UK-1 and structural analogs are potent inhibitors of hepatitis C virus replication

Article abstract of DOI:10.1016/j.bmcl.2013.12.012

The bacterial natural product UK-1 and several structural analogs inhibit replication of the hepatitis C virus in the replicon assay, with IC₅₀ values as low as 0.50 μM. The NS3 helicase has been identified as a possible target of inhibition for several of these compounds, while the remaining inhibitors act via an undetermined mechanism. Gel shift assays suggest that helicase inhibition is a direct result of inhibitor-enzyme binding as opposed to direct RNA binding, and the ATPase activity of NS3 is not affected. The syntheses and biological results are presented herein.

Full text of DOI:10.1016/j.bmcl.2013.12.012

Bioorganic & Medicinal Chemistry Letters 24 (2014) 609–612
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
UK-1 and structural analogs are potent inhibitors of hepatitis C virus
replication
Dawn N. Ward a, , Daniel C. Talley a, Mrinalini Tavag a, Samrawit Menji a, Paul Schaughency a,
Andrea Baier b, Paul J. Smith a,
⇑
a Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, United States
b Department of Molecular Biology, John Paul II Catholic University of Lublin, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
The bacterial natural product UK-1 and several structural analogs inhibit replication of the hepatitis C
Received 19 September 2013
Revised 25 November 2013
Accepted 2 December 2013
Available online 9 December 2013
virus in the replicon assay, with IC50 values as low as 0.50 lM. The NS3 helicase has been identified as
a possible target of inhibition for several of these compounds, while the remaining inhibitors act via
an undetermined mechanism. Gel shift assays suggest that helicase inhibition is a direct result of inhib-
itor–enzyme binding as opposed to direct RNA binding, and the ATPase activity of NS3 is not affected. The
syntheses and biological results are presented herein.
Keywords:
Hepatitis C
NS3
Ó 2013 Elsevier Ltd. All rights reserved.
Helicase
Inhibitor
The hepatitis C virus (HCV), the causative agent of the hepatitis
C infection, has been declared a worldwide health concern by the
World Health Organization (WHO).1,2 Estimates predict that a min-
imum of 3% of the global population, approximately 170 million
people, are currently infected.3 Of those diagnosed with the virus,
70–80% of cases are not suppressed by the body’s immune system
and chronic liver infection develops.4 This acute infection of the
hepatocytes leads to liver cirrhosis, hepatocellular carcinoma,
and eventually liver failure.5 Moreover, HCV infection is currently
the leading cause of liver transplantation.6
Two direct-acting antiviral (DAA) drugs, Boceprevir and Tela-
previr, were approved by the FDA for HCV treatment in 2011.
Although these drugs represent the first DAAs approved for HCV
therapy, during early phases of development it was found that
monotherapy utilizing either of these drugs alone resulted in the
rapid development of resistant strains.7,8 As a result, these drugs
are only FDA approved when given in combination therapy with
two previously approved broad spectrum antivirals, ribavirin and
pegylated interferon (PEG-IFN).9,10 The new treatment regimes
have increased overall response rates, but the severe side effects
and extremely high cost associated with PEG-IFN remain problem-
atic.11 While combination therapy is the most promising approach
for future HCV treatment, replacement of broad spectrum
antivirals with inhibitors specific for HCV will likely result in much
more favorable treatment outcomes.
HCV is a positive sense RNA virus that, following entry into the
host hepatocyte, encodes for a single polyprotein of approximately
3000 amino acids.12–14 The polyprotein is processed co- and post-
translationally by both host and viral proteases to yield ten viral
proteins. The amino terminal third of the peptide contains the four
structural proteins core, E1, E2, and p7. The remaining two-thirds
of the protein is composed of the nonstructural (NS) proteins
NS2, NS3, NS4A, NS4B, NS5A, and NS5B.15 Although each NS pro-
tein possesses a unique function (or functions), as a whole they
are responsible for the synthesis of viral RNA, and thus critical
for viral replication.16 For this reason, the NS proteins are com-
monly pursued as drug targets in HCV research.
NS3 is a protein exhibiting multiple functions and is well char-
acterized both structurally and functionally. NS3 and the adjacent
NS4A function as a complex, with NS4A serving as both a cofactor
for NS3 and as a membrane anchor.17 The amino terminal domain
of NS3 and NS4A form a dimeric serine protease that is responsible
for the intramolecular cleavage of NS3 from NS4A, as well as the
intermolecular cleavage between all downstream non-structural
proteins.18 In addition, the carboxy terminus of NS3 functions as
an ATP dependent helicase, and this activity is facilitated by
NS4A.19 The exact relevance of the helicase activity as it pertains
to HCV viral replication is not known, but it has been shown that
a functional helicase is required for in vivo HCV replication.16,20,21
Several functions for the helicase have been proposed, including
aiding in the polymerase processivity, assisting with RNA folding,
⇑
Corresponding author. Tel.: +1 410 455 2519; fax: +1 410 455 2608.
E-mail address: pjsmith@umbc.edu (P.J. Smith).
Current address: Department of Chemistry, Stevenson University, Stevenson, MD
21153, United States.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.012

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D. N. Ward et al. / Bioorg. Med. Chem. Lett. 24 (2014) 609–612
modulating host gene expression, and involvement in genome
encapsidation.22,23 There are currently no HCV helicase inhibitors
in clinical trials, but numerous strategies for helicase inhibition
have been investigated. Since the NS3 helicase activity is depen-
dent upon ATP hydrolysis, various nucleoside analogs have been
developed to inhibit the NTPase activity of NS3.24 Other helicase
inhibitors include compounds that bind directly to the nucleic acid
binding site of the helicase or to unknown allosteric sites.25,26
UK-1 (Fig. 1) is a Streptomyces metabolite that exhibits broad
spectrum anti-cancer activity and has also been shown to chelate
magnesium and zinc.27–29 It was hypothesized that UK-1 and
structural analogs could potentially inhibit HIV-1 integrase via
magnesium coordination in the enzyme active site. As such, a ser-
ies of UK-1 analogs (1–6) were synthesized and screened against
HIV-1, as well as a number of other viruses. Although no activity
against HIV-1 was observed, all of the compounds screened did
prove to be effective inhibitors of HCV viral replication in replicons,
the benzyl group in 5 (and 6) was not readily determined using 2D
NMR techniques. However, fluorescence experiments confirmed
the product structure (Supplementary data: Scheme 2, Fig. 3).
Briefly, the desired product showed a large Stokes shift consistent
with an excited state intramolecular proton transfer (ESIPT) only
feasible in isomer 5 (and 6), as opposed to the other possible prod-
uct of monodebenzylation.32
Potential inhibition of the helicases of Flaviviridae viruses HCV,
Japanese encephalitis virus (JEV), and dengue virus (DENV) was
investigated using previously described methods.33,34 None of the
compounds inhibit JEV or DENV helicases (IC50 >700 lM), however
several of the compounds did inhibit the activity of the HCV heli-
case (Table 1, Fig. 2). UK-1 itself shows weak inhibition using a
DNA substrate, but no inhibition with an RNA substrate. Impor-
tantly, naphthol derivatives 4–6 show helicase inhibition, with 5
and 6 exhibiting IC50 values in the low micromolar range. None
of the compounds inhibit the ATPase activity of the HCV helicase
with IC50 values as low as 0.50
lM. In an attempt to determine the
(IC50 >1200 lM) eliminating this as a possible mechanism of ac-
mechanism of HCV inhibition, these compounds were also
screened against the HCV NS3 helicase, NS3 NTPase, and NS5B
polymerase.
tion. Compounds 5 and 6 do not affect the gel mobility of an Eco-
RI-digested pT7-7 plasmid, suggesting the inhibition results from
direct helicase interaction, rather than simple nucleic acid binding.
To determine if the compounds are active against HCV replica-
tion in cells, they were screened in the replicon assay (Table 1).
Quite interestingly, all seven compounds were active, with EC50
values in the low- to sub-micromolar range. While the mechanism
of viral inhibition for compounds 5 and 6 may result from helicase
inhibition, this is not the case for 1–3 and seems unlikely for weak
helicase inhibitors UK-1 and 4. This suggests that within this group
of compounds, there is a second, as yet undetermined mechanism
of inhibition. The compounds were then screened against the HCV
RNA-dependent RNA polymerase NS5B, and very little inhibition
The compounds evaluated are shown in Figure 1. UK-1 and ana-
logs 1–3 were synthesized as previously reported.29,30 The synthe-
sis of 5 is shown in Scheme 1 (for the synthesis of 6, the same
methodology was used). This began with carboxylation of 1,5-
dihydroxynaphthalene, using magnesium methyl carbonate as pre-
viously described.31 The resulting acid was reacted with benzyl
chloride, which upon hydrolysis gave 7. The acid was then acti-
vated with 1,10-carbonyldiimidazole (CDI) and coupled to methyl
3-hydroxyanthranilate, giving compound 8. Refluxing 8 in m-xy-
lene with pyridinium p-toluenesulfonate (PPTS) resulted in cyc-
lodehydration as well as monodebenzylation, to give target
compound 5. Compound 4 was synthesized similarly, starting with
1-hydroxy-2-naphthoic acid. It should be noted that the position of
was observed (inhibition 630% at 100 lM). There was no signifi-
cant difference in activities between analogs 1–3, despite expected
differences in cell permeability and susceptibility to cellular
O
O
HO
O
HN
O
HO
HO
HO
HO
N
N
N
O
O
N
O
O
1
O
2
O
3
N
O
UK-1
O
OBn
O
O
O
O
O
HO
HO
HO
N
O
N
O
4
N
O
OBn
5
6
Figure 1. UK-1, truncated analogs (1), acid (2), amide (3), and naphthol analogs 4, 5, and 6.
O
O
N
HO
OH
OBn O
OBn O
OH
a, b, c
e
d
OH
O
N
H
COOMe
OH
5
OBn
7
8
OBn
OBn
Scheme 1. Reagents and conditions: (a) Magnesium methyl carbonate, DMF, 140 °C; (b) BnCl, DMF, K2CO3, reflux; (c) NaOH, MeOH, reflux; (d) (i) CDI, THF, rt, (ii) methyl-3-
hydroxyanthranilate, reflux; (e) PPTS, m-xylene, 140 °C.

D. N. Ward et al. / Bioorg. Med. Chem. Lett. 24 (2014) 609–612
611
Table 1
a direct interaction between 5 (and 6) with purified NS3 is being
sought, as well as potential interactions with other HCV proteins.à
In summary, several novel inhibitors of HCV viral replication
have been identified which exhibit minimal cytotoxicity. Two of
these compounds (5 and 6) inhibit the NS3 helicase with micromo-
lar IC50 values and are highly selective; no inhibition is observed
with the closely related helicases of DENV and JEV. It has been
speculated that viral inhibition may be a result of direct interaction
with the helicase, as neither ATPase inhibition nor direct RNA bind-
ing is observed. A second group of compounds effectively inhibits
HCV RNA replication in the replicon assay via an as yet to be deter-
mined mechanism. Experiments are currently underway to further
investigate and verify the proteins target(s) for all inhibitors.
Helicase inhibition and viral replication inhibition data for UK-1 and analogs 1–6
Compound
Helicasea
RNA
Repliconc,d
DNA
b
b
e
f
g
IC50
(l
M)
IC50
(l
M)
EC50
(lM)
CC50
(l
M)
SI50
UK-1
380
>1200
>1800
>1800
>1800
590
0.77
0.50
0.53
1.4h
0.53
2.1
17
22
11
6.6
1
2
3
4
5
6
>1800
>1800
>1800
540
3.8
2.6
5.4
3.5
6.3h
2.5
>20
>20
4.5h
4.7
>10
>37
20
20
0.54
a
Strand separation of radiolabeled oligonucleotides was monitored using gel
electrophoresis (see Supplementary data).
b
The concentration of compound required to inhibit 50% of helicase activity.
Experiments were carried out at Southern Research Institute, Frederick, MD.
Results are the averages of 2–4 independent dose–response experiments using
Acknowledgments
c
d
This research was financed in part by NIGMS Initiative for Max-
imizing Student Development Grant (r25-GM55036) (D.N.W.), U.S.
Department of Education, Ronald E. McNair Post Baccalaureate
Achievement Program Grant #P217A030141 (S.M.), and EMBARC
Grant #DBI-0453294 (P.S. and M.T.). Thanks to Professor Neerja
Kaushik-Basu, Department of Biochemistry and Molecular Biology,
UMDNJ-New Jersey Medical School for running NS5B assays and a
special thanks to Dr. Ramachandra S. Hosmane for his advice and
guidance.
a combination of luciferase reporter-based and RNA assays unless noted.
e
The concentration of compound that inhibits 50% of viral RNA replication.
The concentration of compound that kills 50% of the cells.
f
g
The ratio of EC50 to CC50
.
h
Result is from a single luciferase reporter-based assay.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.1
2.012.
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