Optimization of potent hepatitis C virus NS3 helicase inhibitors isolated from the yellow dyes thioflavine S and primuline

Article abstract of DOI:10.1021/jm300021v

A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (P4), a benzothiazole tetramer, inhibited unwinding >50% at 2 ± 1 μM, inhibited the subgenomic HCV replicon at 10 μM, and was not toxic at 100 μM. Because P4 also interacted with DNA, more specific analogues were synthesized from the abundant dimeric component of primuline. Some of the 32 analogues prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6 ± 1 μM.

Full text of DOI:10.1021/jm300021v

Article
pubs.acs.org/jmc
Optimization of Potent Hepatitis C Virus NS3 Helicase Inhibitors
Isolated from the Yellow Dyes Thioflavine S and Primuline
Kelin Li,† Kevin J. Frankowski,† Craig A. Belon,‡ Ben Neuenswander,† Jean Ndjomou,§ Alicia M. Hanson,§
Matthew A. Shanahan,§ Frank J. Schoenen,† Brian S. J. Blagg,†,∥ Jeffrey Aube,†,∥ and David N. Frick*,§
́
†University of Kansas Specialized Chemistry Center, University of Kansas, 2034 Becker Drive, Lawrence, Kansas 66047, United States
‡Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, United States
§Department of Chemistry and Biochemistry, University of WisconsinMilwaukee, 3210 North Cramer Street, Milwaukee,
Wisconsin 53211, United States
∥Department of Medicinal Chemistry, University of Kansas, 1251 Wescoe Hall Drive, Lawrence, Kansas 66045, United States
S
* Supporting Information
ABSTRACT: A screen for hepatitis C virus (HCV) NS3
helicase inhibitors revealed that the commercial dye thioflavine
S was the most potent inhibitor of NS3-catalyzed DNA and
RNA unwinding in the 827-compound National Cancer
Institute Mechanistic Set. Thioflavine S and the related dye
primuline were separated here into their pure components, all
of which were oligomers of substituted benzothiazoles. The
most potent compound (P4), a benzothiazole tetramer,
inhibited unwinding >50% at 2
1 μM, inhibited the
subgenomic HCV replicon at 10 μM, and was not toxic at 100
μM. Because P4 also interacted with DNA, more specific
analogues were synthesized from the abundant dimeric component of primuline. Some of the 32 analogues prepared retained
ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors
(compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6 1 μM.
from the ca. 3000-amino-acid-long polypeptide encoded by the
HCV RNA genome. Viral and host proteases cleave the HCV
polyprotein into mature structural (core, E1, E2) and
nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A,
and NS5B). The HCV nonstructural proteins form four
enzymes. NS5B is a polymerase that synthesizes new viral
RNA. The NS2 and NS3 proteins combine to form an
autocatalytic protease. NS3 and NS4A combine to form a
serine protease that cuts itself, cleaves the NS4B/NS5A and
NS5A/NS5B junctions and some cellular proteins. Most
relevant to the present work, NS3 is also an ATP-fueled
helicase that can separate and rearrange RNA/RNA, RNA/
DNA, and DNA/DNA nucleic acid duplexes and displace
nucleic acid bound proteins.6
Helicases have been widely studied as potential drug targets
although progress has been slower than for other viral
enzymes.7,8 Nevertheless, HCV needs a functional helicase to
replicate in cells,9−11 and small molecules that inhibit HCV
helicase-catalyzed reactions also inhibit cellular HCV RNA
replication.12−14 In this paper, we report a new class of
compounds that inhibit the NS3 helicase and also act against
the HCV replicon. We describe the procurement of these
INTRODUCTION
■
The hepatitis C virus (HCV) infects about 170 million people
worldwide, causing profound morbidity and mortality.1 HCV is
typically treated with the nucleoside analogue ribavirin
combined with recombinant human alpha interferon. Although
such treatments are effective, therapy is poorly tolerated,
expensive, and not equally effective against all HCV genotypes.2
Better HCV treatments are therefore being modeled on other
antivirals which, unlike interferon and ribavirin, directly attack
proteins that HCV synthesizes in human cells. Such “direct
acting antivirals” (DAAs) typically are small molecules that
inhibit viral enzymes, with the most common targets being the
HCV RNA polymerase and an HCV protease. Two HCV
protease inhibitors, telaprevir3 and boceprevir,4 were recently
approved for use in HCV patients, but neither alone eradicates
HCV infection because HCV rapidly evolves to become
resistant to these first-generation DAAs.5 Protease inhibitors
need to be administered with interferon and ribavirin and,
consequently, many patients still poorly tolerate the new
therapies. The overall goal of this project is to find new DAAs
for HCV that might be used with telaprevir, boceprevir, or
similar drugs to replace interferon and ribavirin in HCV
therapy.
Telaprevir and boceprevir both inhibit the HCV non-
structural protein 3 (NS3). NS3 is one of 10 proteins derived
Received: January 5, 2012
Published: March 12, 2012
© 2012 American Chemical Society
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Figure 1. Discovery of thioflavine S as a HCV helicase inhibitor. (A) Schematic drawing of the MBHA mechanism. (B) The NCI Mechanistic Set of
827 compounds was screened with an MBHA, each at 20 μM. Fluorescence was read before and 30 min after ATP addition, and compound
inhibition was calculated from F30/F0 ratios. Hits were defined as compounds inhibiting more than 50% and interfering less than 20%. (C) Hits from
the MBHA primary screen were tested for their DNA-binding capacity with an FID counterscreen at 1.5 μM compound concentration, and percent
binding was calculated. Numbers refer to NSC numbers. (D) Concentration response curves for thioflavine S when assayed in MBHAs using either a
DNA or RNA substrate.
inhibitors via isolation from commercial samples of thioflavine
S and primuline, two chemically related dye preparations, and
through the chemical synthesis of congeners based on those
isolated leads.
ATP addition (F30/F0 ratio). In other words, the MBHA can be
used as an “internal” counter-screen to identify compounds that
appear to affect unwinding because they interfere with the
assay. The most common types of interfering compounds are
those that fluoresce or absorb light at the same wavelengths as
the MBHA Cy5-labeled substrate. Alternatively, other com-
pounds may bind the DNA substrate and distort it to change
how the quenching moiety on the beacon is oriented relative to
the Cy5 fluorophore.
To identify HCV helicase inhibitors, the MBHA was used to
screen the National Cancer Institute Developmental Ther-
apeutics Program’s Mechanistic Set Library (http://dtp.nci.nih.
gov/branches/dscb/mechanistic_explanation.html). In total,
827 compounds (at 20 μM) were screened using a MBHA
with a DNA substrate (Table S2, Supporting Information).
When compound interference was plotted versus percent
inhibition (Figure 1B), it was clear that the majority of
compounds that appeared to inhibit HCV helicase also
quenched fluorescence of the MBHA substrate. Compound
interference in the MBHA was evaluated by comparing the
fluorescence of assays containing each compound to the
fluorescence of DMSO-only negative controls before the
addition of ATP. Hits were defined as those compounds that
did not interfere with the assays more than 20%, of which 12
were identified. These 12 hits were then subjected to a
counterscreen that was designed to independently identify
compounds that exhibit DNA-binding properties using a
modified fluorescent intercalator displacement (FID) assay.17
The FID counterscreen used ethidium bromide to determine a
RESULTS
■
Assay Development and Screening. Numerous HCV
helicase inhibitors have been reported in the literature, but
many of these also bind the helicase nucleic acid substrate.
HCV inhibitors that interact with DNA or RNA could also
inhibit cellular nucleic acid enzymes like RNA polymerase,15
therefore such compounds might not act like true DAAs. The
goal of this study was to identify chemical probes that
specifically target NS3. Such compounds are needed to better
understand why the virus encodes a helicase, which may lead to
better candidates for drug development. To facilitate HCV
helicase inhibitor identification, Belon and Frick developed a
new helicase assay that simultaneously identifies compounds
that interact with the helicase substrate and compounds that
inhibit helicase action.16 This molecular beacon-based helicase
assay (MBHA) uses a dual-labeled hairpin-forming DNA
oligonucleotide annealed to a longer oligonucleotide, which
forms a tail for the helicase to load (Figure 1A). Once ATP is
added, the helicase displaces the molecular beacon, resulting in
a decrease in substrate fluorescence. By comparing substrate
fluorescence before ATP is added (F0), one can identify
compounds that bind the MBHA substrate. At the same time,
compounds that inhibit helicase action can be identified by
fluorescence changes in an MBHA before and 30 min after
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Table 1. Activity for Isolated, Pure Compounds from Primuline or Thioflavine S
a
a
a
a
compd
helicase IC50 (μM) DNA binding (ethidium bromide) EC50 (μM) DNA binding (SYBR Green I) EC50 (μM) ATPase IC50 (μM)
thioflavine S
T1
24 1.3
33 24
>100
>100
61 37
ND
50 17
>100
ND
b
T2
26 21
12
70 31
>100
ND
primuline
P1a
1
>100
43 14
>100
>100
ND
67 27
>100
>100
ND
>100
P1b
122
5
>100
b
P2a
49 45
>100
P2b
10 4.6
0.9 0.1
0.9 0.4
73 36
55 20
32
ND
15
3
>100
P3
15
5
4
c
P4
15
3
8
3
a
Helicase (MBHA), DNA binding (FID), and ATP hydrolysis were monitored in the presence of eight different concentrations of each compound
(2-fold dilution series starting at 100 μM). IC50 values were determined from concentration−response curves. All values are means standard
b
c
deviations from three independent titrations with inhibitor. ND, not determined. Average value from three different batches of compound. Average
value from two different batches of compound.
Figure 2. Structures of isolated, pure compounds from thioflavine S (T) and primuline (P) dyes.
molecule’s ability to bind DNA and is based on the assumption
that a DNA-binding compound displaces a fluorescent DNA
intercalating agent, leading to a decrease in observed
fluorescence. Compounds were tested at 1.5 μM in the
presence of the 25 base pair substrate used in the helicase
assays (Figure 1C). Results showed that even at a compound
concentration 13 times lower than that used in the MBHA,
most of the hit compounds decreased the fluorescence of an
ethidium bromide−DNA complex by more than 10%,
indicative of the molecule’s ability to bind DNA. The DNA
was observed. In concentration response experiments (Figure
1D), thioflavine S inhibited HCV helicase-catalyzed DNA-
unwinding with an IC50 of 10 1 μM, and it inhibited HCV
catalyzed RNA unwinding with an IC50 of 12
2 μM. The
related dye thioflavine T, which, like thioflavine S, is used to
specifically stain Alzheimer amyloid plaques,21 had no effect on
either HCV-catalyzed DNA- or RNA-unwinding (data not
shown).
Purification and Characterization of HCV Helicase
Inhibitors. Thioflavine S is not a single compound but rather a
heterogeneous dye that is structurally related to another
heterogeneous yellow dye, primuline.22 Primuline (MP
Biomedicals cat. no. 195454) inhibited HCV helicase in
MBHAs with about twice the potency as thioflavine S (Table
1). To better understand how these dyes inhibit HCV helicase,
both dye mixtures were separated using reverse-phase
preparative HPLC or a combination of normal-phase silica
gel column chromatography and reverse-phase preparative
HPLC.
The structure of commercial samples of thioflavine S is not
reported consistently or is left intentionally vague, creating
confusion over the chemical identity of the screening hit. For
instance the MSDS (Sigma Aldrich) for thioflavine S describes
the compound only as “methylated, sulfonated primuline base.”
In the NCI and PubChem online databases, thioflavine S
(NSC71948, SID550242) was reported as a mixture of
methylated benzothiazoles and Packham’s group reported the
same structure.23 To identify the components of commercially
obtained thioflavine S responsible for the observed activity, we
purified the commercial sample (Sigma cat. no. T1892) via
reverse-phase preparative HPLC to obtain two compounds (T1
and T2), the structures of which were assigned using NMR and
LC/MS (Figure 2).
minor-groove-binding compound Berenil (IC50 = 1.6
0.1
μM) was used a positive control in all FID assays.18
Four compounds decreased the fluorescence of DNA-bound
ethidium bromide less than 8%. The first, CdCl2, was a known
HCV helicase inhibitor that binds in place of the magnesium
ion needed for ATP hydrolysis to fuel unwinding.19 The
second, ellipticine, was found to fully quench DNA-bound
ethidium bromide fluorescence at higher concentrations, and
the IC50 value for ellipticine in MBHAs (5.6 0.8 μM) was
similar to its apparent affinity for DNA, suggesting that it
inhibited the helicase by interacting with the substrate.
Chromomycin A3 inhibited HCV helicase-catalyzed-DNA
unwinding with an IC50 of 0.15 0.03 μM, but had no effect
on HCV helicase-catalyzed RNA unwinding (data not shown).
This false positive can be explained by the fact that
chromomycin A3 functionally resembles ethidium bromide,
and they both represent fluorescent DNA-binding com-
pounds.20 This result also demonstrates that not all DNA
binding compounds will decrease DNA-bound ethidium
bromide fluorescence in an ethidium bromide-based FID
assay. The final sample, thioflavine S, did not affect DNA-
bound ethidium bromide fluorescence until its concentration
exceeded 100 μM, at which point a 20% fluorescence decrease
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Figure 3. Effects of primuline and its components on HCV helicase-catalyzed DNA-unwinding and ATP hydrolysis. (A) MBHAs performed at
various concentrations of primuline. Reactions were initiated by adding ATP at the indicated time. (B) Initial rates of DNA unwinding in MBHAs
◇
○
△
containing indicated concentrations of primuline (*), compound P1a ( ), P3 ( ), or P4 ( ). Data are fit to eq 3 (methods). Individual reaction
time-courses and curve-fits used to calculate initial rates are shown in Figure S1 (Supporting Information). IC50s listed in Table 1 are the averages
□
○
△
)
from three separate titrations with each compound. (C) ATPase assays were performed in the absence of RNA ( ), 15 μM ( ), or 1 mM (
poly(U) RNA (measured as μM UMP). ATP was present at 1 mM, the reactions were initiated by rapidly mixing in NS3h, and the amount of
phosphate released was measured after 15 min at 26 °C. Various concentrations of P4 were present as indicated.
Contrary to our expectation that the isolated compounds
would be methylated primuline derivatives, isolated T1 and T2
were the N,N-diethyl products of the primuline monomeric and
dimeric benzothiazoles. Both T1 and T2 manifested some
inhibitory activity against helicase-catalyzed DNA unwinding
(Figure S1, Supporting Information, Table 1), but neither was
as potent as thioflavine S or primuline, suggesting that a minor
component of the dye was inhibiting HCV helicase action.
In total, six compounds were purified from primuline. The
two major components, P1a and P2a, were separated via
reverse-phase preparative HPLC in 9.2% and 7.6% isolated
yield (by weight for P1a and P2a, respectively). In the MBHA,
P2a was significantly less potent than the primuline mixture,
while P1a was effectively inactive. That the purified major
active component, P2a, did not possess increased potency
compared to the mixture containing the inactive P1a was
unexpected and hinted that, perhaps, highly potent components
could be present in the primuline mixture in small amounts.
The direct isolation of minor components via reverse-phase
preparative HPLC of the dye mixture was not successful.
Hence, the purification procedure was modified, enabling the
isolation of four minor components. Four chromatographic
bands enriched with minor components (UV and LC-MS)
were obtained from silica gel chromatography of commercial
primuline upon elution with 20% DCM/MeOH. Subsequent
reverse-phase preparative HPLC purification afforded the
relatively minor components P1b, P2b, P3, and P4, where
P3 and P4 represented 0.49% and 0.23% isolated yield (by
weight) of the dye, respectively. All purified compounds were
composed of a central benzothiazole oligomer of 1−4 units
terminating with a p-aminobenzene group (Figure 2).
sulfonate group on the terminal benzothiazole rings. FID
assays with the purified compounds revealed that they, indeed,
possessed some ability to bind DNA. Like thioflavine S, all
compounds, except T1, P1a and P1b, decreased the
fluorescence of ethidium bromide-bound DNA by at least
10% when present at 100 μM. However, only compounds P3
and P4 decreased ethidium bromide-bound DNA fluorescence
more than 50% at the highest concentration tested (100 μM).
P3 decreased the fluorescence of ethidium bromide-bound
DNA with an EC50 of 55
20 μM, and P4 decreased
fluorescence with an EC50 of 15 3 μM.
Because P3 and P4 clearly interacted with ethidium bromide-
stained DNA, we suspected that the other benzothiazoles might
also bind DNA but in ways that do not displace the intercalated
ethidium bromide. We therefore examined the effect of each
compound on DNA stained with other dyes, and found that
most compounds decreased the fluorescence of DNA stained
with SYBR Green I. The affinity of primuline, thioflavine S, and
the purified compounds for the MBHA substrate DNA was
therefore estimated using a modified FID assay where ethidium
bromide was replaced with SYBR Green I. The results were less
drastic compared to those seen with ethidium bromide, with P4
binding slightly more tightly than all other compounds (Table
1).
It should be noted that, assuming that IC50 values in the
MBHA reflect dissociation constants for a compound-helicase-
DNA complex and EC50 values reflect the affinity of a
compound for DNA, it appears thioflavine S and primuline
bind the helicase complex more tightly than they bind DNA
and that P4 binds DNA 17 times more weakly. These data
suggest that little, if any, compound was bound to DNA in
MBHAs at concentrations needed to inhibit helicase action,
suggesting that the yellow dye-derived benzothiazoles inhibit
HCV helicase directly.
In the MBHA, all of the compounds purified from primuline
were helicase inhibitors although P1a and P1b only partially
inhibited unwinding at the highest concentrations tested
(Figure S1, Supporting Information). Potency correlated with
the length of the benzothiazole chain. For P3 or P4, only about
1 μM of either was needed to reduce the rate of helicase-
catalyzed DNA-unwinding by 50% (Figure 3B, Table 1).
These purified compounds resemble molecules known to
bind DNA, typically along the minor groove, such as the
cyanine dye known as BEBO,24,25 but unlike many DNA-
binding benzothiazoles, these helicase inhibitors are not
positively charged. Instead they are anionic, due to the
If the purified benzothiazoles inhibit helicase-catalyzed
nucleic acid unwinding by directly binding NS3, then they
might also inhibit other functions of NS3, namely ATP
hydrolysis in the presence and absence of stimulating nucleic
acids. The most potent compound, P4, was therefore added to
NS3 ATPase assays. The compound inhibited both assays in a
dose-dependent manner (Figure 3C). P4 inhibited ATP
hydrolysis both in the presence and absence of RNA, indicating
that the compound is not simply sequestering RNA and
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Figure 4. Effect of various compounds on Huh7.5 hepatoma cells harboring a stably transfected subgenomic rLuc HCV replicon. All compounds
were tested at 10 μM such that cell media contained 0.5% (v/v) DMSO. Percent Renilla luciferase, which is proportional to HCV RNA content, is
expressed with regard to cells grown in media and 0.5% DMSO. Cell viability was measured with the Titer-Glo luminescent cell viability kit
(Promega) and is also expressed compared to DMSO controls.
preventing activation of ATP hydrolysis. It is also interesting to
note that far more P4 is needed to inhibit ATP hydrolysis in
the absence of RNA, indicating that the presence of nucleic acid
might enhance the enzyme’s affinity for inhibitors (Figure 3A).
It is not uncommon for helicase inhibitors to inhibit the
protein’s ability to hydrolyze ATP because ATP hydrolysis is
needed to fuel unwinding. When ATPase assays were
performed with compounds isolated from thioflavine S and
primuline (Table 1), the same pattern was observed as
previously seen in the MBHAs and FIDs (i.e., the longer
benzothiazole oligomers were always more potent in all assays
than the shorter oligomers).
Most of the compounds isolated from the two yellow dyes
are fluorescent, absorbing light around 340 nm and emitting
near 420 nm. Their extinction coefficients and peak absorption
wavelengths increase as the length of the benzothiazole
oligomer increases. Their relative fluorescence decreases with
the length of the benzothiazole chain. None of the compounds
absorbed light near the excitation or emission wavelengths of
the Cy5-labeled MBHA substrate or the wavelengths where
ethidium bromide-stained DNA, SYBR Green I-stained DNA,
NS3-catalyzed peptide cleavage, or ATP hydrolysis were
measured.
To test if the above compounds might be useful as HCV
antiviral agents, they were added to cells harboring a HCV
subgenomic RNA replicon. The HCV replicon chosen was
derived from the same HCV strain (genotype 1b) as the NS3
protein used for screening and enzyme assays and was a variant
of the replicon first reported by Lohmann et al.26 with two cell
culture adaptive mutations (E1202G and S2204I).27,28 The
subgenomic replicon used here also had a Renilla luciferase
gene fused to the 5′-end of the neomycin phosphotransferase
gene used for selection, so that the cellular levels of Renilla
luciferase correlated directly with the amount of HCV RNA
present in cells.29 After replicon transfection and selection, cells
were treated in parallel with one of the compounds purified
from thioflavine S and primuline or one of four recently
reported HCV helicase inhibitors 1−414,30−32 in two triplicate
sets (Figure 4, and Table 2). One set of cells was used for
Renilla luciferase assays, and the other set was used to
determine cell viability using a firefly luciferase-based assay,
and all compounds were tested at 10 μM. While none of the
compounds isolated from the yellow dyes were notably toxic to
Table 2. Activity of Reference Inhibitors under Comparison
Assays
a
a
helicase
IC50
(μM)
DNA binding
(ethidium bromide)
EC50 (μM)
DNA binding
(SYBR Green I)
EC50 (μM)
ATPase
IC50
(μM)
a
a
compd
1
2
3
4
>100
>100
>100
>100
>100
>100
>100
>100
>100
25
17
19
6
7
4
2
20
2
8
>100
74 21
a
Helicase (MBHA), DNA binding (FID), and ATP hydrolysis were
monitored in the presence of eight different concentrations of each
compound (2-fold dilution series starting at 100 μM). IC50 values were
determined from concentration−response curves. All values are means
standard deviations from three independent titrations with inhibitor.
ND, not determined.
cells, only P3 and P4 showed any ability to decrease the
amount of HCV RNA present in the cultures. The ability of P3
and P4 to inhibit HCV replication was similar to that of the
comparison helicase inhibitors tested. None of the comparison
helicase inhibitors were particularly toxic at 10 μM except for 1.
Only compound 3 inhibited MBHAs with a potency similar to
the yellow dyes, although the precise effects of compounds 3
and 4 on HCV helicase action were difficult to assess because
both interfered with the MBHA (Figure S2, Supporting
Information).
Synthesis of Primuline Analogues. The positive results
obtained with some of the higher-order components of
thioflavine S and primuline led us to undertake a structure−
activity relationship study based on them. However, difficulties
with both isolation of sufficient quantities from the commercial
sources and with fully synthetic approaches necessitated that we
consider more accessible bioisosteric equivalents. Accordingly,
we focused on modification of the amino group at the terminus
of P2a, which was isolable from commercial primuline in
sufficient quantities to serve as a starting material. We
envisioned that an appropriately rigid linker could substitute
for the third benzothiazole ring of P3. Thus, the terminal amine
of P2a was acylated to give amides and reacted with isocyanates
or sulfonyl chlorides to give urea analogues and sulfonamides,
respectively (Scheme 1). Table 3 and Figure S3 (Supporting
Information), illustrate the effect of various functional groups
on substituted phenyl amide derivatives. These included
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a
Scheme 1
a
Reagents: (a) Substituted benzoyl chloride, pyridine, 80 °C; (b) arylisocyanate, DMF, 80 °C; (c) arylsulfonyl chloride, pyridine, 80 °C.
Table 3. Activity of Phenyl Amide Derivatives of P2a
DNA binding
d
helicase IC50
(SYBR Green I)
HCV replication
a
cell viability
a
assay matrix solubility
a
ab
,
compd
R
(μM)
(% displaced at 100 μM)
(% inhibited)
(% viable)
(μM)
e
5
H
11 1.5
10 2.4
5.2 0.6
10 2.6
9.7 4.6
3.4 0.3
3.3 0.3
1.8 0.4
31 13
63 15
35 15
35 10
28 10
67 17
50 15
45
33
50
64
40
42
5
1
5
4
1
9
88
93
2
4
2
5
8
6
4
4
4
5
5
4
4
7
1
3
2
1
2
5
ND
6
4-NH2
ND
129.4
ND
ND
ND
ND
ND
ND
ND
ND
ND
29.2
2.6
7
4-F
94
8
4-OCH3
4-CO2CH3
4-Cl
85
9
101
84
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
4-CH3
52 12
44 12
87
4-CF3
69
9
90
4-t-Bu
8.2
1
72 19
51
22
7
9
87
4-N(CH3)2
4-Br
11 6.7
44
70
76
4
9
5
2
94
5.2
5.4
2.6
3.7
14
4
1
1
1
1
18
113
92
4-NHFmoc
3-Cl
57 21
54 10
43 15
41 11
67 12
30 15
46 10
66 40
49 27
66 18
43 13
35 26
35 21
48 17
112
114
112
121
122
122
129
122
3,4-di-Cl
2-CF3
0
41
55
9
8
7
ND
ND
ND
ND
ND
180.1
ND
ND
ND
3-CF3
20 12
2-F,6-CF3
2-F,3-CF3
3-F,4-CF3
3,5-di-CF3
2-F,5-CF3
3-F,6-CF3
3-F,5-CF3
17
6
3
9.2
48 18
17 17
48
60
39
4
4
4
22
4
2
6.4
132 14
19 15
28
61 14
51
118
113
4
1
7
9
a
Helicase (MBHA), DNA binding (SGI-FID) were monitored in the presence of eight different concentrations of each compound (2-fold dilution
series starting at 100 μM). IC50 values were determined from concentration−response curves. All values are means standard deviations from three
b
c
independent titrations with inhibitor. Average ( SD) percent bound at 100 μM. Cell viability and HCV replicon assays were performed in
d
triplicate in the presence of 10 μM compound. Average ( SD) percent inhibition or viability is reported. Solubility measurements were performed
using a mock assay matrix (25 mM MOPS, 1.25 mM MgCl2, 0.05 mM DTT, 5 μg/mL BSA, 0.01% v/v final [Tween 20], and 5% v/v final [DMSO])
e
at pH 6.5. ND, not determined.
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Table 4. Activity of Additional P2a Derivatives
a
Helicase (MBHA), DNA binding (SGI-FID) were monitored in the presence of eight different concentrations of each compound (2-fold dilution
series starting at 100 μM). IC50 values were determined from concentration−response curves. All values are means standard deviations from three
b
c
independent titrations with inhibitor. Average ( SD) percent bound at 100 μM. Cell viability and HCV replicon assays were performed in
d
triplicate in the presence of 10 μM compound. Average ( SD) percent inhibition or viability is reported. Solubility measurements were performed
using a mock assay matrix (25 mM MOPS, 1.25 mM MgCl2, 0.05 mM DTT, 5 μg/mL BSA, 0.01% v/v final [Tween 20], and 5% v/v final [DMSO])
e
f
at pH 6.5. Solubility measurement was performed using MOPS buffer (25 mM MOPS, 1.25 mM MgCl2, 2% v/v final [DMSO]) at pH 6.5. ND, not
determined.
electron-donating or -withdrawing groups as well as aliphatic
moieties. While replacing the para substitution RH with
NH2, OCH3, CO2CH3, t-Bu, and N(CH3)2 had no significant
effect on the potency of helicase inhibition, replacement with F,
Br, and NHFmoc groups led to slightly more potent analogues
(2-fold, compared to RH). Even better analogues (3−5-fold)
were obtained when Cl, CH3, CF3, and 2-naphthalene (33,
Table 4, replacement of phenyl group of 5) groups were used.
The increased size of alkyl substitution from Me to t-Bu
resulted in an ca. 3-fold activity loss. Altering the Cl position
from para to meta had no effect on helicase inhibition, although
the compound with meta substitution displayed weaker DNA
binding. Increasing the number of chloro groups as in 18
showed no improvement. Moving the CF3 group from the para
position to the ortho or meta position resulted in a loss of
inhibitory activity (7−10-fold, compounds 12, 19, and 20).
Introduction of additional fluorines to the derivatives also
exhibited no effect on the helicase inhibition when compared to
12 (see 21−27). In fact, depending on the position of fluorine
substitution, a significant decrease in potency was observed. In
DNA-binding assays, none of the P2a derivatives decreased the
fluorescence of DNA-bound ethidium bromide by more than
10%, even at 100 μM. Like P3 and P4, many of the derivatives
appeared to bind SYBR Green I-stained DNA. However, unlike
P3 and P4, many of the derivatives did not displace more than
50% SYBR Green at the highest concentration tested (100
μM). Therefore, to compare the DNA-binding potential of all
derivatives, the percent SYBER Green I displaced at the highest
concentration tested (100 μM) was compared rather than EC50
values (Tables 3 and 4). Most of the amide derivatives were at
least a 10-fold more potent in the MBHA than the DNA-
binding assay.
Two additional classes of chemical linkers were also explored
to mimic the benzothiazole moiety (Table 4). The urea
analogues were synthesized from P2a by reacting P2a with
different isocyanates and the sulfonamide analogues prepared
via the sulfonation of P2a with sulfonyl chlorides. The urea
analogue 28 has comparable potency in the MBHA to the
amide analogue 11, although increased DNA binding was
observed (13 μM for 28 compared to >100 μM for 11). Less
potent analogues were achieved via sulfonation (e.g., 29, 2-fold
activity drop) compared to 11. Replacing substituted phenyl
with methyl resulted in complete loss of activity (30).
Analogues targeting improved solubility by replacing the
phenyl ring of 5 with the pyridine ring, produced less potent
analogues (2−5-fold decrease in helicase activity, 31, 32). N-
Methylation of the naphthyl analogue 33 also caused a
significant drop in activity (5-fold, 34), which could indicate
the loss of a key hydrogen bond interaction.
In an effort to mimic the tetrameric structure of P4, the more
elaborate amide derivatives 35 and 36 were synthesized. No
improvements in potency were observed for the tetrameric
analogues 35 and 36 over the previous trimeric analogues. The
simple one-step synthesis of the trimeric analogues compared
to the tetrameric analogues prompted us to focus on the former
for future studies targeting more potent inhibitors of helicase
function and HCV replication.
When all derivatives (5−36) were compared with the
purified compounds and the recently reported helicase
inhibitors (1−4), it is clear that most compounds that bound
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Figure 5. Comparative effects of benzothiazoles in HCV helicase assays. (A) Potency of each compound in MBHAs is plotted against the
interference observed in an MBHA performed in the presence of 10 μM of each compound. Points are labeled with compound number, although
some numbers are omitted for clarity. Compounds that bind the helicase DNA substrate and quench its fluorescence lie on the left side of the plot,
while those that enhance substrate fluorescence are on the right. (B) Effects of various concentrations of the most potent compound that does not
interfere with the MBHA (compound 17) in standard MBHAs where Cy5 fluorescence was monitored.
Table 5. PK Evaluation of Compound 17
plasma protein binding
(% bound)
plasma
stability
hepatic microsome
a
e
g
aqueous solubility (μg/mL) (at pH)
stability
PAMPA Pe
(× 10−6 cm/s)
(@ pH)
hepatic
h
d
Prisma HT
assay
human
mouse
aqueous
f
toxicity
a
b
c
buffer
PBS
matrix
1 μM/10 μM 1 μM/10 μM human/mouse stability
human
83.57
mouse
83.11
LC50 (μM)
36.7 (5.0)
>60 (6.2)
>60 (7.4)
0 (5.0)
0.22 (6.2)
0 (7.4)
0.12 (7.4)
17.8 (6.5)
98/99
98/99
96.6/95.0
100
>50
a
b
c
In aqueous pION’s Prisma HT buffer, pHs 5.0/6.2/7.4. In aqueous PBS, pH 7.4. In a mock assay matrix (25 mM MOPS, 1.25 mM MgCl2, 0.05
d
mM DTT, 5 μg/mL BSA, 0.01% v/v final [Tween 20], and 5% v/v final [DMSO]) at pH 6.5. In aqueous buffer; donor compartment pH’s 5.0/6.2/
7.4; acceptor compartment pH 7.4. Percent remaining at 3 h. In aqueous PBS buffer with 50% acetonitrile, pH 7.4; % remaining after 48 h at room
temperature. Percent remaining at 1 h. Toward Fa2N-4 immortalized human hepatocytes
e
f
g
h
DNA in the FID assay also interfered with the MBHA by
quenching substrate fluorescence (Figure 5A). The most potent
benzothiazoles were notably more effective than the recently
reported helicase inhibitors used for comparison, two of which
appeared to function primarily by interacting with the DNA
substrate (compounds 3 and 4). Compound 17 (Figure 5A)
was the most potent compound that did not interfere with the
MBHA, and it eliminated the HCV replicon without apparent
toxicity, similar to both P3 and P4. Also, like P4, compound 17
inhibited HCV helicase-catalyzed RNA-unwinding and ATP
hydrolysis (data not shown).
The pharmacokinetic (PK) properties of compound 17 were
profiled using a standard panel of assays (Table 5). The most
striking result is the solubility variation depending on the buffer
system used. While the aqueous solubility is low in the PBS-
based solvent system, in both the detergent-containing (Tween
20) assay matrix and the proprietary Prisma HT buffer system,
the compound is readily soluble. The unknown identity of the
components in the Prisma HT buffer system complicates
further speculation into the solubility discrepancy. The
solubility results have inspired us to pursue improving
compound solubility through formulation with other solubiliz-
ing agents, and these experiments are currently in progress. The
low solubility and permeability of compound 17 were not
surprising for a polycyclic aromatic compound of molecular
weight 592 g/mol. Encouragingly, compound 17 was highly
stable under the various conditions screened and possessed no
detectable hepatic toxicity. Analogues possessing improved
solubility and PAMPA properties will be the aim of future
efforts.
DISCUSSION
■
One of the challenges in developing chemical probes that target
helicases is that potent helicase inhibitors often exert their
actions by binding nucleic acid helicase substrates. Such
compounds lack specificity because they can inhibit any protein
that needs to access the genetic material. We attempted to
discover more specific helicase inhibitors that do not target
nucleic acids using high-throughput helicase and DNA-binding
assays. However, even the most promising compounds, which
were purified from the most active compound-library sample,
interacted with the DNA substrate in the absence of protein.
We have, nevertheless, been able to engineer potent analogues
that interact with DNA less tightly yet still retain an ability to
inhibit helicase-catalyzed nucleic-acid-strand rearrangements.
Some of these compounds retain the important ability to inhibit
HCV replication in cells and could therefore prove useful for
antiviral drug development.
To discover helicase inhibitors that do not bind nucleic acids,
we screened a compound library using a helicase assay that
simultaneously detects a compound’s ability to interact with the
helicase substrate and its ability to interfere with the movement
of HCV helicase through DNA. The results of the screen
confirmed that most compound library samples that block
helicase movements also interact with the helicase substrate.
Using a DNA binding assay, we confirmed that most of the
newly uncovered HCV helicase inhibitors interact with DNA
(Figure 1C). The binding assay was based on a FID assay17 that
monitors fluorescence changes that occur when a compound
interacts with DNA stained with ethidium bromide. The most
active sample in the screened compound library that least
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affected the fluorescence of ethidium bromide-stained DNA
was a yellow dye called thioflavine S, and potent benzothiazole
oligomers were purified from this dye and its relative,
primuline. When the most active benzothiazole oligomers
purified from primuline were found to interact with SYBR
Green-stained DNA, we learned that the ethidium bromide-
based FID failed to detect the interaction of thioflavine S with
DNA. A more sensitive SYBR Green I assay was therefore
developed and used to chemically optimize more specific P2a
derivatives. The observation that DNA interactions escaped
detection in the ethidium bromide-based FID reinforces the
notion that care needs to be taken when using FID assays
because DNA-binding compounds might not displace the
bound intercalator. There is likewise still some uncertainty as to
whether or not the P2a derivatives that failed to influence the
fluorescence of SYBR Green-stained DNA interact with nucleic
acids. Preliminary results using isothermal titration calorimetry
support the relative affinities reported here, but more extensive
DNA- and RNA-binding experiments clearly need to be done
with these compounds.
The compounds reported here are more potent and specific
than other recently reported HCV helicase inhibitors (Table 2,
Figure 5). However, we have not been able to reproduce all of
the results previously reported for the comparison helicase
inhibitors. For example, the nucleotide mimic (compound 1),
which had been reported to have a Ki of 20 nM,30 had almost
no detectable effect on the MBHA at 5000 times higher
concentration (Table 2 and Figure S2, Supporting Informa-
tion). Compound 1 had some antiviral activity, and it was
coupled with notable toxicity at 10 μM (Figure 4). The
acridone (compound 2) was about 17-fold less potent in our
helicase assay than it was in a previously reported assay, yet it
still was one of the most effective compounds in eliminating the
HCV replicon. Similarly, although its interaction with the
helicase substrate obscured effects in our MBHA, the
triphenylmethane (compound 3) displayed no inhibition at
its previously reported IC50 of 12 μM and the compound did
not appear as cytotoxic as had been reported.32 Compound 4
had antiviral activity as previously reported, but again,
compound interference in the MBHA made its effect on the
helicase in vitro difficult to judge.31 Our different results could
be due to several factors, including the use of different
recombinant HCV helicase proteins and assay conditions
(Figure S2, Supporting Information).
suggest that some of the derivatives might enter cells. Effects of
the most potent replicon inhibitors on HCV replication (e.g.,
11, 17, 24, and 26) are presently being examined in more
detail. The results of these studies will be used to inform
additional chemistry efforts toward helicase inhibitors, which is
an ongoing concern of our laboratories.
EXPERIMENTAL SECTION
■
Materials. Thioflavine S and primuline were purchased from Sigma
(cat. no. T1892, lot no. 048K1656) and MP Biomedicals (cat. no.
195454, lot no. 7792J), respectively. The Mechanistic Diversity Library
was obtained from the National Cancer Institute (NCI, http://dtp.
cancer.gov/repositories.html). All other reagents were purchased from
commercial suppliers and used as received. Methylene chloride,
acetonitrile, toluene, ethyl ether, and THF were dried by being passed
through two packed columns of anhydrous, neutral alumina prior to
use. HPLC/MS analysis was carried out with gradient elution (5%
CH3CN to 100% CH3CN) on an Agilent 1200 RRLC with a
photodiode array UV detector and an Agilent 6224 TOF mass
spectrometer. Compound purity was determined using RP HPLC and
was measured on the basis of peak integration (area under the curve)
from UV/vis absorbance (at 214 nm), and compound identity was
determined on the basis of exact mass analysis. All compounds used
for biological studies have purity >95% (Table S1, Supporting
Information) except for the following compounds: P3 (89.2%), P4
batch 2 (85.6%), 6 (90.0%), 11 (89.1%), 14 (80.4%), 17 (93.6%), 28
(93.2%), 32 (94.4%), and 36 (88.6%).
All oligonucleotides were purchased from Integrated DNA
Technologies (IDT, Coralville, IA). The partially duplex DNA
substrates used in MBHAs consisted of a helicase substrate forming
25 base pairs and consists of a 45-mer bottom strand 5′-GCT CCC
CGT TCA TCG ATT GGG GAG C(T)20-3′and the 25-mer HCV top
strand 5′-Cy5-GCT CCC CAA TCG ATG AAC GGG GAG C-IBRQ-
3′. The 19 base pair RNA substrate used in MBHAs consisted of a 39
nucleotide long bottom strand 5′-AGU GCC UUG ACG AUA CAG
C(U)20-3′ and the 24 nucleotide long top strand 5′-Tye665-AGU GCG
CUG UAU CGU CAA GGC ACU-IBRQSp-3′. Underlined areas
denote hairpin-forming regions. DNA and RNA substrates were
annealed and purified as described previously.16
The cloning, expression, and purification of His-tagged recombinant
HCV NS3 protein have been described previously.33−36
Helicase Assays. All molecular beacon-based helicase assays
(MBHAs) were performed as described before.16,36 For screening the
NCI library, MBHAs contained 25 mM MOPS pH 6.5, 1.25 mM
MgCl2, 5.0 nM MBHA substrate, 12.5 nM NS3h_1b(con1), 5 μg/mL
BSA, 0.01% (v/v) Tween20, 0.05 mM DTT with 20 μM each test
compound (2% v/v final DMSO). In each flat, black 384-well plate, 56
compounds were tested, in triplicate, along with three negative
controls (DMSO only), three positive controls (500 nM dT20), and
two wells with no enzyme. Fluorescence was read before ATP (F0)
addition and 30 min after ATP was added to 1 mM (F30) using a
Tecan Infinite M200 fluorescence microplate reader with excitation
and emission wavelengths set to 643 and 670 nm, respectively. Percent
inhibition was calculated with eq 1, and compound interference in the
MBHA was calculated with eq 2.
CONCLUSIONS
■
In conclusion, we have found that the commercial dyes
thioflavine S and primuline contain potent compounds for the
inhibition of the NS3 helicase of HCV. We show here that
minor components of primuline inhibit both the HCV helicase
and HCV replicon replication. The antiviral potential of these
trimeric and tetrameric benzothiazoles inspired the derivatiza-
tion of the more abundant dimeric constituent. Several
derivatives were found to be close in potency to the isolated
trimer or tetramer in the MBHA and to possess improved DNA
binding profiles. Importantly, the antiviral potential of this class
of helicase inhibitors does not appear to depend entirely on
either their ability to inhibit HCV helicase or bind DNA. We
speculate that the ability to inhibit HCV replication results from
a compound’s ability to enter Huh7.5 cells, which can be
monitored by examining compound fluorescence when they are
administered to cells. In cultures, both primuline and
thioflavine S mainly stain membranes, but preliminary data
inhibition(%) = Fc Fc − F( − ) F( − )
((
)
(
)
0
30
0
30
1 − F( − ) F( − )
× 100
(
(
)))
30
(1)
(2)
0
interference (ratio) = Fc F( − )
(
)
0
0
In eqs 1 and 2, Fc0 is the fluorescence of the reactions containing
the test compound before adding ATP, Fc30 is the fluorescence of the
test compound reaction 30 min after adding ATP. F(−)0 is the average
of three DMSO-only negative control reactions before adding ATP
and F(−)30 is the average of three DMSO-only reactions 30 min after
adding ATP.
To monitor helicase reaction kinetics and to calculate IC50 values,
MBHAs were performed in 60 μL in white 1/2 area 96-well plates and
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measured in a Thermo Varioscan Multimode reader (Thermo
Scientific) using the 643 nm excitation wavelength and 667 nm
emission wavelengths. Reactions were again performed by first
incubating all components except for ATP for two minutes, then
initiated by injecting in 1/10 volume of ATP such that the final
concentration of all components was as noted above. Conditions were
as described above except that 5% v/v DMSO was present in each
assay. Initial reaction velocities were calculated by fitting first-order
decay equation to data obtained after ATP addition and calculating an
initial velocity from the resulting amplitude and rate constant. The
concentration at which a compound causes a 50% reduction in
reaction velocity (IC50) was calculated by fitting compound
concentration to initial velocity using eq 3:
generous gift from Seng-Lai Tan. In HCV RLuc, the HCV internal
ribosome entry site (IRES) drives the translation of the neomycin and
Renilla luciferase genes, while the HCV nonstructural proteins (NS3 to
NS5B) are translated from the encephalomyocarditis virus IRES.29
The plasmid DNA was cleaved with Sca I, purified by phenol/
chloroform extraction followed by ethanol precipitation, and used as
template for RNA transcription using MEGAscript T7 RNA
transcription kit (Ambion, Austin, TX). The RNA transcripts were
treated with 2 U DNase I (Ambion) at 37 °C for 30 min, purified by
acid phenol/chloroform extraction, followed by isopropyl alcohol
precipitation, and suspended in diethylpyrocarbonate-treated water.
RNA concentration was determined by spectrophotometry by
measuring the A260. RNA integrity and size was checked on 1%
agarose gel. Transcribed RNA was stored in aliquots at −80 °C until
needed.
Huh-7.5 cells RNA were transfected with HCV RNA by
electroporation. Briefly, subconfluent Huh7.5 cells were trypsinized,
suspended in complete growth medium, and centrifuged at 1000g for 5
min at 4 °C. The cell pellets were then washed twice with ice-cold
phosphate-buffered saline (PBS) and suspended at 1.75 × 107 cells/
mL in ice-cold PBS. Replicon RNA (5 μg) was mixed with 0.4 mL of
cell suspension and transferred to 2 mm gap width electroporation
cuvette (Eppendorf AG, Germany) and pulsed with five times for 99
μs at 820 V over 1.1 s intervals using the ECM 830 electroporator
instrument (BTX Havard Apparatus, Holliston, MA). After 5 min
recovery period at room temperature, cells were transferred to 10 mL
complete growth medium and seeded into 10 cm diameter cell culture
dishes. Twenty-four hours after transfection, the medium was replaced
with fresh complete DMEM supplemented with 1 mg/mL Geneticin
(Invitrogen) and the medium was replaced every three to four days
with fresh medium containing 1 mg/mL Geneticin. Geneticin-resistant
colonies were selected for a period of two weeks and expanded in the
presence of 250 μg/mL Geneticin.
h
ν = ν / 1 + [I]/IC
(
)
i
0
(
50
)
(3)
where ν0 is the velocity observed in DMSO-only controls-inhibition, h
is the Hillslope coefficient, and [I] is the concentration of test
compound.
DNA Binding Assays. Fluorescent intercalation displacement
(FID) assays18 were used to measure the ability of a compound to
bind the MBHA substrate. The concentration at which half the
ethidium bromide is displaced (EC50), was determined using the
different conditions as above to more closely mimic the conditions of a
standard helicase assay. Each 100 μL reaction contained 25 mM
MOPS pH 6.5, 0.16 μM MBHA DNA substrate (lacking Cy5 and
IBQ-RQ modifications), 2 μM ethidium bromide, and various
concentrations of test compound. Fluorescence of ethidium bromide
was monitored using excitation and emission wavelengths of 545 and
595 nm, respectively, on a Cary Eclipse fluorescence spectropho-
tometer in white 96-well plates. The amount of ethidium bromide-
DNA complex fluorescence was used to estimate the ability of
compound to bind DNA, and therefore displace the fluorescent
intercalator (ethidium bromide).
HCV RLuc replicon cells were seeded at a density of 10 × 103 cells
per well in 96-well plates and incubated for 4−5 h to allow the cells to
attach to the plate. The compounds dissolved in dimethyl sulfoxide
(DMSO) were added at a final concentration of 10 μM (DMSO
solvent final concentration was 0.5%), and the cells were incubated for
72 h at 37 °C under 5% CO2 atmosphere. The effects of compounds
on HCV replication were then assessed by measuring the Renilla
luciferase activity in compound-treated versus DMSO-treated cells. At
the end of the incubation period, the medium was aspirated and the
cells were washed with 1× PBS. The Renilla luciferase reporter gene
assay was performed using the Renilla luciferase assay kit (Promega,
Madison, WI) according to the manufacturer’s instructions. Briefly, the
cells were lysed by addition of 50 μL of 1× Renilla luciferase lysis
buffer followed by two cycles of freeze/thaw. The luciferase activity
content of the lysate was measured with a FLUOstar Omega
microplate reader instrument (BMG Labtech, Germany) after
injecting 50 μL of luciferase substrate and reading for 5 s.
binding = (1 − ((Fc − F( + ))/(F( − ) − F( + )))) × 100
(4)
In eq 4, Fc is the fluorescence in the presence of the compound,
F(−) is the average DMSO-only negative controls, and F(+) is the
average positive controls (100 μM berenil). EC50 values were from a
normalized concentration−response curve.
A modified FID assay in which ethidium bromide was replaced with
SYBR Green I (Invitrogen) was used to estimate a compound’s affinity
for the MBHA substrate. Reactions were performed as described above
except that the DNA substrate was present at 0.32 μM, ethidium
bromide was absent, and SYBR Green was present at (0.68 μM). Data
were normalized as described above and fit to concentration−response
equation using GraphPad Prism software. Titrations with each
compound were performed in triplicate, and EC50 values from three
Cell Viability Assay. To assess compound toxicity toward Huh-7.5
cells, cells were plated and treated as above and cell viability was
assessed using the CellTiter-Glo luminescent cell viability kit
(Promega) following the manufacturer’s instructions. Briefly, at the
end of a 72 h incubation period, the medium was aspirated and the
cells were washed with 1× PBS, and then an equal volume of growth
medium and CellTiter-Glo reagent was added and the lysis was
initiated by mixing on an orbital shaker. The plate was incubated at 23
°C for 30 min, and the luciferase activity was measured for 1 s using
the FLUOstar Omega microplate reader (BMG Labtech).
independent titrations are reported
standard deviations. Average
percent bound at 100 μM is reported for compounds that did not
decrease the fluorescence of SYBR Green stained DNA more than
50% at the highest concentration tested.
ATP Hydrolysis Assays. A modified “malachite green” assay was
used to measure ATP hydrolysis.37 The 50 μL reactions contained 25
mM MOPS pH 6.5, 5 mM MgCl2, 2 mM ATP, 5 μg/mL BSA, 0.01%
(v/v) Tween20, 0.05 mM DTT, and poly(U) RNA (Saint Louis, MO)
as indicated. Reactions were initiated by adding NS3h_1b(con1).
Reactions were incubated for 15 min at 37 °C then stopped by mixing
40 μL of the reaction into 200 μL of the malachite green reagent (3
volumes 0.045% (w/v) malachite green, 1 volume 4.2% ammonium
molybdate in 4N HCl, 0.05 volume of 20% Tween 20). Then 25 μL of
34% sodium citrate was quickly added to each reaction and color
allowed to develop for 15 min. Absorbance at 630 nm was
proportional to the concentration of phosphate produced; free
phosphate produced was measured against a standard curve.
ASSOCIATED CONTENT
* Supporting Information
■
S
HPLC purity analysis and screening results of the NCI
Mechanistic Set. Sample helicase assays with various concen-
trations of each compound. General protocols for the PK
assays, experimental details for the isolation of primuline and
thioflavine S components, the synthesis of compounds 5−36,
and compound characterization data, including HPLC purity
HCV Subgenomic Replicon Assay. An HCV Renilla luciferase
(HCV RLuc) reporter construct was used to measure the effect of
each compound on cellular HCV RNA levels. The replicon was a
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and NMR spectra. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 414-229-6670. E-mail: frickd@uwm.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by National Institutes of Health
grants U54 HG005031, R03 MH085690-01, and R01
AI088001 and a Research Growth Initiative Award from the
UWM Research Foundation. We thank Dmitriy Minond, Peter
Hodder, Jill Ferguson, and Katharine Emery of the Scripps
Research Institute, Florida, for help with assay development
and for uploading the screening results into PubChem
BioAssay. We are also grateful to Doreen Badheka, Yoji High,
William Shadrick, and John J. Hernandez for their help with
various enzymatic assays, and to Seng-Lai Tan for providing the
HCV replicon. Finally, we thank Arianna Mangravita-Novo,
Michael Vicchiarelli, and Layton H. Smith of the Sanford−
Burnham Medical Research Institute at Lake Nona for
providing the solubility and PK profiling measurements.
ABBREVIATIONS USED
■
HCV, hepatitis C virus; DAA, direct acting antiviral; NS3,
nonstructural protein 3; MBHA, molecular beacon-based
helicase assay; FID, fluorescent intercalator displacement; PK,
pharmacokinetics
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