1.0 Buspirone hydrochloride B 7148 0 1.0 Altretamine A 8723 0 0.9 Cephalothin sodium C 4520 0 1.0 N-(4-Amino-2-chlorophenyl)phthalimide A 9345 0 1.0 N-Acetyl-5-hydroxytryptamine A 1824 0 1.0 BRL 50481 B 0936 0 1.0 Adenosine 3',5'-cyclic monophosphate A 9501 0 0.9 Clemizole hydrochloride C 5040 0 1.0 (±)-HA-966 A 9699 0 1.0 Benoxathian hydrochloride B-016 0 1.0 Roscovitine R 7772 0 1.0 1,3-Dipropyl-8-p-sulfophenylxanthine A-022 0 1.0 Gabaculine&hydrochloride A&3539 0 1.0 DAPH D 3943 0 1.0 BU224 hydrochloride B-154 0 1.0 N-(2-[4-(4-Chlorophenyl)piperazin-1-yl]ethyl)-3-methoxybenzamide C 7230 0 1.0 Psora-4 P 9872 0 1.0 AS-252424 A8981 0 1.0 PK 11195 C 0424 0 1.0 Acetylthiocholine chloride A 5626 0 1.0 Cantharidin C 7632 0 1.0 Chelidamic acid C 8011 0 1.0 CGP-7930 C 0862 0 1.0 L-Cycloserine C 1159 0 1.0 Arecoline hydrobromide A 6134 0 1.0 A-315456 A 6351 0 1.0 (+)-Catechin Hydrate C 1251 0 1.0 Cephradine C 8395 0 1.0 Actinonin A&6671 0 1.1 Chlorprothixene hydrochloride C 1671 0 1.0 Corticosterone C 2505 0 1.0 Centrophenoxine hydrochloride C 8773 0 1.0 p-Benzoquinone B 1266 0 1.0 TBBz T6951 0 1.0 CPCCOEt C 9611 0 1.0 Bromoenol lactone B 1552 0 1.0 Benzamide B 2009 0 1.0 ML-7 I 2764 31 0.9 Mifepristone M 8046 31 0.9 (±)-Ibotenic acid I 2765 31 0.9 L-alpha-Methyl-p-tyrosine M 8131 31 0.9 SB-215505 S 1068 31 0.9 Isotharine mesylate I 3639 31 0.9 Methapyrilene hydrochloride M 9125 31 1.0 Naltriben methanesulfonate N-156 31 1.0 3-Isobutyl-1-methylxanthine I 5879 31 0.9 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride I 7016 31 0.9 (+)-MK-801 hydrogen maleate M-107 31 1.0 N-Oleoylethanolamine O 0383 31 1.0 Oxolinic acid O 0877 31 1.0 Stevioside S3572 31 1.0 Ethopropazine hydrochloride E5406 31 1.0 Iofetamine hydrochloride I-120 31 1.0 Mevastatin M 2537 31 0.9 Oxotremorine methiodide O-100 31 1.0 Niflumic acid N 0630 31 0.9 3-(1H-Imidazol-4-yl)propyl di(p-fluorophenyl)methyl ether hydrochloride I-160 31 1.0

5 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( (±)-Octoclothepin maleate O-111 31 1.0 Pentamidine isethionate P 0547 31 1.0 O-Phospho-L-serine P 0878 31 1.0 Gossypol G8761 31 0.9 L-162,313 L 1415 31 1.0 Papaverine hydrochloride P 3510 31 1.0 LY-310,762 hydrochloride L 2536 31 1.0 Lomefloxacin hydrochloride L 2906 31 1.0 6-Methyl-2-(phenylethynyl)pyridine hydrochloride M 5435 31 1.0 (±)-Methoxyverapamil hydrochloride M 5644 31 1.0 Nifedipine N 7634 31 1.0 7-Nitroindazole N 7778 31 1.0 CI-976 C3743 31 1.0 GW405833 hydrochloride G1421 31 1.1 2-methoxyestradiol M 6383 31 1.0 alpha,beta-Methylene adenosine 5'-triphosphate dilithium M 6517 31 1.0 Propafenone hydrochloride P 4670 31 1.0 5alpha-Pregnan-3alpha-ol-11,20-dione P 5052 31 1.0 PNU-282987 P6499 31 1.0 (-)-Tetramisole hydrochloride L 9756 31 1.0 Piroxicam P 5654 31 1.0 3-n-Propylxanthine P 5679 31 1.0 Phenylephrine hydrochloride P 6126 31 1.0 Perphenazine P 6402 31 1.0 Cyclothiazide C 9847 31 1.0 N6-Cyclohexyladenosine C 9901 31 1.0 Efaroxan hydrochloride E 3263 31 1.0 OXA-22 C-011 31 1.0 8-Cyclopentyl-1,3-dimethylxanthine C-102 31 1.0 Disopyramide phosphate D 6035 31 1.0 Diclofenac sodium D 6899 31 1.0 Glipizide G-117 31 1.0 Staurosporine aglycone S 3939 31 1.0 EGTA E 4378 31 1.0 Cetirizine dihydrochloride C3618 31 1.0 Cilnidipine C1493 31 1.0 Y-27632 dihydrochloride Y 0503 31 1.0 Dephostatin D 8065 31 1.0 MHPG piperazine H 1377 31 1.0 2-Chloro-2-deoxy-D-glucose C-203 31 1.0 Felbamate F 0778 31 1.0 CNQX disodium C-239 31 1.0 CX 546 C-271 31 1.0 Diazoxide D 9035 31 1.0 DL-threo-beta-hydroxyaspartic acid H 2775 31 1.0 3,4-Dihydroxyphenylacetic acid D 9128 31 1.0 Lithium Chloride L 4408 31 1.0 Hydrochlorothiazide H 4759 31 1.0 SB 218795 S 8817 31 1.0 Hispidin H 5257 31 1.0 WB-4101 hydrochloride D 0411 31 1.0 Fluvoxamine maleate F 2802 31 1.0 Dihydroouabain D 0670 31 1.0 Dobutamine hydrochloride D 0676 31 1.0 p-Fluoro-L-phenylalanine F 4646 31 1.0 Doxycycline hydrochloride D 9891 31 1.0 6,7-ADTN hydrobromide D-002 31 1.0 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole H 6036 31 1.0 Harmane H 7258 31 1.0 Debrisoquin sulfate D 1306 31 1.1 5-Fluorouracil F 6627 31 1.0 R(+)-SCH-23390 hydrochloride D-054 31 1.0 Diltiazem hydrochloride D 2521 31 1.1 Dextromethorphan hydrobromide monohydrate D 2531 31 1.1 Flunarizine dihydrochloride F 8257 31 1.0 Diphenhydramine hydrochloride D 3630 31 1.0 5-fluoro-5'-deoxyuridine F 8791 31 1.0 Flutamide F 9397 31 1.0

6 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( L-Hyoscyamine H 9002 31 1.1 BU99006 B 8433 31 0.9 Propofol D126608 31 1.0 MNS M 7445 31 1.0 Serotonin hydrochloride H 9523 31 1.0 L-165,041 L 2167 31 1.0 (S)-3,5-Dihydroxyphenylglycine D 3689 31 1.1 Fexofenadine hydrochloride F 9427 31 1.1 Formoterol F 9552 31 1.1 Felodipine F 9677 31 1.0 Desipramine hydrochloride D 3900 31 1.0 cis-(Z)-Flupenthixol dihydrochloride F-114 31 1.0 Furafylline F-124 31 1.0 FPL 64176 F-131 31 1.0 D(-)-2-Amino-5-phosphonopentanoic acid A 8054 31 1.1 3,7-Dimethyl-1-propargylxanthine D-134 31 1.0 5,7-Dichlorokynurenic acid D-138 31 1.0 Histamine, R(-)-alpha-methyl-, dihydrochloride H-128 31 1.0 GW2974 G 0668 31 1.0 L-Glutamic acid hydrochloride G 2128 31 1.0 2,3-Dimethoxy-1,4-naphthoquinone D 5439 31 1.0 Ibudilast I 0157 31 1.0 Venlafaxine hydrochloride V7264 31 1.1 Imidazole-4-acetic acid hydrochloride I 0375 31 1.0 Imazodan I 0782 31 1.0 DM 235 D 5689 31 1.1 GW5074 G 6416 31 1.0 Genistein G 6649 31 1.0 1,1-Dimethyl-4-phenyl-piperazinium iodide D 5891 31 1.0 Iodoacetamide I 1149 31 1.1 Idarubicin I 1656 31 0.8 GR 55562 dihydrobromide G 0419 31 1.0 Phenelzine sulfate P 6777 31 0.9 Pheniramine maleate P 6902 31 0.9 SKF 89976A hydrochloride S 9066 31 0.9 1-(1-Naphthyl)piperazine hydrochloride S-003 31 1.0 Taxol T 7402 31 0.9 Retinoic acid R 2625 31 0.9 Pirenzepine dihydrochloride P 7412 31 1.0 Prazosin hydrochloride P 7791 31 0.9 L-Beta-threo-benzyl-aspartate B6436 31 1.0 SQ 22536 S-153 31 1.0 Rotenone R 8875 31 1.0 ARP 101 A8356 31 0.9 Tranylcypromine hydrochloride P 8511 31 1.0 Ro 8-4304 R 8900 31 0.9 U-75302 U 1508 31 0.9 Piribedil maleate P 9233 31 1.0 Prilocaine hydrochloride P 9547 31 1.0 Pyridostigmine bromide P 9797 31 1.0 Tolazamide T 2408 31 1.0 Uridine 5'-diphosphate sodium U 4125 31 1.0 U-69593 U-103 31 1.0 (-)-Scopolamine methyl nitrate S 2250 31 1.0 Phaclofen P-118 31 1.0 IC 261 I 0658 31 1.0 U0126 U-120 31 1.0 Spermine tetrahydrochloride S 2876 31 1.0 A-134974 dihydrochloride hydrate A2846 31 0.9 SC 19220 S 3065 31 1.0 PD 98,059 P-215 31 1.0 Tyrphostin AG 835 T 5568 31 1.0 AMG 9810 A 2731 31 1.0 Wortmannin from Penicillium funiculosum W 1628 31 1.0 Quinidine sulfate Q 0875 31 1.0 BAY 61-3606 hydrochloride hydrate B 9685 31 1.0 N,N,N-trimethyl-1-(4-trans-stilbenoxy)-2-propylammonium iodide T 6692 31 1.0 (-)-Sulpiride S 7771 31 1.0

7 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( (-)-Scopolamine,n-Butyl-, bromide S 7882 31 1.0 Quipazine, 6-nitro-, maleate Q-109 31 1.0 Quinelorane dihydrochloride Q-110 31 1.0 (±)-Quinpirole dihydrochloride Q-111 31 1.0 Aprindine hydrochloride A 7606 31 1.0 (±)-Sulpiride S 8010 31 1.1 YS-035 hydrochloride Y-101 31 1.0 Zaprinast Z 0878 31 1.0 Olprinone hydrochloride O 7389 31 1.0 DL3alpha3Methyl3p3tyrosine 120693 31 0.9 3-Bromo-7-nitroindazole B 2050 31 0.9 53Aminovaleric&acid&hydrochloride 194336 31 0.9 N-Bromoacetamide B 2377 31 0.9 Benzamil hydrochloride B 2417 31 1.0 L-2-aminoadipic acid A 7275 31 0.9 (±)-Atenolol A 7655 31 0.9 Cyproterone acetate C 3412 31 0.9 5-Bromo-2'-deoxyuridine B 5002 31 1.0 GR 113808 G 5918 31 1.0 Carbamazepine C 4024 31 1.0 (±)-Baclofen B 5399 31 0.9 Betamethasone B 7005 31 1.0 4-Amino-1,8-naphthalimide A 0966 31 1.0 Carbachol C 4382 31 0.9 Adenosine A 9251 31 0.9 Astaxanthin A 9335 31 1.0 Amantadine&hydrochloride A&1260 31 0.9 N-arachidonylglycine A 1977 31 1.0 BP 897 B 9308 31 1.0 3'3Azido33'3deoxythymidine A&2169 31 0.9 (E)-5-(2-Bromovinyl)-2'-deoxyuridine B 9647 31 1.0 5-azacytidine A 2385 31 0.9 Bromoacetyl alprenolol menthane B-015 31 1.0 Apigenin A 3145 31 1.0 Phenoxybenzamine hydrochloride B-019 31 1.0 Paroxetine hydrochloride hemihydrate (MW = 374.83) P 9623 31 0.9 SB 204741 S 0693 31 0.9 Bupropion hydrochloride B-102 31 1.0 1-Aminobenzotriazole A 3940 31 1.0 3-Amino-1-propanesulfonic acid sodium A 4147 31 1.0 Cyproheptadine hydrochloride C 6022 31 1.0 Arecaidine propargyl ester hydrobromide A-140 31 1.0 O3(Carboxymethyl)hydroxylamine&hemiHCl A&4508 31 1.0 Sandoz 58-035 S 9318 31 1.0 3-Morpholinosydnonimine hydrochloride M5793 31 1.0 Diacylglycerol kinase inhibitor I D 5919 31 1.0 (±)323Amino373phosphonoheptanoic&acid A&5157 31 1.0 Ciprofibrate C 0330 31 1.0 6-Chloromelatonin C 0331 31 1.0 Cilostazol C 0737 31 1.0 N6-2-(4-Aminophenyl)ethyladenosine A-202 31 1.0 Cilostamide C 7971 31 1.0 gamma-Acetylinic GABA A-230 31 1.0 Alloxazine A-242 31 1.0 Acetazolamide A 6011 31 1.0 GR 46611 G 8543 31 1.0 Chlorpropamide C 1290 31 1.0 cis-Azetidine-2,4-dicarboxylic acid A-243 31 1.1 trans-Azetidine-2,4-dicarboxylic acid A-244 31 1.0 AIDA A-254 31 1.0 Imipenem monohydrate I 0160 31 1.0 ATPA A-263 31 1.0 ARL 67156 trisodium salt A-265 31 1.0 Carisoprodol C 8759 31 1.0 Methotrexate&hydrate A&6770 31 1.0 Amperozide hydrochloride A 6976 31 1.0 Aminoguanidine hemisulfate A 7009 31 1.0 3-Aminopropylphosphonic acid A 7162 31 1.0

8 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( 2,3-Butanedione monoxime B 0753 31 1.1 Pyrocatechol C 9510 31 1.0 Colchicine C 9754 31 1.0 1-Methylimidazole M 8878 32 0.9 (±)-Ibuprofen I 4883 32 1.0 Memantine hydrochloride M 9292 32 1.0 Nitrendipine N-144 32 0.9 BW 723C86 B175 32 0.9 SB 242084 dihydrochloride hydrate S 8061 32 0.9 Naftopidil dihydrochloride N-158 32 1.0 Fenobam F 0430 32 1.0 (±)-Muscarine chloride M-104 32 1.0 DFB D 0943 32 1.0 Ivermectin I 8898 32 1.0 Methysergide maleate M-137 32 1.0 Olomoucine O 0886 32 0.9 Oxiracetam O 3011 32 1.0 Indatraline hydrochloride I-119 32 0.9 S15535 S 5321 32 1.0 Orphenadrine hydrochloride O 3752 32 0.9 Mexiletene hydrochloride M 2727 32 1.0 IB-MECA I-146 32 0.9 Isonipecotic acid I18008 32 1.0 Nomifensine maleate N 1530 32 1.0 N-Methyl-D-aspartic acid M 3262 32 1.0 JL-18 J-102 32 1.0 nor-Binaltorphimine dihydrochloride N 1771 32 1.0 Niclosamide N 3510 32 1.0 Metergoline M 3668 32 1.0 L-701,324 L 0258 32 1.0 Nordihydroguaiaretic acid from Larrea divaricata (creosote bush) N 5023 32 1.0 3-alpha,21-Dihydroxy-5-alpha-pregnan-20-one P 2016 32 1.0 (±)-alpha-Methyl-4-carboxyphenylglycine M 4796 32 1.0 PRE-084 P 2607 32 1.0 NG-Nitro-L-arginine N 5501 32 1.0 NG-Nitro-L-arginine methyl ester hydrochloride N 5751 32 1.0 Mibefradil dihydrochloride M 5441 32 1.0 N6-Methyladenosine M 5501 32 1.0 NU2058 N 4286 32 1.0 Leflunomide L 5025 32 1.0 Lidocaine N-ethyl bromide quaternary salt L 5783 32 1.0 4-Amidinophenylmethanesulfonyl fluoride hydrochloride A 6664 32 1.0 NS 521 oxalate N 7904 32 1.0 LE 300 L 8401 32 1.0 Lansoprazole L 8533 32 1.0 6-Nitroso-1,2-benzopyrone N 8403 32 1.0 Nilutamide N 8534 32 1.0 Promethazine hydrochloride P 4651 32 1.0 Cysteamine hydrochloride M 6500 32 1.0 Methoxamine hydrochloride M 6524 32 1.0 O-Methylserotonin hydrochloride M 6628 32 1.0 Phosphomycin disodium P 5396 32 1.0 Norcantharidin N 8784 32 1.0 Noscapine hydrchloride N 9007 32 1.0 Pentylenetetrazole P 6500 32 1.0 (+)-Pilocarpine hydrochloride P 6503 32 1.0 2,6-Difluoro-4-[2-(phenylsulfonylamino)ethylthio]phenoxyacetamide D 8941 32 1.0 SB 415286 S 3567 32 1.0 S-Ethylisothiourea hydrobromide E 3149 32 1.0 Epinastine hydrochloride E 5156 32 1.0 Ebselen E 3520 32 1.0 Diethylenetriaminepentaacetic acid D 6518 32 1.0 R-(-)-Desmethyldeprenyl hydrochloride D 6940 32 1.0 (±)-Vanillylmandelic acid H 0131 32 1.0 6-Hydroxymelatonin H 0627 32 1.0 Hexamethonium bromide H 0879 32 1.0 (-)-Physostigmine E 8375 32 1.0 NBI 27914 N 3911 32 1.0

9 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( 8-(3-Chlorostyryl)caffeine C-197 32 1.0 Imperatorin I6659 32 1.0 CGS-15943 C-199 32 1.0 Fusidic acid sodium F 0881 32 1.0 S-(+)-Fluoxetine hydrochloride F 1553 32 1.0 DCEBIO D 9190 32 1.0 1-Deoxynojirimycin hydrochloride D 9305 32 1.0 Dipyridamole D 9766 32 1.0 Furegrelate sodium F 3764 32 1.0 Furosemide F 4381 32 1.0 Fluphenazine dihydrochloride F 4765 32 0.9 R(-)-2,10,11-Trihydroxyaporphine hybrobromide D-029 32 1.0 2',3'-didehydro-3'-deoxythymidine D 1413 32 1.0 Droperidol D 1414 32 1.0 L-3,4-Dihydroxyphenylalanine methyl ester hydrochloride D 1507 32 1.0 GBR-12909 dihydrochloride D-052 32 1.0 MHPG sulfate potassium H 8759 32 1.0 (±)-2,3-Dichloro-alpha-methylbenzylamine hydrochloride D-103 32 1.0 5-Hydroxyindolacetic acid H 8876 32 1.0 Carvedilol C3993 32 1.0 4-DAMP methiodide D-104 32 1.1 Doxylamine succinate D 3775 32 1.1 N^G,N^G-Dimethylarginine hydrochloride D 4268 32 1.0 Clodronic acid D 4434 32 1.0 N-Desmethylclozapine D5676 32 1.0 Hexahydro-sila-difenidol hydrochloride, p-fluoro analog H-127 32 1.0 AL-8810 A 3846 32 1.0 Phenytoin sodium D 4505 32 1.1 S(-)-Pindolol P-152 32 1.0 Ganaxolone G 7795 32 1.0 Ganciclovir G 2536 32 1.0 Sematilide monohydrochloride monohydrate S0323 32 1.1 Epibestatin hydrochloride E 0381 32 1.0 Bezafibrate B 7273 32 1.1 5,5-Dimethyl-1-pyrroline-N-oxide D 5766 32 1.0 N-Methyldopamine hydrochloride D 5886 32 1.0 Gallamine triethiodide G 8134 32 1.0 Ipratropium bromide I 1637 32 1.0 Etazolate hydrochloride E 1896 32 1.0 SU 4312 S 8567 32 0.9 BRL 52537 hydrochloride B 5559 32 0.9 Tetracaine hydrochloride T 7508 32 0.9 Phorbol 12-myristate 13-acetate P 8139 32 1.0 NU6027 N 4411 32 0.9 D-609 potassium T 8543 32 1.0 Phosphoramidon disodium R 7385 32 1.0 Picotamide P 8477 32 1.0 Ammonium pyrrolidinedithiocarbamate P 8765 32 1.0 Tamoxifen citrate T 9262 32 0.9 Ibandronate sodium I5784 32 1.0 (±)-cis-Piperidine-2,3-dicarboxylic acid P 8782 32 1.0 Tetramisole hydrochloride T 1512 32 1.0 S(+)-Raclopride L-tartrate R-121 32 1.0 Procainamide hydrochloride P 9391 32 1.0 Tetradecylthioacetic acid T 1698 32 1.0 (±)-Sotalol hydrochloride S 0278 32 1.0 SB-366791 S 0441 32 1.0 (±)-Synephrine S 0752 32 1.0 SKF 95282 dimaleate S 5317 32 1.0 4-Imidazoleacrylic acid U 7500 32 1.0 N2-Ethyl-2'-deoxyguanosine N 3289 32 1.0 Terbutaline hemisulfate T 2528 32 1.0 Triamterene T 4143 32 1.0 Auranofin A 6733 32 1.0 DL-Stearoylcarnitine chloride S 2381 32 1.0 BF-170 hydrochloride B 4311 32 1.0 Tyrphostin AG 494 T 4318 32 0.9 A3 hydrochloride A 1980 32 1.0

10 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( Spermidine trihydrochloride S 2501 32 1.0 U-99194A maleate U-116 32 1.0 Vinblastine sulfate salt V 1377 32 1.0 Vinpocetine V 6383 32 1.0 Phenamil methanesulfonate P-203 32 1.0 Tyrphostin AG 555 T 4818 32 1.0 (±)-PD 128,907 hydrochloride P-216 32 1.0 Stattic S7947 32 1.0 ICI 63,137 I 8283 32 1.0 Sanguinarine chloride S 5890 32 1.0 LY2183240 L 9044 32 1.0 N-Succinyl-L-proline S 6633 32 1.0 SU 6656 S 9692 32 1.0 Quinolinic acid P63204 32 1.0 Triamcinolone T 6376 32 1.0 Quinine sulfate Q 1250 32 1.0 S(-)-Timolol maleate T 6394 32 1.0 (+)-Quisqualic acid Q 2128 32 1.0 IRAK-1/4 Inhibitor I I 5409 32 1.1 Ara-G hydrate A 4233 32 1.0 Xylazine hydrochloride X 1251 32 1.0 Xanthine amine congener X-103 32 1.0 Tyrphostin AG 112 T 6943 32 1.0 (-)-Quinpirole hydrochloride Q-102 32 1.0 Tyrphostin 1 T 7040 32 1.0 O6-benzylguanine B 2292 32 0.9 L-Buthionine-sulfoximine B 2515 32 1.0 53Fluoroindole323carboxylic&acid 265128 32 1.0 Chlormethiazole hydrochloride C1240 32 1.0 CGP-74514A hydrochloride C 3353 32 0.9 S3(43Nitrobenzyl)363thioguanosine 861669 32 0.9 1-Aminocyclopropanecarboxylic acid hydrochloride A 0430 32 1.0 D-Cycloserine C 3909 32 0.9 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride A 8456 32 0.9 Org&24598&lithium&salt O7639 32 0.9 Benzamidine hydrochloride B 6506 32 1.0 Cyclobenzaprine hydrochloride C 4542 32 1.0 Aminophyllineðylenediamine A&1755 32 0.9 Ro 20-1724 B 8279 32 1.0 Chlorothiazide C 4911 32 0.9 Bethanechol chloride C 5259 32 1.0 Acetyl-beta-methylcholine chloride A 2251 32 0.9 5-(N-Ethyl-N-isopropyl)amiloride A 3085 32 1.0 (±)-AMT hydrochloride A 9834 32 1.0 Ceftriaxone sodium C 5793 32 1.0 HEMADO H3288 32 1.0 (-)-Cotinine C 5923 32 1.0 Clotrimazole C 6019 32 1.0 YM&976 Y&4877 32 1.0 AA-861 A 3711 32 1.0 9-Amino-1,2,3,4-tetrahydroacridine hydrochloride A 3773 32 1.0 Bromoacetylcholine bromide B-121 32 1.0 R(+)-6-Bromo-APB hydrobromide B-135 32 1.0 S(-)-Atenolol A-143 32 1.0 53(N,N3Dimethyl)amiloride&hydrochloride A&4562 32 1.0 Azathioprine A 4638 32 1.0 Acyclovir A 4669 32 1.0 BRL 54443 maleate B-173 32 1.0 (±)-2-Amino-3-phosphonopropionic acid A 4910 32 1.0 Supercinnamaldehyde S3322 32 0.9 DL-Cycloserine C 7005 32 1.0 1-Amino-1-cyclohexanecarboxylic acid hydrochloride A-162 32 1.0 McN-A-343 C 7041 32 1.0 Cystamine dihydrochloride C 7255 32 1.0 SB 200646 hydrochloride S 0568 32 1.0 Caffeine C 0750 32 1.0 Acetohexamide A-178 32 1.0 AB-MECA A-236 32 1.0

11 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( ML-9 C 1172 32 1.0 2-Hydroxysaclofen A 6566 32 1.0 Chlorpromazine hydrochloride C 8138 32 1.0 CBIQ C 6617 32 1.0 Caffeic acid phenethyl ester C 8221 32 1.0 DSP-4 hydrochloride C 8417 32 1.0 CB 1954 C 2235 32 1.0 4-Aminobenzamidine dihydrochloride A 7148 32 1.0 1-benzoyl-5-methoxy-2-methylindole-3-acetic acid B 1183 32 1.0 8-Bromo-cGMP sodium B 1381 32 1.0 (±)-Isoproterenol hydrochloride I 5627 33 1.0 R(-)-Me5 M 0814 33 1.0 Iproniazid phosphate I 7627 33 0.9 (-)-MK-801 hydrogen maleate M-108 33 0.9 Cibenzoline succinate C1618 33 0.9 L-alpha-Methyl DOPA M-129 33 1.0 4-Methylpyrazole hydrochloride M 1387 33 0.9 Oxybutynin Chloride O 2881 33 0.9 MRS 1523 M 1809 33 1.0 ZM 39923 hydrochloride Z 4626 33 0.9 SB-525334 S 8822 33 1.0 Mianserin hydrochloride M 2525 33 0.9 SB 216763 S 3442 33 1.0 Imetit dihydrobromide I-135 33 1.0 1,5-Isoquinolinediol I-138 33 0.9 Nimesulide N 1016 33 1.0 Molsidomine M 2901 33 1.0 BBMP B7936 33 0.9 Mizoribine M 3047 33 1.0 MG 624 M 3184 33 1.0 alpha-Methyl-DL-tyrosine methyl ester hydrochloride M 3281 33 1.0 Kainic acid K 0250 33 1.0 Ketorolac tris salt K 1136 33 1.0 Ketotifen fumarate K 2628 33 1.0 NAN-190 hydrobromide N 3529 33 1.0 Pimozide P 1793 33 1.0 U-73343 U 6881 33 1.0 S-Nitrosoglutathione N 4148 33 1.0 Milrinone M 4659 33 1.0 1-Methylhistamine dihydrochloride M 4910 33 1.0 1,3-Dimethyl-8-phenylxanthine P 2278 33 1.0 S-Methyl-L-thiocitrulline acetate M 5171 33 1.0 Melatonin M 5250 33 1.0 L-Methionine sulfoximine M 5379 33 1.0 Naphazoline hydrochloride N 5504 33 1.0 3-Nitropropionic acid N 5636 33 1.0 (±)-Normetanephrine hydrochloride N 7127 33 1.0 1-Phenyl-3-(2-thiazolyl)-2-thiourea P 4015 33 1.0 Podophyllotoxin P 4405 33 1.0 VER-3323 hemifumarate salt V 1889 33 1.0 Lidocaine hydrochloride L 5647 33 1.0 L-687,384 hydrochloride L 8539 33 1.0 Praziquantel P 4668 33 1.0 LY-294,002 hydrochloride L 9908 33 1.0 Loxapine succinate L-106 33 1.0 GR 127935 hydrochloride hydrate G 5793 33 0.9 Edrophonium chloride E 3256 33 1.0 Ellipticine E 3380 33 1.0 Isoguvacine hydrochloride G-002 33 1.0 Guvacine hydrochloride G-007 33 1.0 Demeclocycline hydrochloride D 6140 33 1.0 Muscimol hydrobromide G-019 33 1.0 2,2'-Bipyridyl D 7505 33 1.0 CGS-12066A maleate C-106 33 1.0 N-Ethylmaleimide E 3876 33 1.0 2-Cyclooctyl-2-hydroxyethylamine hydrochloride C-108 33 1.0 5-Carboxamidotryptamine maleate C-117 33 1.0 (±)-CGP-12177A hydrochloride C-125 33 1.0

12 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( DL-Homatropine hydrobromide H 0126 33 1.0 3,4-Dichloroisocoumarin D 7910 33 1.0 beta-Estradiol E 8875 33 1.0 Phenserine P0111 33 1.0 Hypotaurine H 1384 33 1.0 Haloperidol H 1512 33 1.0 Cytidine 5'-diphosphocholine sodium salt hydrate C0256 33 1.0 Hydralazine hydrochloride H 1753 33 1.0 S-(-)-Eticlopride hydrochloride E-101 33 1.0 Fenoterol hydrobromide F 1016 33 1.0 Ciproxifan hydrochloride H 3132 33 1.0 Dantrolene sodium D 9175 33 1.0 Hydrocortisone H 4001 33 1.0 R(-)-Propylnorapomorphine hydrochloride D-027 33 1.0 Histamine dihydrochloride H 7250 33 1.0 JS-K J 4137 33 1.1 Retinoic acid p-hydroxyanilide H 7779 33 1.0 Fenspiride hydrochloride F 6145 33 1.0 Genipin G4796 33 1.0 1,4-Dideoxy-1,4-imino-D-arabinitol D 1542 33 1.0 Fusaric acid F 6513 33 1.1 Esomeprazole magnesium dihydrate E 7906 33 1.0 Fenoldopam bromide F 6800 33 0.9 R(-)-N-Allylnorapomorphine hydrobromide D-042 33 1.1 (±)-SKF-38393 hydrochloride D-047 33 1.0 Hydroxyurea H 8627 33 1.0 (+)-Hydrastine H 8645 33 1.0 (±)-DOI hydrochloride D-101 33 1.0 Famotidine F 6889 33 1.0 FSCPX F 7927 33 1.0 Dihydroergotamine methanesulfonate D 2763 33 1.0 NS8593 hydrochloride N 2538 33 1.0 2,3-Butanedione D 3634 33 1.0 Flupirtine maleate F 8927 33 1.0 1,3-Dipropyl-7-methylxanthine D-108 33 1.1 Domperidone D-122 33 1.0 Eliprodil E 2031 33 1.0 5-Hydroxy-L-tryptophan H 9772 33 1.0 N-Methyl-1-deoxynojirimycin M 1777 33 1.0 Fluoxetine hydrochloride F-132 33 1.0 Dihydro-beta-erythroidine hydrobromide D-149 33 1.0 Dilazep hydrochloride D 5294 33 1.0 1,7-Dimethylxanthine D 5385 33 1.0 L-Glutamine G 3126 33 1.0 DL-alpha-Difluoromethylornithine hydrochloride D-193 33 1.0 ATPO A 7845 33 1.0 NSC 95397 N 1786 33 1.0 Etodolac E 0516 33 1.0 alpha-Guanidinoglutaric acid G 7788 33 1.0 HA-100 I 1392 33 1.0 Phosphonoacetic acid P 6909 33 0.9 Ropinirole hydrochloride R 2530 33 1.0 Resveratrol R 5010 33 1.0 Putrescine dihydrochloride P 7505 33 0.9 PAPP S-009 33 0.9 SR-95531 S-106 33 0.9 Pargyline hydrochloride P 8013 33 1.0 Rottlerin R 5648 33 0.9 Ro 25-6981 hydrochloride R 7150 33 1.0 Thioridazine hydrochloride T 9025 33 0.9 Thapsigargin T 9033 33 1.0 Sepiapterin S-154 33 0.9 Amisulpride A 2729 33 0.9 SB 206553 hydrochloride S-180 33 0.9 (S)-Propranolol hydrochloride P 8688 33 1.0 RX 821002 hydrochloride R 9525 33 0.9 Terfenadine T 9652 33 0.9 Ranitidine hydrochloride R-101 33 0.9

13 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( Trifluperidol hydrochloride T-103 33 1.0 Ro 41-1049 hydrochloride R-107 33 1.0 Tiapride hydrochloride T 0410 33 0.9 Protriptyline hydrochloride P 8813 33 0.9 6(5H)-Phenanthridinone P 8852 33 1.0 Tetraethylthiuram disulfide T 1132 33 1.0 TCPOBOP T 1443 33 1.0 K114 K 1015 33 1.0 Prochlorperazine dimaleate P 9178 33 1.0 Telenzepine dihydrochloride T-122 33 1.0 Thioperamide maleate T-123 33 0.9 Riluzole R-116 33 0.9 Steviol H8664 33 1.0 A-68930 hydrochloride A 8852 33 0.9 Tizanidine hydrochloride T 6950 33 1.0 Theophylline T 1633 33 0.9 1,10-Phenanthroline monohydrate P 9375 33 1.0 Ro 04-6790 dihydrochloride R-140 33 1.0 4-Hydroxyphenethylamine hydrochloride T 2879 33 1.0 R(+)-3PPP hydrochloride P-102 33 1.0 S(-)-3PPP hydrochloride P-103 33 1.0 L-765,314 L 3040 33 1.0 UK 14,304 U-104 33 1.0 Tetrabenazine T2952 33 1.0 (+)-Norfenfluramine hydrochloride N3288 33 1.0 Semicarbazide hydrochloride S 2201 33 1.0 U-101958 maleate U-115 33 1.0 Tetrahydrozoline hydrochloride T 4264 33 1.0 1-Phenylbiguanide P-120 33 1.0 SID7969543 S1323 33 1.0 Pirenperone P-126 33 1.0 cDPCP C 0996 33 0.9 (±)-Taxifolin T 4512 33 1.0 SNC80 S 2812 33 1.0 (±)-Verapamil hydrochloride V 4629 33 1.0 Terazosin hydrochloride T 4680 33 1.0 N-Oleoyldopamine O 2139 33 1.0 L-Mimosine from Koa hoale seeds M0253 33 1.0 D-Serine S 4250 33 1.0 PD 168,077 maleate P-233 33 1.0 SB 269970 hydrochloride S 7389 33 1.0 Spiperone hydrochloride S 7395 33 1.0 WAY-100635 maleate W-108 33 1.0 SB 205384 S 7936 33 1.0 Na-p-Tosyl-L-lysine chloromethyl ketone hydrochloride T 7254 33 1.0 Chelerythrine chloride C 2932 33 0.9 N-Acetyltryptamine A 7342 33 0.9 Cortisone 21-acetate C 3130 33 0.9 L-allylglycine A 7762 33 1.0 Bumetanide B 3023 33 0.9 Benazoline oxalate B 4555 33 0.9 Atropine&methyl&nitrate A&0382 33 1.0 Arcaine&sulfate A&0384 33 1.0 ATPO A 7845 33 0.9 Calmidazolium chloride C 3930 33 0.9 SB 202190 S 7067 33 0.9 (±)-Norepinephrine (+)bitartrate A 0937 33 0.9 Brefeldin A from Penicillium brefeldianum B 7651 33 0.9 (±)-2-Amino-4-phosphonobutyric acid A 1910 33 1.0 Alfuzosin hydrochloride A 0232 33 1.0 Cefazolin sodium C 5020 33 0.9 (±)-p-Aminoglutethimide A 9657 33 1.0 Aniracetam A 9950 33 0.9 CL 316,243 C 5976 33 1.0 7-Chloro-4-hydroxy-2-phenyl-1,8-naphthyridine C 5982 33 1.0 (-)-Bicuculline methbromide, 1(S), 9(R) B-103 33 1.0 10058-F4 F3680 33 1.0 BTCP hydrochloride B-138 33 1.0

14 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( 5'-Amino-5'-deoxyadenosine p-toluenesulfonate salt A283 33 1.0 GR 79236X G5794 33 1.0 Clozapine C 6305 33 1.0 Aminobenztropine A-138 33 1.0 (±)-p-Chlorophenylalanine C 6506 33 1.0 Chloroquine diphosphate C 6628 33 1.0 B-HT 933 dihydrochloride B-161 33 1.0 Biperiden hydrochloride B 5311 33 1.0 Chlorambucil C 0253 33 1.0 L-Arginine A 5006 33 1.0 1-Allyl-3,7-dimethyl-8-p-sulfophenylxanthine A-145 33 1.0 (±)323Amino353phosphonopentanoic&acid A&5282 33 1.0 L-732,138 A 5330 33 0.9 Acetylsalicylic acid A 5376 33 1.0 4-Androsten-4-ol-3,17-dione A 5791 33 1.0 2-(2-Aminoethyl)isothiourea dihydrobromide A 5879 33 1.0 Cyclophosphamide monohydrate C 0768 33 1.0 SKF-89145 hydrobromide S 3316 33 1.0 cis-4-Aminocrotonic acid A-201 33 1.0 N3Acetylprocainamide&HCl A&5909 33 1.0 Amifostine A 5922 33 1.0 A-77636 hydrochloride A-255 33 0.9 Beclomethasone B 0385 33 1.0 Choline bromide C 1754 33 1.0 Carboplatin C 2538 33 1.0 Clemastine fumarate C 8903 33 1.0 beta-Chloro-L-alanine hydrochloride C 9033 33 1.0 Ifenprodil tartrate I 2892 34 0.9 TMPH hydrochloride T5576 34 0.9 Nimodipine N-149 34 0.9 cis(+/-)-8-OH-PBZI hydrobromide P 0618 34 0.9 Levallorphan tartrate L-121 34 1.0 AFMK A 2355 34 1.0 Minocycline hydrochloride M 9511 34 0.9 Proglumide M-001 34 0.9 Metoclopramide hydrochloride M 0763 34 1.0 NS 2028 N-211 34 1.0 (±)-Octopamine hydrochloride O 0250 34 1.0 L-N6-(1-Iminoethyl)lysine hydrochloride I 8021 34 0.9 3-Iodo-L-tyrosine I 8250 34 0.9 Imiloxan hydrochloride I 9531 34 1.0 Oleic Acid O 1008 34 0.9 Moxonidine hydrochloride M 1559 34 0.9 Ouabain O 3125 34 1.0 ICI 204,448 hydrochloride I-122 34 1.0 p-MPPI hydrochloride M-204 34 0.9 Metaproterenol hemisulfate M 2398 34 0.9 TG003 T 5575 34 0.9 Oxotremorine sesquifumarate salt O 9126 34 0.9 8-Methoxymethyl-3-isobutyl-1-methylxanthine M 2547 34 0.9 Trandolapril T4827 34 1.0 Oxaprozin O 9637 34 1.0 Molindone hydrochloride M 1818 34 0.9 p-MPPF dihydrochloride M-226 34 0.9 Levetiracetam L8668 34 1.0 Nialamide N 1392 34 1.0 JWH-015 J 4252 34 1.0 Progesterone P 0130 34 0.9 S-Methylisothiourea hemisulfate M 3127 34 1.0 S-(4-Nitrobenzyl)-6-thioinosine N 2255 34 0.9 S-Nitroso-N-acetylpenicillamine N 3398 34 1.0 (±)-Propranolol hydrochloride P 0884 34 1.0 MRS 2179 M 3808 34 1.0 Meloxicam sodium M 3935 34 1.0 Morin M 4008 34 1.0 Pentoxifylline P 1784 34 1.0 Minoxidil M 4145 34 1.0 L-Glutamic acid, N-phthaloyl- P 1801 34 1.0

15 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( NCS-382 N 4159 34 1.0 loxoprofen L 0664 34 1.0 Labetalol hydrochloride L 1011 34 1.0 5-Nitro-2-(3-phenylpropylamino)benzoic acid N 4779 34 1.0 AMN082 A6605 34 1.0 Pirfenidone P 2116 34 1.0 Olvanil O 0257 34 1.0 Loperamide hydrochloride L 4762 34 1.0 Lonidamine L 4900 34 1.0 NADPH tetrasodium N 7505 34 1.0 Thiolactomycin T 9567 34 1.0 (S)-MAP4 hydrochloride M 5560 34 1.0 Palmitoyl-DL-Carnitine chloride P 4509 34 1.0 Naloxone hydrochloride N 7758 34 1.0 Piracetam P 5295 34 1.0 Naltrindole hydrochloride N-115 34 1.0 Sertraline hydrochloride S 6319 34 1.0 (±)-AMPA hydrobromide G-017 34 0.9 S-(-)-Carbidopa C1335 34 1.0 (±)-Chloro-APB hydrobromide C-130 34 1.0 Paliperidone P0099 34 1.0 Daidzein D 7802 34 1.0 GR-89696 fumarate G-133 34 1.0 Dicyclomine hydrochloride D 7909 34 1.0 (±) trans-U-50488 methanesulfonate D 8040 34 1.0 1-(m-Chlorophenyl)-biguanide hydrochloride C-144 34 1.0 Estrone E 9750 34 1.0 3-deazaadenosine D 8296 34 1.0 Bendamustine hydrochloride B 5437 34 1.0 erythro-9-(2-Hydroxy-3-nonyl)adenine hydrochloride E-114 34 1.0 Cirazoline hydrochloride C-223 34 1.0 Alinidine A7230 34 1.0 Hydroxytacrine maleate H 3146 34 1.0 Doxazosin mesylate D 9815 34 1.0 DNQX D 0540 34 1.0 R(-)-Apocodeine hydrochloride D-003 34 1.0 1-(4-Hydroxybenzyl)imidazole-2-thiol H 6892 34 1.0 Flumazenil F 6300 34 1.0 SANT-1 S4572 34 1.0 Mephetyl tetrazole M 7945 34 1.1 Icilin I 9532 34 1.0 Dopamine hydrochloride H 8502 34 1.0 (±)-7-Hydroxy-DPAT hydrobromide H 8653 34 1.0 N,N,N',N'-Tetramethylazodicarboxamide D 3648 34 1.0 BNTX maleate salt hydrate B 8312 34 1.0 N-Methylhistaprodifen dioxalate salt M 7320 34 1.0 5-hydroxydecanoic acid sodium H-135 34 1.0 1-(2-Methoxyphenyl)piperazine hydrochloride S-008 34 0.9 Propionylpromazine hydrochloride P 7780 34 0.9 (±)-6-Chloro-PB hydrobromide S-143 34 0.6 Phloretin P 7912 34 1.0 BIA 2-093 B 5435 34 1.0 (±)-SKF 38393, N-allyl-, hydrobromide S-168 34 0.9 Phenylbutazone P 8386 34 1.0 Taurine T 0625 34 0.9 CCT007093 C 9369 34 1.0 Sodium nitroprusside dihydrate S 0501 34 1.0 Urapidil hydrochloride U-100 34 1.0 Sulindac sulfone S 1438 34 1.0 2-Phenylaminoadenosine P-101 34 1.0 Trimipramine maleate T 3146 34 1.0 (±)-PPHT hydrochloride P-105 34 1.0 SKF 86466 S 1563 34 1.0 (-)-Scopolamine hydrobromide S 1875 34 1.0 S(-)-UH-301 hydrochloride U-108 34 1.0 (-)-trans-(1S,2S)-U-50488 hydrochloride U-111 34 1.0 N-p-Tosyl-L-phenylalanine chloromethyl ketone T 4376 34 1.0 (6R)-5,6,7,8-Tetrahydro-L-biopterin hydrochloride T 4425 34 1.0

16 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( SKF 83959 hydrobromide S 2816 34 1.0 S(+)-PD 128,907 hydrochloride P-183 34 1.0 Spironolactone S 3378 34 1.0 (±)-Vesamicol hydrochloride V-100 34 1.0 Tulobuterol hydrochloride T 6050 34 1.0 1-(2-Chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane C 3010 34 0.9 TMB38&hydrochloride 861804 34 0.9 43aminopyridine A&0152 34 0.9 Allopurinol A 8003 34 0.9 Alprenolol hydrochloride A 8676 34 1.0 Bay 11-7085 B 5681 34 0.9 p-Aminoclonidine hydrochloride A 0779 34 0.9 Reserpine R 0875 34 1.0 BW 284c51 A 9013 34 0.9 Aminopterin A 1784 34 0.9 Bestatin hydrochloride B 8385 34 1.0 Cephalexin hydrate C 4895 34 0.9 L(-)-Norepinephrine bitartrate A 9512 34 0.9 BRL 15572 B 9929 34 0.9 Chloroethylclonidine dihydrochloride B-003 34 1.0 Antozoline hydrochloride A 9899 34 0.9 PNU-37883A P 0248 34 1.0 Amoxapine A-129 34 1.0 Cytosine-1-beta-D-arabinofuranoside hydrochloride C 6645 34 0.9 BRL 37344 sodium B-169 34 1.0 Amiprilose hydrochloride A 4687 34 1.0 Alaproclate hydrochloride A-164 34 1.0 Clomipramine hydrochloride C 7291 34 1.0 Carmustine C 0400 34 1.0 Aconitine A 8001 34 1.0 Citalopram hydrobromide C 7861 34 1.0 Clonidine hydrochloride C 7897 34 1.0 CP55940 C 1112 34 1.0 Cinoxacin C 8645 34 1.0 Atropine methyl bromide A 6883 34 1.0 BTO-1 B 6311 34 1.0 Lorglumide sodium L-109 35 0.9 Nylidrin hydrochloride N-153 35 0.9 (-)-Isoproterenol hydrochloride I 6504 35 0.9 Indomethacin I 7378 35 1.0 Isoxanthopterin I 7388 35 1.0 Dihydrocapsaicin M 1022 35 1.0 S(+)-Isoproterenol (+)-bitartrate I 8005 35 0.9 Nocodazole M 1404 35 0.9 Melphalan M 2011 35 1.0 m-Iodobenzylguanidine hemisulfate I 9890 35 0.9 Methiothepin mesylate M-149 35 0.9 Nemadipine-A N4163 35 0.9 3-Morpholinosydnonimine hydrochloride M-184 35 1.0 3-Methoxy-morphanin hydrochloride M-187 35 1.0 Methylergonovine maleate M 2776 35 1.0 Sivelestat sodium salt hydrate S 7198 35 0.9 ML 10302 M 7319 35 1.0 Pindolol P 0778 35 1.0 Neostigmine bromide N2001 35 1.0 Ketoconazole K 1003 35 1.0 Ketoprofen K 1751 35 1.0 (-)-cis-(1S,2R)-U-50488 tartrate U-106 35 1.0 Picrotoxin P 1675 35 1.0 LP44 L9793 35 1.0 Rufinamide R 8404 35 1.0 Nalidixic acid sodium N 4382 35 1.0 CyPPA C5493 35 1.0 beta-Lapachone L 2037 35 1.0 Lamotrigine L 3791 35 1.0 Nortriptyline hydrochloride N 7261 35 1.0 Metrazoline oxalate M 5685 35 1.0 GW9662 M 6191 35 1.0

17 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( Se-(methyl)selenocysteine hydrochloride M 6680 35 1.0 Ro 90-7501 R 0529 35 0.6 (-)-Epinephrine bitartrate E 4375 35 1.0 DBO-83 D 7938 35 1.0 Tamoxifen T 5648 35 1.0 Tocainide hydrochloride T0202 35 1.0 AS 604850 A 0231 35 1.0 Fiduxosin hydrochloride F 4303 35 0.9 Diphenyleneiodonium chloride D 2926 35 1.0 Nefiracetam N 2288 35 1.0 R-(+)-8-Hydroxy-DPAT hydrobromide H-140 35 1.0 Doxepin hydrochloride D 4526 35 1.0 Tomoxetine T 7947 35 0.9 1,4-PBIT dihydrobromide P 8352 35 0.9 THIP hydrochloride T-101 35 1.0 XCT790 X4753 35 1.0 Tolbutamide T 0891 35 1.0 Tetraisopropyl pyrophosphoramide T 1505 35 1.0 TPMPA T-200 35 1.0 (E)-4-amino-2-butenoic acid T 1694 35 1.0 U-74389G maleate U 5882 35 0.9 Urapidil, 5-Methyl- U-101 35 1.0 Triflupromazine hydrochloride T 2896 35 1.0 Enalaprilat dihydrate E9658 35 1.0 U-62066 U-105 35 1.0 CGP 57380 C0993 35 1.0 Theobromine T 4500 35 1.0 Pinacidil P-154 35 1.0 SKF 75670 hydrobromide S 2941 35 1.0 Vancomycin hydrochloride from Streptomyces orientalis V 8138 35 1.0 Phenylbenzene-omega-phosphono-alpha-amino acid P-204 35 1.0 Thio-NADP sodium T 5515 35 1.0 Betaine hydrochloride B 3501 35 0.9 Betaine aldehyde chloride B 3650 35 0.9 Amitriptyline hydrochloride A 8404 35 0.9 Ancitabine hydrochloride A 8598 35 1.0 L-azetidine-2-carboxylic acid A 0760 35 0.9 Chlorzoxazone C 4397 35 0.9 Aminoguanidine hydrochloride A 8835 35 0.9 L-Cysteinesulfinic Acid C 4418 35 0.9 Budesonide B 7777 35 0.9 S-(p-Azidophenacyl)glutathione A 1782 35 0.9 8-Bromo-cAMP sodium B 7880 35 0.9 Benztropine mesylate B 8262 35 1.0 5-(N,N-hexamethylene)amiloride A 9561 35 0.9 4-Chloromercuribenzoic acid C 5913 35 1.0 (±)-Bay K 8644 B-112 35 0.9 BMY 7378 dihydrochloride B-134 35 1.0 Tracazolate T-112 35 1.0 Cefmetazole sodium C 6048 35 1.0 Clofibrate C 6643 35 1.0 (±)-Butaclamol hydrochloride B-168 35 1.0 trans-(±)-ACPD A-155 35 1.0 Opipramol dihydrochloride O 5889 35 1.0 N6-Cyclopentyladenosine C 8031 35 1.0 Cantharidic Acid C 8088 35 1.0 Sodium&Taurocholate&hydrate T&9034 35 1.0 Nisoxetine hydrochloride N-151 36 0.9 Maprotiline hydrochloride M 9651 36 0.9 1-(5-Isoquinolinylsulfonyl)-3-methylpiperazine dihydrochloride I 6391 36 0.9 H-8 dihydrochloride M 9656 36 0.9 Naloxone benzoylhydrazone N-165 36 0.9 NS-1619 N-170 36 0.9 Naloxonazine dihydrochloride N-176 36 0.9 NBQX disodium N-183 36 1.0 (-)-Naproxen sodium M 1275 36 1.0 2-Methyl-5-hydroxytryptamine maleate M-109 36 0.9 Metolazone M-116 36 1.0

18 Table&S2

(Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No. %(Inhibition Interference( N-omega-Methyl-5-hydroxytryptamine oxalate salt M 1514 36 0.9 Oxymetazoline hydrochloride O 2378 36 0.9 S(+)-Ibuprofen I-106 36 0.9 p-Iodoclonidine hydrochloride I-114 36 0.9 Moclobemide M3071 36 0.9 R(+)-IAA-94 I-117 36 1.0 Ofloxacin O 8757 36 0.9 Piceatannol P 0453 36 0.9 TBB T 0826 36 1.0 Parthenolide P 0667 36 1.0 CR 2249 N 2034 36 1.0 Naltrexone hydrochloride N 3136 36 1.0 Kynurenic acid K 3375 36 1.0 Clorgyline hydrochloride M 3778 36 1.0 SKF-525A hydrochloride P 1061 36 0.9 Moxisylyte hydrochloride M 5154 36 1.0 Gabapentin G-154 36 1.0 ABT-702 dihydrochloride A2721 36 0.9 Guanidinyl-naltrindole di-trifluoroacetate G 3416 36 0.9 Raloxifene hydrochloride R 1402 36 0.9 Phentolamine mesylate P 7561 36 0.9 Spiroxatrine S-103 36 0.9 Trifluoperazine dihydrochloride T 8516 36 0.9 SDZ-205,557 hydrochloride S-174 36 1.0 Ribavirin R 9644 36 0.9 Tropicamide T 9778 36 0.9 Pergolide methanesulfonate P 8828 36 1.0 FAUC 213 F 4429 36 0.9 (±)-Thalidomide T-144 36 1.0 SR 57227A S 1688 36 1.0 (±)-Chlorpheniramine maleate C 3025 36 0.9 ABT-418 hydrochloride A 6476 36 1.0 Betaxolol hydrochloride B 5683 36 0.9 9-cyclopentyladenine C 4479 36 0.9 (+)-Chlorpheniramine maleate C 4915 36 0.9 Apomorphine hydrochloride hemihydrate A 4393 36 1.0 CHM-1 hydrate C1244 37 0.9 Idazoxan hydrochloride I 6138 37 0.9 Imipramine hydrochloride I 7379 37 1.0 Linopirdine L-134 37 0.9 alpha-Methyl-5-hydroxytryptamine maleate M-110 37 0.9 Sodium Oxamate O 2751 37 0.9 MDL 26,630 trihydrochloride M-166 37 0.9 Palmitoylethanolamide P 0359 37 1.0 Ranolazine dihydrochloride R 6152 37 0.9 3-Tropanyl-3,5-dichlorobenzoate T 8160 37 0.9 Rolipram R 6520 37 1.0 L-741,626 L-135 38 0.9 L-703,606 oxalate salt hydrate L119 38 0.9 R(-)-N6-(2-Phenylisopropyl)adenosine P 4532 38 1.0 ( R)-(+)-WIN 55,212-2 mesylate W-102 38 1.0 8-(4-Chlorophenylthio)-cAMP sodium C 3912 38 0.9 Amsacrine hydrochloride A 9809 38 0.9 Promazine hydrochloride P 6656 39 0.9 AGK2 A 8231 312 0.8 Ruthenium red R 2751 398 0.0

19 Table&S3

DNA*Unwinding* *Cy5;dT15;SSB*Binding* Structure CID*No. 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

DNA*Unwinding* *Cy5;dT15;SSB*Binding* Structure CID*No. 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

DNA*Unwinding* *Cy5;dT15;SSB*Binding* Structure CID*No. 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

DNA*Unwinding* *Cy5;dT15;SSB*Binding* Structure CID*No. 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

DNA*Unwinding* *Cy5;dT15;SSB*Binding* Structure CID*No. 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

DNA*Unwinding* *Cy5;dT15;SSB*Binding* Structure CID*No. 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

DNA*Unwinding* *Cy5;dT15;SSB*Binding* Structure CID*No. IC50,*µM IC50,*µM

49849290 10&±&5 2&±&0

44251434 7&±&4 1&±&0