Antiviral Activity of (+)-Rutamarin against Kaposi's Sarcoma-Associated Herpesvirus by Inhibition of the Catalytic Activity of Human Topoisomerase II

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is an etiological agent of several AIDS-associated malignancies, including Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). Its lytic replication cycle has been proven to be critical for the pathogenesis of KSHV-associated diseases. In KS lesions, lytic viral replication, production of virion particles, and reinfection of endothelial cells are essential to sustain the population of infected cells that otherwise would be quickly lost as spindle cells divide. Thus, antivirals that block KSHV replication could be a strategy in the treatment of KSHV-associated diseases. However, there is no effective anti-KSHV drug currently available. Our previous work showed that human topoisomerase II (Topo II) is indispensable for KSHV lytic replication and is suggested to be an effective target for antiviral drugs. Here, we report the discovery and characterization of a novel catalytic inhibitor of human Topo IIα, namely, (+)-rutamarin. The binding mode of (+)-rutamarin to the ATPase domain of human Topo IIα was established by docking and validated by molecular dynamics (MD) simulations. More importantly, (+)-rutamarin efficiently inhibits KSHV lytic DNA replication in BCBL-1 cells with a half-maximal inhibitory concentration (IC50) of 1.12 μM and blocks virion production with a half-maximal antiviral effective concentration (EC50) of 1.62 μM. It possesses low cytotoxicity, as indicated by the selectivity index (SI) of 84.14. This study demonstrated great potential for (+)-rutamarin to become an effective drug for treatment of human diseases associated with KSHV infection.

Full text

Antiviral Activity of (ⴙ)-Rutamarin against Kaposi’s Sarcoma-
Associated Herpesvirus by Inhibition of the Catalytic Activity of
Human Topoisomerase II
Bo Xu,
a,b
Ling Wang,
c
Lorenzo González-Molleda,
d
Yan Wang,
a,e
Jun Xu,
c
Yan Yuan
a,b,d
‹Institute of Human Virology
a
and Key Laboratory of Tropical Disease Control,
b
Ministry of Education, Sun Yat-Sen University, Guangzhou, Guangdong, China; Research
Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, Guangdong, China
c
; Department of Microbiology, University of
Pennsylvania School of Dental Medicine, Philadelphia, Pennsylvania, USA
d
; Guanghua School of Stomatology, Sun Yat-Sen University, Guangzhou, Guangdong, China
e
Kaposi’s sarcoma-associated herpesvirus (KSHV) is an etiological agent of several AIDS-associated malignancies, including Ka-
posi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD). Its lytic replication cycle
has been proven to be critical for the pathogenesis of KSHV-associated diseases. In KS lesions, lytic viral replication, production
of virion particles, and reinfection of endothelial cells are essential to sustain the population of infected cells that otherwise
would be quickly lost as spindle cells divide. Thus, antivirals that block KSHV replication could be a strategy in the treatment of
KSHV-associated diseases. However, there is no effective anti-KSHV drug currently available. Our previous work showed that
human topoisomerase II (Topo II) is indispensable for KSHV lytic replication and is suggested to be an effective target for antivi-
ral drugs. Here, we report the discovery and characterization of a novel catalytic inhibitor of human Topo II␣, namely, (ⴙ)-rut-
amarin. The binding mode of (ⴙ)-rutamarin to the ATPase domain of human Topo II␣was established by docking and vali-
dated by molecular dynamics (MD) simulations. More importantly, (ⴙ)-rutamarin efficiently inhibits KSHV lytic DNA
replication in BCBL-1 cells with a half-maximal inhibitory concentration (IC
50
) of 1.12 ␮M and blocks virion production with a
half-maximal antiviral effective concentration (EC
50
) of 1.62 ␮M. It possesses low cytotoxicity, as indicated by the selectivity
index (SI) of 84.14. This study demonstrated great potential for (ⴙ)-rutamarin to become an effective drug for treatment of hu-
man diseases associated with KSHV infection.
Kaposi’s sarcoma-associated herpesvirus (KSHV), also known
as human herpesvirus 8 (HHV-8), is a member of the human
gammaherpesvirus family. It has been proven to be the etiologic
agent of Kaposi’s sarcoma (KS), a multicentric malignant neo-
plasm of endothelial origin (1–5). Although classic KS is a rare
disease and in general nonfatal, it often occurs in immunocom-
promised patients who are under immunosuppression treatment
after organ transplantation. In the AIDS epidemic, KS has become
the most common AIDS-associated malignancy and has led to
significant mortality (6). Approximately 20% of AIDS patients
develop KS during the course of their disease, and AIDS-associ-
ated KS is estimated to contribute to 10% of the deaths of AIDS
patients (7). Besides KS, two B cell-associated lymphoproliferative
disorders, namely, primary effusion lymphoma (PEL) and multi-
centric Castleman’s disease (MCD), are also related to KSHV in-
fection and mainly occur in AIDS patients (6,8). The incidence of
KSHV-associated MCD has risen since the advent of highly active
antiretroviral therapy (HAART) (9).
The contemporary treatment modalities for KS and other
KSHV-associated malignancies include conventional cancer ther-
apies, such as radiation and chemotherapy, or AIDS treatment,
such as HAART (6). These cancer therapies in general have serious
side effects, and the response of tumors to them is only transient.
The success of human papillomavirus (HPV) vaccine in prevent-
ing cervical cancer has proven that antiviral-oriented therapies
have the potential to be used to treat human malignant diseases
that are caused by viruses. Thus, an antiviral targeting KSHV
could prove successful at treating KSHV-associated tumors (10).
However, currently, there is no therapy for KSHV-associated dis-
ease based on targeting of KSHV.
Like all herpesviruses, KSHV has two types of replication cycle,
latent and lytic replication. In KS lesions, most KSHV-trans-
formed spindle-shaped cells are in the latent replication phase, but
a small percentage of infected cells undergo spontaneous lytic rep-
lication (11–13). Increasing evidence suggests that the lytic repli-
cation in these cells is necessary for maintaining stable infection
and viral pathogenicity. The majority of KSHV genes, including
those responsible for malignant cell growth and alteration of the
microenvironment, are expressed only in the lytic phase (14). Fur-
thermore, lytic replication is necessary for sustaining the popula-
tion of latently infected cells that otherwise would be quickly lost
by segregation of latent viral episomes as spindle cells divide (15).
Thus, KSHV lytic replication and constant primary infection of
fresh cells are crucial, not only for viral propagation, but also for
viral pathogenesis. Blockade of the lytic replication of KSHV could
be a good strategy for the treatment of KS or other KSHV-associ-
ated human diseases.
Received 14 June 2013 Returned for modification 8 July 2013
Accepted 11 October 2013
Published ahead of print 2 December 2013
Address correspondence to Yan Yuan, yuan2@pobox.upenn.edu, or Jun Xu,
xujun9@mail.sysu.edu.cn.
B.X., L.W., and L.G.-M. contributed equally to this article.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AAC.01259-13.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.01259-13
January 2014 Volume 58 Number 1 Antimicrobial Agents and Chemotherapy p. 563–573 aac.asm.org 563

Viral DNA replication is considered an ideal target for antivi-
rals. Our laboratory has been studying the mechanisms that con-
trol KSHV lytic DNA replication (16–19). One of our findings was
that several host cellular proteins, including topoisomerase I
(Topo I) and Topo II, MSH2/6, RecQL, and poly(ADP-ribose)
polymerase 1 (PARP-1), are involved in KSHV lytic DNA replica-
tion (19). We also demonstrated that both Topo I and Topo II are
indispensable for KSHV lytic replication and that specific inhibi-
tors of Topo I and II can effectively inhibit KSHV lytic replication.
One category of Topo II inhibitors, namely, catalytic Topo II in-
hibitor, was found to provide marked inhibition of KSHV repli-
cation with minimal cytotoxicity, as indicated by their high selec-
tivity indices (e.g., 31.6 for novobiocin) (20). As such, Topo II can
serve as an effective target for antivirals, and catalytic inhibitors of
Topo II represent promising antiviral agents for the treatment of
malignancies associated with KSHV infection.
In this study, we attempted to search for new compounds with
higher Topo II inhibition properties and lower cytotoxicities.
With a ligand-based virtual screening approach, we screened
more than 7,200 compound structures in our compound reposi-
tory and identified a set of coumarin derivatives that showed ac-
tivities in inhibition of KSHV DNA replication equal to or greater
than that of novobiocin. In particular, (⫹)-rutamarin exhibits the
highest efficiency in blocking KSHV lytic replication. We demon-
strated that (⫹)-rutamarin has human Topo II inhibition activity
and belongs to the category of catalytic Topo II inhibitors.
MATERIALS AND METHODS
Cells and plasmids. BCBL-1 and JSC-1 are two primary effusion lym-
phoma cell lines that are latently infected with KSHV (21,22). The BJAB
cell line is a KSHV-negative B cell line isolated from Burkitt’s lymphoma.
These B cells were grown in RPMI 1640 medium (Gibco-BRL, Gaithers-
burg, MD) supplemented with 10% fetal bovine serum (Gibco-BRL),
penicillin-streptomycin (50 units/ml), and 1.25 g/ml amphotericin
B-1.25g/ml sodium deoxycholate (Fungizone). Peripheral blood mono-
nuclear cells (PBMC) were isolated from whole blood of a healthy donor
and cultured in RPMI 1640 medium supplemented with 10% fetal bovine
serum, penicillin-streptomycin, and amphotericin B-sodium deoxy-
cholate.
Plasmid pOri-A contains an EcoRI-PstI fragment (nucleotides 22409
to 26491) of KSHV DNA in pBluescript at the EcoRI/PstI site, as previ-
ously described (16). pCR3.1-ORF50 is an RTA expression vector de-
scribed previously (16).
Chemicals and treatment of cells. The chemicals used in this study
were from the Guangdong Small Molecule Tangible Library (GSMTL)
(23). The compounds were dissolved in dimethyl sulfoxide (DMSO) and
serially diluted before being added to BCBL-1, JSC-1, or BJAB cell cultures
(4 ⫻10
5
cells/ml). The final DMSO concentration in the culture medium
was maintained at 1%. For lytic replication, BCBL-1 cells were induced by
tetradecanoylphorbol acetate (TPA) as previously reported (20). JSC-1
cells were induced by 3 mM sodium butyrate (22). Three hours postin-
duction, the compounds in dilutions were added to BCBL-1 or JSC-1 cells,
and subsequently, the antiviral effects and cytotoxicity were assayed at
different time points.
Analysis of intracellular KSHV genomic DNA content and chemical
effects. TPA-induced and uninduced BCBL-1 cells were harvested at 48 h
postinduction, and total DNA was purified using a DNeasy kit according
to the manufacturer’s protocol (Qiagen). The KSHV genomic copy num-
ber was quantified by real-time PCR on a Roche LightCycler instrument
using the LightCycler FastStart DNA MasterPlus SYBR green kit with
primers for the detection of LANA (forward, 5=-CGCGAATACCGCTAT
GTACTCA-3=; reverse, 5=-GGAACGCGCCTCATACGA-3=). The intra-
cellular viral genomic DNA in each sample was normalized to GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) using primers directed to
GAPDH (forward, 5=-ACATCATCCCTGCCTCTAC-3=; reverse, 5=-TCA
AAGGTGGAGGAGTGG-3=).
The half-maximal inhibitory concentration (IC
50
) values of com-
pounds were determined from a dose-response curve of KSHV DNA con-
tent values from TPA-induced and chemical-treated cells. The viral DNA
contents with those of uninduced cells subtracted were divided by those of
the control cells with no drug treatment and then represented on the y
axes of dose-response curves: yaxis value ⫽(TPA
X
⫺no TPA
X
)/(TPA
0
⫺
no TPA
0
), where Xis any concentration of the drug and 0 represents
nondrug treatment. The IC
50
on viral DNA synthesis for each compound
was calculated with the aid of GraphPad Prism software.
Analysis of extracellular KSHV virion production and chemical ef-
fects. Five days postinduction with TPA, BCBL-1 culture media were
collected and extracellular virions were pelleted from the medium super-
natant. To remove contaminating DNA outside viral particles, the con-
centrated viruses were treated with Turbo DNase I (Ambion) at 37°C for
1 h, followed by proteinase K digestion. Virion DNA was extracted with
phenol-chloroform, precipitated with ice-cold ethanol, and then dis-
solved in Tris-EDTA (TE) buffer. The KSHV genomic copy numbers were
determined by real-time PCR, and the values were corrected as described
above. The half-maximal antiviral effective concentration (EC
50
) values
were calculated from dose-response curves with GraphPad Prism soft-
ware.
Cytotoxicity assay. The viabilities of BCBL-1 cells treated or not with
chemicals were assessed by counting Trypan blue-stained cells 2 or 5 days
posttreatment using a light microscope. Cell viabilities were defined rela-
tive to control cells (non-drug treated). The half-maximal cytotoxic con-
centration (CC
50
) was calculated from dose-response curves with Graph-
Pad Prism software.
Cell proliferation assay. BCBL-1 and BJAB cells (both starting with
2⫻10
5
cells/ml) were treated with (⫹)-rutamarin for 5 days at two
different concentrations: the IC
50
and 5 times the IC
50
. The cells were
stained with Trypan blue and counted every day for 5 days. To maintain
the exponential growth, fresh medium (supplemented or not with the
drug) was added to the cultures every 2 days.
Cell cycle assay. BCBL-1 and BJAB cells (both starting with 4 ⫻10
5
cells/ml) were treated with (⫹)-rutamarin and novobiocin at two differ-
ent concentrations: the IC
50
and 5 times the IC
50
. Two days posttreat-
ment, cells were collected and fixed with cold 70% ethanol for 15 min,
permeabilized, and stained with propidium iodide (PI) solution (50 g/ml
of PI, 0.1 mg/ml of RNase A, and 0.05% Triton X-100) for 2 h. Cell cycle
progression was measured using a FACStar Plus cell sorter flow cytometer
(Becton, Dickinson).
ori-Lyt-dependent DNA replication assay. To assess the effect of (⫹)-
rutamarin on ori-Lyt-dependent DNA replication, BCBL-1 cells were
cotransfected with plasmids pOri-A (2.5 ␮g) and pCR3.1-ORF50 (2.5 ␮g)
by nucleoporation (Amaxa) and cultured with different concentrations of
(⫹)-rutamarin. Seventy-two hours posttransfection, extrachromosomal
DNA was prepared from cells using the Hirt DNA extraction method as
previously described (20). The extracted extrachromosomal DNA was
treated with RNase A at 25°C for 30 min, followed by proteinase K at 50°C
for 30 min. Five micrograms of DNA was digested with KpnI/SacI or
KpnI/SacI/DpnI (New England Bio-Labs), separated on a 1% agarose gel,
and transferred onto GeneScreen membranes (PerkinElmer, Boston,
MA). The Southern blots were hybridized with a
32
P-labeled pBluescript
plasmid in 5⫻SSC (1⫻SSC is 0.15 M NaCl plus 0.015 M sodium citrate),
2⫻Denhardt’s solution, 1% SDS, and 50 ␮g/ml denatured salmon sperm
DNA at 68°C.
Kinetoplast DNA decatenation assay. The decatenation assay is de-
signed to measure type II topoisomerase activity and inhibition of the
enzyme by testing compounds. The kinetoplast DNA (kDNA) (200 ng)
was incubated in a 20-␮l reaction mixture containing 50 mM Tris-HCl
(pH 7.5), 85 mM KCl, 10 mM MgCl
2
, 0.5 mM Na
2
EDTA, 0.5 mM dithio-
threitol, 1 mM ATP, 30 ␮g/ml bovine serum albumin (BSA), and 2 units
Xu et al.
564 aac.asm.org Antimicrobial Agents and Chemotherapy

of Topo II␣or Topo II␤for 30 min at 37°C in the absence or the presence
of (⫹)-rutamarin or control compounds (novobiocin and etoposide).
The reactions were analyzed by electrophoresis on agarose gels. The de-
catenated kDNAs were measured with the Gel Dox XR⫹imaging system
(Bio-Rad).
Malachite green assay for ATPase activity of Topo II␣.Malachite
green reagent was prepared by mixing malachite green (0.0812% [wt/
vol]), polyvinyl alcohol (2.32% [wt/vol]), ammonium molybdate (5.72%
[wt/vol] in 6 M HCl), and ultrapure water in a ratio of 2:1:1:2. An ATP
hydrolysis reaction with a mixture (20 ␮l) containing 50 mM Tris-HCl,
pH 7.4, 85 mM KCl, 10 mM MgCl
2
, 0.5 mM dithiothreitol (DTT), 0.5 mM
Na
2
EDTA, 30 ␮g/ml BSA, 200 ng pBR322 DNA, 2 units Topo II␣, 500 ␮M
ATP, and various concentrations of (⫹)-rutamarin was initiated by add-
ing ATP to the reaction mixture and incubated at 37°C for 2 h. The reac-
tion was stopped by adding 80 ␮l of the malachite green reagent to the
reaction mixture, followed by adding 10 ␮l of 34% sodium citrate to
develop a blue-green color change. The absorbance at 620 nm was mea-
sured using a plate reader. The optical density at 620 nm (OD
620
) was used
to represent the ATP hydrolysis level.
Molecular docking study. The X-ray crystal structure of the ATPase
domain of human Topo II␣in complex with ADPNP (Protein Data Bank
[PDB] ID, 1ZXM; resolution, 1.87Å) was retrieved from the Protein Data
Bank (24). Only chain A was kept for docking study. The missing loop
(residues 345 to 350) was added and optimized using Prepare Protein
Module encoded in Discovery Studio 3.5 (Accelrys Inc.) (25). The initial
structures of (⫹)-rutamarin and ADPNP were optimized using MMFF
force field (26), and the Powell method was used for energy minimization
by default parameters in Discovery Studio 3.5.
The docking program FlexX encoded in SYBYL 7.3 (Tripos Inc.) was
applied to identify the potential binding of (⫹)-rutamarin to the Topo II␣
ATPase domain. A previous study suggested that two conserved water
molecules were not neglected to distinguish the active, moderate, and
inactive ligands (27). In the present study, the receptor for docking sim-
ulation consisted of protein chain A, two conserved water molecules, and
Mg
2⫹
. To validate the molecular-docking protocol, ADPNP was re-
docked into the crystal structure of the ATPase domain of Topo II␣. The
docked ADPNP has a binding pose similar to that of the cocrystallized
ligand, with a root mean square deviation (RMSD) of about 1.6 Å. The
active sites were defined as all residues within a 6.5-Å radius of the bound
ADPNP. Other FlexX parameters were set to default values. Thirty poses
were retained during the docking process. After running FlexX, all the
poses were visually inspected, and the most suitable docking pose was
selected on the basis of the score and interactions with key residues of the
active site. The complex of Topo II␣and the most suitable docking con-
former of (⫹)-rutamarin was used as the initial coordinates for the sub-
sequent molecular dynamics (MD).
MD simulations. MD simulations were performed in AMBER 12 (28)
with the AMBER ff99SB force field (29,30). The docked structure of the
Topo II␣ATPase domain with (⫹)-rutamarin was used as the initial
coordinates for MD simulations. Parameters for (⫹)-rutamarin were ob-
tained from the ANTECHAMBER module using the Generalized Amber
Force Field (GAFF) (31) with the RESP charge-fitting procedure with
input from Hartree-Fock calculations at the 6- to 31-G* level by using the
Gaussian03 program (32). All hydrogen atoms of the protein were added
using the tleap module, considering ionizable residues set at their default
protonation states at a neutral pH. The system was solvated in a truncated
octahedron box of TIP3P water molecules with a margin distance of 10 Å.
Neutralizing counterions were added to the simulation system. The de-
tailed MD simulations can be found in the supplemental material. The
molecular-mechanics Poisson-Boltzmann surface area (MM-PBSA) and
molecular-mechanics generalized born surface area (MM-GBSA) meth-
ods (33) in the AMBER 12 suite were used to calculate the binding free
energies (see the supplemental material).
RESULTS
(ⴙ)-Rutamarin inhibits the lytic replication of KSHV. To search
for a novel Topo II inhibitor(s) that effectively blocks KSHV lytic
DNA replication, a ligand-based virtual screening using an in-
house three-dimensional (3D) molecular superimposing algo-
rithm, WEGA (34), was conducted against the molecular struc-
tures in our compound repository, GSMTL (23). Using the
novobiocin structure as the template, we screened approximate
7,200 compounds from the GSMTL. The top 33 compounds that
are three-dimensionally most similar to novobiocin were selected
and subsequently tested for antiviral activities. Exponentially
growing BCBL-1 cells were induced into lytic replication with
TPA. Three hours after induction, the cells were exposed to each of
the compounds, as well as novobiocin, at a concentration of 20
␮M. Forty-eight hours postinduction, the KSHV genomic DNA
contents of the cells treated with these compounds were analyzed
by quantitative real-time PCR. Treatment with novobiocin re-
sulted in 42% inhibition of viral replication compared to the con-
trol cells treated with no drug. Seven compounds showed inhibi-
tory effects similar to or better than those of novobiocin on viral
DNA replication (Fig. 1A). Four of these, C28, C31, C32, and C33,
also displayed strong activities in blocking virion production (Fig.
1B). With the exception of C31, the compounds possess signifi-
cant cytotoxicity, as demonstrated by low cell viability rates with
the treatment (Fig. 1C). C31, which was identified as the natural
product (⫹)-rutamarin (Fig. 2A), exhibited significant inhibition
of both viral DNA synthesis and virion production, with relatively
low cytotoxicity at a concentration of 20 ␮M. Therefore, it was
chosen for further investigation.
The IC
50
values of (⫹)-rutamarin were determined from the
dose-response curve of KSHV DNA content in TPA-induced
BCBL-1 cells and were found to be 1.12 ␮M(
Fig. 2B). The effect of
(⫹)-rutamarin on progeny virion production was also deter-
mined by quantification of encapsidated viral DNA in BCBL-1 cell
culture media. The EC
50
calculated from the dose-response curve
of extracellular virions is 1.62 ␮M(
Fig. 2B). The cytotoxicity of
(⫹)-rutamarin on BCBL-1 cells was examined in parallel with
inhibition of KSHV DNA replication and virion production. Cells
treated with (⫹)-rutamarin at differing concentrations were
subjected to the trypan blue exclusion method to determine cell
viability in response to (⫹)-rutamarin treatment. The CC
50
was
determined to be 94.24 ␮M(
Fig. 2B). Low cytotoxicity of (⫹)-
rutamarin results in an appreciable selectivity index (SI) (SI ⫽
CC
50
/IC
50
) of 84.14.
To make sure that (⫹)-rutamarin inhibits KSHV lytic replica-
tion regardless of host cells and the means of lytic cycle induction,
we tested the effect of (⫹)-rutamarin on KSHV replication in
JSC-1 cells induced by sodium butyrate for lytic replication. The
IC
50
,EC
50
, and CC
50
of (⫹)-rutamarin on butyrate-induced
JSC-1 cells were found to be 2.29 ␮M, 1.40 ␮M, and 94.34 ␮M,
respectively, and are very similar to those obtained with BCBL-1.
In addition, the cytotoxicity of (⫹)-rutamarin to primary lym-
phocytes was also assessed with PBMC. The CC
50
for PBMC was
calculated to be 60.91 ␮M (see Fig. S1 in the supplemental mate-
rial).
Evaluation of (ⴙ)-rutamarin for its effects on host cell pro-
liferation and cell cycle progression. To further investigate the
potential of (⫹)-rutamarin to become an effective and safe anti-
viral, the effect of the compound on cell proliferation was assessed.
(⫹)-Rutamarin Inhibits KSHV Replication
January 2014 Volume 58 Number 1 aac.asm.org 565

KSHV-carrying BCBL-1 and virus-free BJAB cells were cultured
in the presence and absence of (⫹)-rutamarin, and live cells were
counted over 5 days. (⫹)-Rutamarin did not exhibit a cell growth-
inhibitory effect in the two cell lines at the IC
50
. At an excess
concentration (5 times the IC
50
), (⫹)-rutamarin showed an ad-
verse effect on cell growth, but the extent of inhibition was less
than that with novobioicin (Fig. 3). The effect of (⫹)-rutamarin
on cell cycle progression was also examined. BCBL-1 cells treated
with (⫹)-rutamarin were stained with propidium iodide and sub-
jected to flow cytometric analysis. At the IC
50
, no effect on the cell
cycle pattern was observed with either (⫹)-rutamarin or novobio-
cin in comparison with the vehicle control. At an excess concen-
tration (5 times the IC
50
)of(⫹)-rutamarin, a slight increase in the
S phase was observed in comparison to untreated cells. However,
the influence of (⫹)-rutamarin on cell cycle progression was ob-
served to be less than that of novobiocin (Fig. 4). Overall, these
results demonstrated that (⫹)-rutamarin has minimal effects on
host cell proliferation and cell cycle progression in its pharmaceu-
tical dose range.
(ⴙ)-Rutamarin inhibits KSHV lytic replication by blocking
viral ori-Lyt-dependent DNA replication. We have demon-
strated that (⫹)-rutamarin is capable of inhibiting KSHV DNA
synthesis and virion production. To confirm whether the inhibi-
tory effect of (⫹)-rutamarin on KSHV lytic replication is directly
due to the blocking of ori-Lyt-dependent DNA replication, we
conducted an ori-Lyt DNA replication assay. BCBL-1 cells were
cotransfected with an ori-Lyt-containing plasmid (pOri-A) and an
RTA expression vector. Lytic DNA replication was induced by
RTA expression (35). The transfected cells were cultured in the
presence of (⫹)-rutamarin at various concentrations, and the ef-
fect of (⫹)-rutamarin on ori-Lyt-dependent DNA replication was
measured by a DpnI assay (16,17). In brief, DNA was isolated
from the treated cells 72 h posttransfection and digested with
KpnI/SacI and KpnI/SacI/DpnI. Replicated plasmid DNA was dis-
tinguished from input plasmids by DpnI restriction digestion,
which cleaves input DNA that has been Dam
⫹
methylated in Esch-
erichia coli but leaves intact the DNA that has been replicated in at
least one round in eukaryotic cells. Thus, only newly replicated
plasmid DNA in BCBL-1 cells, which is resistant to DpnI diges-
tion, can be detected in Southern blot analysis. Replicated pOri-A
DNA was detected as a DpnI-resistant DNA band in the cells that
were cotransfected with pOri-A and an RTA expression vector.
However, the replicated DNA decreased with elevated concentra-
tions of (⫹)-rutamarin in the cell culture (Fig. 5). This result
FIG 1 Compound screening. (A) Thirty-three compounds (see Table S2 in the supplemental material) derived from a virtual screening were assayed for their
effects on KSHV lytic DNA replication. BCBL-1 cells were induced with TPA and treated with each of the compounds at a concentration of 20 ␮M. Forty-eight
hours postinduction, the intracellular KSHV genomic copy numbers were determined using quantitative real-time PCR. Seven compounds (marked with
asterisks) exhibited considerable inhibitory effects on viral lytic DNA replication. (B) Effects of the compounds on KSHV virion production. Five days
postinduction, the KSHV virions in supernatants were collected and measured by quantifying encapsidated viral DNA in the preparations. (C) Effects of the
compounds on host cell viability. Uninduced BCBL-1 cells were treated with each of the compounds at 20 ␮M for 48 h, and cell viability was assessed by trypan
blue staining as described in Materials and Methods. The compounds are shown with ascending molecular weights. The error bars indicate standard deviations.
Xu et al.
566 aac.asm.org Antimicrobial Agents and Chemotherapy

supports the idea that (⫹)-rutamarin inhibits KSHV ori-Lyt-de-
pendent DNA replication.
To rule out the possibility that (⫹)-rutamarin blocks KSHV
DNA replication by inhibiting RTA expression, the effect of (⫹)-
rutamarin on RTA expression in TPA-induced BCBL-1 cells was
examined by Western analysis. The result showed no adverse ef-
fect of the compound on RTA expression up to 25 ␮M (22-fold
excess over the IC
50
) (see Fig. S2 in the supplemental material).
The reduced levels of RTA in the treatments with 50 and 100 ␮M
(⫹)-rutamarin may result from the cytotoxicity of the compound
in a concentration near its IC
50
(94.24 ␮M).
(ⴙ)-Rutamarin is an inhibitor of human topoisomerase II␣.
The next question was whether (⫹)-rutamarin is a Topo II inhib-
itor that blocks KSHV ori-Lyt-dependent DNA replication by in-
hibiting the catalytic activity of host cell Topo II. Furthermore,
there are two Topo II isoforms in mammals, Topo II␣and Topo
II␤. We wanted to know which isoform (⫹)-rutamarin affects if it
is a Topo II inhibitor. To accomplish this, a DNA decatenation-
based topoisomerase II assay was established. In this system,
kinetoplast DNA consisting of a large network of interlocked
DNA minicircles can be decatenated into separate minicircles in
the presence of ATP and Topo II in either isoform. (⫹)-Rutama-
rin in a wide range of concentrations was added to the reaction
mixture, and the effect of the compound on Topo II activity was
measured by determining decatenated kDNA levels using agarose
gel electrophoresis. The results showed that (⫹)-rutamarin is able
to inhibit the enzymatic activity of Topo II␣in a dose-dependent
manner (Fig. 6A and B). The IC
50
of (⫹)-rutamarin in Topo II␣
inhibition was calculated as 28.22 ⫾6.2 ␮M(Fig. 6C and D). At a
concentration of 100 ␮M, the inhibition rate of (⫹)-rutamarin is
significantly higher than that of novobiocin and close to that of the
Topo II poison etoposide (Fig. 6A). In contrast, (⫹)-rutamarin
did not exhibit any inhibitory effect on Topo II␤activity (Fig. 6E).
To confirm if (⫹)-rutamarin is a catalytic inhibitor of Topo
II␣and acts in blocking the ATPase activity of the enzyme, as
novobiocin does, we studied the inhibition effect of rutamarin on
ATP hydrolysis of Topo II␣using the malachite green assay. As
shown in Fig. 7, the ATPase activity of Topo II␣was inhibited by
(⫹)-rutamarin in a dose-dependent manner, suggesting that, like
novobiocin, (⫹)-rutamarin inhibits human Topo II␣by binding
to the ATP pocket of the enzyme and blocking its ATP hydrolysis.
A binding model of (ⴙ)-rutamarin in the ATP binding
pocket of Topo II␣.The mode of (⫹)-rutamarin binding in the
ATPase domain of Topo II␣was investigated by docking and fur-
ther defined by MD simulations. Figure 8A illustrates the time
dependence of the RMSD values for the X-ray reference enzyme
structure of backbone atoms (C, C
151
, N, and O) over the produc-
tion phase of simulation. The RMSD values of simulation con-
verged after ⬃3 ns, indicating that the system is stable and equil-
ibrated. The RMSD value of ligand compared docking pose is
swinging within 1.8 Å, which illustrates that the docking pose is
reliable. Previous studies indicated that Mg
2⫹
is necessary for the
ATPase domain to hydrolyze the ATP molecule and is stabilized
by two conserved water molecules and residues Asn91 and Glu87
(24,27). The time evolution of oxygen- and nitrogen-Mg
2⫹
dis-
tances for these residues and the water molecules is shown in Fig.
8C. The oxygen- and nitrogen-Mg
2⫹
distances for these residues
and the water molecules stabilized at 1.9 to 2.0 Å during the sim-
ulation, suggesting that Mg
2⫹
is stabilized by these factors. Our
simulation results are consistent with previous experimental re-
sults, suggesting that our model is reliable and reasonable.
The details of binding of (⫹)-rutamarin to the ATPase domain
of Topo II␣at an atomic level were revealed in a conformation
clustering analysis. The results suggest that (⫹)-rutamarin forms
a hydrogen bond with the side chain of Asn95, Ala167, and con-
served water molecule 2 and hydrophobically interacts with
Ile125, Ile141, Phe142, Ala167, Thr215, Gly166, and Lys123 in the
catalytic site of Topo II␣(Fig. 8D). The distance distribution anal-
ysis also showed that the hydrogen bonds between conserved wa-
ter molecule 2- and Ala167–(⫹)-rutamarin are comparatively sta-
ble, whereas the hydrogen bond of Asn95–(⫹)-rutamarin is
unstable (Fig. 8B). Solvent-accessible-surface area (SASA) analy-
sis suggests that the polar surface of (⫹)-rutamarin is comple-
FIG 2 Effect of (⫹)-rutamarin on KSHV replication and its associated cyto-
toxicity. (A) Chemical structure of (⫹)-rutamarin. (B) Effect of (⫹)-rutama-
rin on KSHV lytic replication and its associated cytotoxicity in BCBL-1 cells
that were treated with a wide range of concentrations of (⫹)-rutamarin 3 h
after lytic replication was induced by TPA. Intracellular KSHV genomic DNA
replication (blue), extracellular virion production (green), and cell viability
(orange) were determined as described in Materials and Methods. The values
were compared to those from the control cells (nondrug treatment). The mean
values of results from three independent experiments and standard deviations
are presented on the yaxis of dose-response curves. (C) Effect of (⫹)-rutama-
rin on KSHV lytic replication and its associated cytotoxicity in JSC-1 cells that
were induced with 3 mM sodium butyrate. Intracellular KSHV genomic DNA
replication (blue), extracellular virion production (green), and cell viability
(orange) were determined as described for panel B.
(⫹)-Rutamarin Inhibits KSHV Replication
January 2014 Volume 58 Number 1 aac.asm.org 567

mentary to the polar surface of the binding pocket of Topo II␣
(Fig. 8E). Superimposition of (⫹)-rutamarin and cocrystallized
ADPNP suggests that (⫹)-rutamarin occupies the ATP binding
pocket in a fashion similar to that of an ATP molecule (Fig. 8F).
The acetyl groups of (⫹)-rutamarin, like the three phosphate
groups of ATP, is involved in interaction with the highly con-
served Walker A consensus motif GXXGXG at residues 161 to 166
of Topo II␣(Fig. 8D and F)(36). This conserved motif is impor-
tant for the binding of ATP, as well as for the catalytic inhibitors
acting on the ATPase domain (27). This notion is supported by a
site-directed mutagenesis result showing that the mutations in
Arg162Gln and Tyr165Ser in the ATP binding site lead to drug
resistance (37,38). These observations, consistent with the results
of the DNA decatenation assay and the malachite green assay,
support the idea that (⫹)-rutamarin functions as a Topo II␣cat-
alytic inhibitor.
The MM-PBSA and MM-GBSA methods were employed to
calculate the binding free energies and to gain information on
the different components of interaction energy that contribute
to (⫹)-rutamarin binding. The results are listed in Table 1.
⌬G
MM-PBSA
and ⌬G
MM-GBSA
for the Topo II␣-(⫹)-rutamarin
complex are ⫺29.90 and ⫺24.66 kcal/mol, respectively. ⌬G
MM-
PBSA
, accounting for the total relative binding free energy, equal to
⌬G
gas
plus ⌬G
solv
, comes mainly from the van der Waals compo-
nent, whereas the electrostatic contribution is much less. This no-
tion is also reflected in the results of the MM-GBSA method
(Table 1), which show that (⫹)-rutamarin is a comparatively hy-
drophobic molecule that can form favorable hydrophobic inter-
actions with residues of the Topo II␣catalytic pocket.
DISCUSSION
In the current study, we identified (⫹)-rutamarin as an effective
antiviral agent that inhibits KSHV ori-Lyt-dependent DNA repli-
cation with low cytotoxicity. It blocks KSHV DNA replication by
FIG 3 Effects of (⫹)-rutamarin and novobiocin on BCBL-1 cell proliferation. BCBL-1 cells (starting with 2 ⫻10
5
cells/ml) were exposed to (⫹)-rutamarin and
novobiocin at their IC
50
s and 5 times their IC
50
s. Data were obtained from three independent determinations and are presented as means and standard
deviations.
FIG 4 Effects of (⫹)-rutamarin and novobiocin on BCBL-1 cell cycle progression. BCBL-1 cells (starting with 2 ⫻10
5
cells/ml) were exposed to (⫹)-rutamarin
and novobiocin at their IC
50
s and 5 times their IC
50
s for 48 h. Cell cycle progressions were measured by PI staining, followed by flow cytometric analysis. The data
are presented as means obtained from three independent experiments.
Xu et al.
568 aac.asm.org Antimicrobial Agents and Chemotherapy

inhibiting the catalytic activity of human Topo II␣. The salient
features and implications of this finding are as follows.
There is a need for effective drugs targeting KSHV. KSHV has
been proven to be the etiological cause of KS and other KSHV-
associated malignancies. However, the current treatment modal-
ities for KSHV-associated diseases include only traditional cancer
therapies. For classic KS, the chemotherapeutics that have been
approved by the FDA include liposomal anthracycline products
(liposomal doxorubicin or liposomal daunorubicin), paclitaxel,
and alpha interferon (39). However, the majority of these agents
have serious side effects, and the tumor responses to the chemo-
therapeutic regimens are only transient. In the current AIDS epi-
demic, even though KS has become a major AIDS-associated ma-
lignancy and remains an important cause of morbidity and
mortality in AIDS patients, little effort has been made to develop
more effective drugs for KS, as there was a general belief that
AIDS-associated KS would disappear if AIDS were under control
FIG 5 Inhibition of KSHV ori-Lyt-dependent DNA replication with (⫹)-
rutamarin. BCBL-1 cells were transfected with a KSHV ori-Lyt-containing
plasmid (pOri-A) and an RTA expression vector (pCR3.1-ORF50). The trans-
fected cells were treated with increasing concentrations of (⫹)-rutamarin and
incubated for 72 h. Hirt DNAs were extracted and digested with KpnI/SacI or
KpnI/SacI/DpnI. DpnI-resistant viral replicated DNA (Rep’d DNA) was de-
tected by Southern blotting with
32
P-labeled pBluescript plasmid.
FIG 6 Identification of (⫹)-rutamarin as a catalytic inhibitor of Topo II␣. (A) A Topo II-mediated kinetoplast DNA decatenation assay was performed to
evaluate the effects of testing compounds for Topo II␣activity at a concentration of 100 ␮M. The catenated (cat) and decatenated (decat) DNA positions in gels
are indicated. (A and B) The Topo II␣activities in the reactions were quantitated (A), and inhibition rates of compounds were calculated (B). (C and E) Inhibition
of Topo II␣(C) and Topo II␤(E) activities by (⫹)-rutamarin in a wide range of concentrations. (D) The IC
50
of (⫹)-rutamarin on Topo II␣inhibition was
calculated as 28.22 ⫾6.2 ␮M. The data are means and standard deviations for three experimental replicates. (E) (⫹)-Rutamarin exhibits no obvious inhibitory
effect on human Topo II␤. Rut, (⫹)-rutamarin; Eto, etoposide; Nov, novobiocin.
FIG 7 Effect of (⫹)-rutamarin on ATPase activity of Topo II␣. The inhibitory
effect of (⫹)-rutamarin on Topo II␣ATPase activity was measured using the
malachite green assay as described in Materials and Methods. The OD
620
rep-
resents the ATP hydrolysis level and reflects the inhibition of ATPase activity
by (⫹)-rutamarin. The error bars indicate standard deviations.
(⫹)-Rutamarin Inhibits KSHV Replication
January 2014 Volume 58 Number 1 aac.asm.org 569

FIG 8 Stability properties of the simulation system and binding model of (⫹)-rutamarin with Topo II␣. (A) RMSD plot for the backbone atoms and
(⫹)-rutamarin during MD simulations after equilibration. (B) Distance distributions between Asn95, Ala167, and conserved water molecule 2 and (⫹)-
rutamarin in water at 310 K. The cutoff value for the formation of a hydrogen bond is 3.5 Å. (C) Time dependence of the distance between the carboxyl oxygen
of Glu87, the amide nitrogen of Asn91, the two conserved water molecules, and Mg
2⫹
during the 10-ns MD simulations. (D) Detailed binding model between
(⫹)-rutamarin and the residues in the ATPase domain of Topo II␣. Hydrogen bonds are shown by red dashed lines, and magenta spheres represent Mg cations.
(E) Polar and hydrophobic surface profile of the ATPase domain of Topo II␣with (⫹)-rutamarin. Red and gray represent polar and hydrophobic areas,
respectively. (F) Superposition of (⫹)-rutamarin with ADPNP in the ligand binding pocket.
Xu et al.
570 aac.asm.org Antimicrobial Agents and Chemotherapy

with the advent of HAART. However, despite its dramatic de-
crease in frequency since the advent of HAART, KS remains the
most common AIDS-associated cancer (40). In addition, as expe-
rience with HAART has grown, a new HAART-associated syn-
drome has emerged. In a subset of HIV-seropositive individuals,
starting HAART in the setting of advanced HIV infection results
in a paradoxical clinical worsening of existing infection or the
appearance of a new condition, including KS, in a process known
as immune reconstitution inflammatory syndrome (IRIS) (41).
IRIS is thought to be the direct result of a reconstituted im-
mune system recognizing pathogens or antigens that were pre-
viously present but clinically asymptomatic. The current regi-
men for IRIS-associated KS is a combination therapy with
HAART plus chemotherapeutics, such as liposomal doxorubicin.
Since IRIS-KS is the result of responses by a recovered immune
system to the KS-causing pathogen, i.e., KSHV, treatment of
KSHV-seropositive, HIV-positive patients with a combination of
antiretroviral (HAART) and anti-KSHV therapeutics is expected
to yield positive results. However, at this time, there are no effec-
tive drugs targeting KSHV available. Earlier studies on cohorts of
HIV-seropositive subjects suggested that treatment with the anti-
herpesviral drug ganciclovir or foscarnet could reduce the inci-
dence of KS in AIDS patients (42,43). However, severe side effects
(renal impairment and bone marrow suppression), as well as
rapid development of drug resistance, have limited the use of these
drugs in the treatment of KS. Therefore, there is a need for effec-
tive drugs targeting KSHV.
(ⴙ)-Rutamarin is an antiviral-drug candidate for KSHV-as-
sociated diseases. Recently, we reported that host cellular Topo II
is absolutely required for KSHV DNA replication, and thus, Topo
II inhibitors effectively block KSHV DNA synthesis (20). Topo II
inhibitors are divided into two categories: Topo II poisons, which
target the topoisomerase-DNA intermediate (cleavable complex),
and Topo II catalytic inhibitors, which disrupt catalytic turnover
of the enzyme. Although both categories possess inhibitory activ-
ities against KSHV DNA replication, Topo II poisons in general
exhibit very strong cytotoxicity to host cells. In contrast, Topo II
catalytic inhibitors show less cytotoxicity (such as novobiocin,
with a CC
50
value of 871 ␮M, and merbarone, with a CC
50
value of
212.9 ␮M for BCBL-1 cells). Both novobiocin and merbarone are
effective in halting KSHV DNA synthesis, with IC
50
s of 27.55 ␮M
and 19.54 ␮M, respectively. The low cytotoxicities and high inhi-
bition rates for viral replication of catalytic Topo II inhibitors
suggest the potential for Topo II catalytic inhibitors to become
effective anti-KSHV drugs for treatment of KS and other KSHV-
associated diseases (20). In addition, given that viral polymerases
may introduce mutations which subsequently can give rise to drug
resistance, targeting host cellular proteins that viruses rely on for
their replication offers the advantage of minimizing drug resis-
tance and thus constitutes a novel therapeutic strategy.
In the current study, we attempted to search for new Topo II
catalytic inhibitors that are potent in inhibiting viral replication
and that have low cytotoxicity. This effort led to identification of
(⫹)-rutamarin as a promising lead that efficiently inhibits KSHV
lytic DNA replication in BCBL-1 cells with an IC
50
of 1.12 ␮M and
an EC
50
of 1.62 ␮M, 25- and 17-fold lower than those of novobio-
cin. In addition, (⫹)-rutamarin exhibits very low cytotoxicity,
and the SI of KSHV lytic replication was calculated to be 84.14,
three times better that that of novobiocin. In addition, the molec-
ular weight of (⫹)-rutamarin (356.42) is smaller than that of
novobiocin (634.61), and the mass concentration differences of
their IC
50
s and EC
50
s would be greater. These data demonstrate
great potential for (⫹)-rutamarin to become an effective antiviral
agent with low cytotoxicity.
(⫹)-Rutamarin is a natural product found in plants, such as
Ruta graveolens L (common rue). It has been reported that (⫹)-
rutamarin possesses antiproliferation and anticancer activities
against a variety of tumor cell lines (44). It has also been found to
induce the expression and translocation of glucose transporter 4
(GLUT4). As impaired translocation or decreased expression
of GLUT4 in response to insulin is one of the major patholog-
ical features of type 2 diabetes, the function of (⫹)-rutamarin in
ameliorating glucose homeostasis suggests a potential for the
compound in anti-type 2 diabetes drug development (45). Our
study is the first to demonstrate the capability of the compound in
inhibition of viral replication with the potential to be an antiviral.
Anti-proliferation potential of (ⴙ)-rutamarin. At a dose
equal to the IC
50
for inhibiting KSHV replication, (⫹)-rutamarin
did not show a perceptible effect on cell proliferation and cell cycle
progression in BCLB-1 and BJAB cells. At an excess dose (5 times
the IC
50
,), (⫹)-rutamarin exhibited a degree of suppression of cell
proliferation with a delay in the S phase.
The effects of (⫹)-rutamarin on cell proliferation in a variety
of tumor cell lines were estimated, and inhibitory activities against
A549, Bel7402, HepG2, and HCT8 cell proliferation were reported
(44). The antiproliferation activity of (⫹)-rutamarin against these
tumor cell lines appears to be similar to the effects on B cells
observed in our study. Although (⫹)-rutamarin has an extent of
antiproliferative ability that may provide an additional benefit if it
becomes a drug to treat KSHV-associated malignancies, the mod-
erate antiproliferation ability may not be potent enough to pro-
vide antitumor activity. This may also be true of all Topo II cata-
lytic inhibitors. This notion is supported by studies and
observations of some Topo II catalytic inhibitors, including novo-
biocin and merbarone, which exhibit antiproliferation activities
in cell cultures but show no significant effects on tumor develop-
ment in clinical trials (46,47).
The moderate effect of (⫹)-rutamarin on cell proliferation, as
well as its low cytotoxicity, may result from redundancy of Topo II
activities in mammalian cells. Mammals express two isoforms of
TABLE 1 Binding free energy for Topo II␣–(⫹)-rutamarin complex
and its different energy components based on MM-PBSA and MM-
GBSA methods
Energy term
Binding free energy (kcal/mol) (SEM)
MM-PBSA MM-GBSA
⌬E
vdwa
⫺40.12 (2.14) ⫺40.22 (2.26)
⌬E
eleb
⫺0.35 (0.22) ⫺0.42 (0.15)
⌬E
pol,solvc
39.99 (1.93) 22.09 (1.66)
⌬E
nonpol,solvd
⫺29.43 (1.37) ⫺6.11 (0.34)
⌬G
gase
⫺40.47 (2.41) ⫺40.64 (2.28)
⌬G
solvf
10.56 (1.28) 15.98 (1.14)
⌬G
bindingg
⫺29.91 (1.38) ⫺24.66 (1.69)
a
Nonbonded van der Waals.
b
Nonbonded electrostatics.
c
Polar component to solvation.
d
Nonpolar component to solvation.
e
Total gas phase energy.
f
Sum of nonpolar and polar contributions to solvation.
g
Final estimated binding free energy calculated from the terms above.
(⫹)-Rutamarin Inhibits KSHV Replication
January 2014 Volume 58 Number 1 aac.asm.org 571

Topo II, alpha and beta, which are highly homologous but display
differences in expression and subcellular localization at the time of
mitosis. The similar functions and domain structures of the two
isoforms make it possible that Topo II␤may be able to partially
compensate for the loss of Topo II␣function for cell survival in
the presence of an inhibitor, such as (⫹)-rutamarin. However,
KSHV may be dependent on only Topo II␣for its lytic replication,
explaining the sensitivity of KSHV replication to Topo II␣cata-
lytic inhibitors.
(ⴙ)-Rutamarin is a novel human Topo II␣catalytic inhibi-
tor. Topo II is an ATPase and uses the energy derived from ATP
hydrolysis to resolve the winding problem of double-stranded
DNA. The detailed binding mode of (⫹)-rutamarin and human
Topo II␣was identified using molecular docking and MD simu-
lation. The significance of these studies is 2-fold. First, the study
provided insight into the binding of (⫹)-rutamarin to Topo II␣,
and the results confirmed that the compound binds to the ATP
pocket of Topo II␣. Second, the results of the study will be of great
help in our next effort to modify the compound for more potent
antiviral candidates. Energy decomposition analysis led to identi-
fication of key residues contributing to binding affinity at the ac-
tive site. As shown in Fig. 9, the major contributing residues can be
divided into three clusters, which are consistent with the three
binding pocket sites, i.e., (i) the Walker A consensus motif (resi-
dues 161 to 167), (ii) the Mg
2⫹
binding area (residues 87, 91, and
94), and (iii) the hydrophobic chamber (key residues, Ile 125,
Ile141, Phe142, Lys123, and Thr215). All of these residues except
Glu87 positively contribute to the binding affinity for (⫹)-
rutamarin. The incompatibility between the carboxyl group of
Glu87 and the acetyl groups of (⫹)-rutamarin suggests that Glu87
is unfavorable for binding affinity to (⫹)-rutamarin (Fig. 8D).
These results provide a theoretical basis for (⫹)-rutamarin lead
optimization.
ACKNOWLEDGMENTS
We thank Manunya Nuth at the University of Pennsylvania for construc-
tive discussion, suggestions, and critical reading of the manuscript.
This work was supported by Natural Science Foundation of China
research grants (no. 81271805 and 81171575), the Guangdong Innovative
Research Team program (no. 2009010058), and a research grant from the
U.S. National Institutes of Health (R01AI052789).
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