ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2009, p. 1727–1734
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 53, No. 5
Novel Inhibitor of Plasmodium Histone Deacetylase That Cures
P. berghei-Infected Mice?
S. Agbor-Enoh,1,4C. Seudieu,1E. Davidson,1A. Dritschilo,2,3and M. Jung2*
Departments of Biochemistry and Molecular Biology,1Radiation Medicine,2and Oncology,3Georgetown University Medical Center,
Vincent T. Lombardi Comprehensive Cancer Center, Washington, DC 20057, and Biotechnology Center, University of Yaounde I,
Nkol-Bisong, Yaounde, Cameroon4
Received 3 June 2008/Returned for modification 10 August 2008/Accepted 4 February 2009
Histone deacetylases (HDAC) are potential targets for the development of new antimalarial drugs. The
growth of Plasmodium falciparum and other apicomplexans can be suppressed in the presence of potent HDAC
inhibitors in vitro and in vivo; however, in vivo parasite suppression is generally incomplete or reversible after
the discontinuation of drug treatment. Furthermore, most established HDAC inhibitors concurrently show
broad toxicities against parasites and human cells and high drug concentrations are required for effective
antimalarial activity. Here, we report on HDAC inhibitors that are potent against P. falciparum at subnano-
molar concentrations and that have high selectivities; the lead compounds have mean 50% inhibitory concen-
trations for the killing of the malaria parasite up to 950 times lower than those for the killing of mammalian
cells. These potential drugs improved survival and completely and irreversibly suppressed parasitemia in P.
Malaria parasites infect hundreds of millions of people and
lead to more than 1 million deaths annually, predominantly in
sub-Saharan Africa and Asia. The principal malarial parasite
in humans responsible for malaria-associated mortality is Plas-
modium falciparum. Recently, a number of drug-resistant
strains of P. falciparum have evolved, and these strains are
resistant to the majority of the existing antimalarial drugs (4,
19). Even though antimalarials like quinine, artemisinin, and
their derivatives offer effective treatment, the need to develop
new drugs for the treatment of infections caused by resistant
parasite strains has become a clear, current priority. Initiatives
to identify new molecular targets, to structurally modify exist-
ing antimalarials, and to identify agents that can be used in
combination to overcome parasite drug resistance are in
progress (4, 19).
Histone acetylation is a reversible process and is controlled
by histone acetyltransferases (HATs) and histone deacetylases
(HDACs). HATs are divided into three families on the basis of
the number of conserved motifs and include the GNAT,
MYST, and p300/CBP families. Of these, homologues of the
first two families (P. falciparum GCN5 [PfGCN5] and P. fal-
ciparum MYST [PfMYST]) have been described in P. falcipa-
rum (11) and may play critical roles in parasite development
and virulence. Recently, the inhibition of PfGCN5 has been
shown to alter the expression of approximately 5% of plasmo-
dial genes and blocked the growth of chloroquine-sensitive and
-resistant P. falciparum strains (7).
A genomic search revealed five putative deacetylase-encod-
ing genes, including the partially characterized P. falciparum
HDAC1 (PfHDAC1) gene (13) and the P. falciparum Sir2 gene
(23). Sequence analyses of PfHDAC1 showed that it has a high
degree of homology among apicomplexans, raising the possi-
bility that inhibitors with the potential for broad clinical appli-
cation against apicomplexan diseases may be developed. The
HDACs offer attractive molecular targets since they are in-
volved in the regulation of the chromatin structure and tran-
It is believed that acetylation on histone lysine residues
changes the conformation of chromatin from a condensed,
closed form to an open, transcriptionally active form. HDAC
inhibitors affect the balance of reversible acetylation to modify
the expression of genes involved in cell growth, cell cycle pro-
gression, and apoptosis. Human cells contain at least 18
HDACs with functional redundancy capabilities (5, 15). The
HDACs are distributed among four classes, with HDACs 1, 2,
3, and 8 belonging to class I; HDACs 4, 5, 6, 7, 9, and 10
belonging to class II, sirtuins (NAD?-dependent deacetylases)
1 to 7 belonging to class III; and HDAC 11 belonging to class
IV. The presence of only five putative HDACs in P. falciparum
potentially offers fewer redundant targets for inhibition than is
the case for human cells.
HDACs have been targets for antimalarial drug discovery
for the past 10 years, and some inhibitors have shown broad
antiprotozoal growth-inhibitory activities (8, 20). Generally,
high concentrations of drugs have been required to inhibit
parasite growth. Furthermore, inhibitors show little selectivity
for Plasmodium cells compared with their selectivity for human
cells and incompletely and/or reversibly suppress parasite
growth in vivo after drug treatment. For example, treatment of
P. berghei-infected mice with apicidin, a broad-spectrum
HDAC inhibitor, resulted in a significant reduction in the level
of parasitemia but no cures (8).
The compounds evaluated in the present study were devel-
oped for isoform-specific HDAC inhibition (6, 14). Here, we
report on novel potent HDAC inhibitors that suppress the
growth of P. falciparum with 50% inhibitory concentration
* Corresponding author. Mailing address: Department of Radiation
Medicine, Georgetown University Medical Center, Research Building,
Room E211, Box 571482, 3970 Reservoir Rd., NW, Washington, DC
20057-1482. Phone: (202) 687-8352. Fax: (202) 687-0400. E-mail:
?Published ahead of print on 17 February 2009.
(IC50s) that are up to 950 times lower than the concentration
required for inhibition of the growth of mammalian cells. In
vivo, the lead compound YC-II-88 cured mice infected with P.
berghei and showed no clinically apparent toxicity to the mice.
MATERIALS AND METHODS
Materials. Type O-positive human blood and heat-inactivated serum were
obtained from BiocheMed (Winchester, VA), Sybr green was obtained from
Invitrogen (Carlsbad, CA), an HDAC fluorescent activity assay kit was obtained
from BioMol (San Diego, CA), and all other reagents were from Sigma (St.
Louis, MO). ICR mice were purchased from Charles River Laboratories (Wil-
Culture and synchronization of P. falciparum. Geographically representative
strains of P. falciparum banked in the Departments of Biochemistry and Molec-
ular Biology, Georgetown University Medical Center, or obtained from the MR4
malaria parasite depository resource (ATCC, Manassas, VA) or Carole Long
(NIAID, NIH, Rockville, MD) were used in these assays. All parasite strains
were maintained as described previously (1, 25). In brief, parasites were main-
tained in complete medium (RPMI 1640 medium supplemented with 25 mM
HEPES, 29 mM sodium bicarbonate, 0.005% hypoxanthine, p-aminobenzoic
acid [2 mg/liter], gentamicin sulfate [50 mg/liter], 10% type O-positive human
serum with O-positive human red blood cells [RBCs] at a 2% hematocrit). The
cultures were maintained at 37°C in an atmosphere of 90% N2, 5% O2, and 5%
CO2. The culture medium was exchanged every 2 days.
The cultures were synchronized by using a modified Percoll-sorbitol method
(18). Briefly, infected RBCs with mature schizonts were separated on a 55%
Percoll solution, washed twice with serum-free medium, and mixed with 20
volumes of noninfected RBCs (NIRBCs) and maintained in culture. Thin-film
slides of the culture were made every 30 min to observe the first signs of schizont
rupture, the presence of free merozoites, or newly invaded rings. Two hours later
(the time for merozoite release and the invasion of new RBCs), the cells were
harvested by centrifugation, diluted with 5 volumes of prewarmed 5% sorbitol,
and incubated at 37°C for 5 min. The cultures were washed with serum-free
medium and maintained as described above. The cultures were synchronized
every 2 to 3 weeks.
Inhibition of Plasmodium HDAC activity. The compounds were provided by
Alan Kozikowski (Department of Medicinal Chemistry and Pharmacognosy,
University of Illinois at Chicago). The activities of the compounds against P.
falciparum HDAC (PfHDAC) were assessed with an HDAC fluorescent activity
assay kit, according to the manufacturer’s protocol (BioMol) (14). Trophozoite
or schizont crude whole-cell extracts were used in the assays for PfHDAC-
inhibitory activity. Trophozoites and schizonts were separated on a 55% Percoll
solution, washed several times, and diluted in a reaction solution (0.1 M KCl, 20
mM HEPES, pH 7.9, 20% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.5
mM phenylmethylsulfonyl fluoride). The cells were lysed by freezing-thawing and
were stored at ?80°C until use. To verify the anti-PfHDAC activities, various
amounts of crude parasite extracts were mixed with Fluor de Lys substrate and
1 ?M trichostatin A or 0.1% dimethyl sulfoxide (Me2SO), and the mixtures were
incubated for 15 min at 37°C in HDAC buffer. The reactions were stopped by the
addition of Fluor de Lys developer, and fluorescence measurements were made
at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. All
samples were assayed in triplicate and included a blank (an RBC extract with
0.1% Me2SO). After comparison of the fluorescence intensities for samples with
different amounts of parasite extracts, 1 ? 106infected RBCs were determined
to be optimal for this assay. To assess the PfHDAC-inhibitory activities of the
compounds, we repeated the experiments with the indicated concentrations of
selected compounds (K.2, I.2, W.2, YC-II-84, YC-II-88, YC-II-90, AG-THIA-01,
AG-b, and suberoylanilide hydroxamic acid). Fluorescent readings were normal-
ized and were used to calculate the percent anti-PfHDAC activities of the
compounds by comparing the readings to those for the controls (RBC extracts).
Dose-response curves of the anti-PfHDAC activities versus the concentrations
were used to determine the IC50s. These values were calculated by using the
nonlinear regression curve-fitting option within Prism (version 3.0) software
(GraphPad Software Inc., San Diego, CA).
Screening of drugs for antimalarial activities. A total of 22 compounds were
screened for their antimalarial growth-inhibitory activities with two strains of P.
falciparum, 3D7 (chloroquine sensitive) and FCB1 (chloroquine resistant), by a
standard growth inhibition assay (GIA) (24). In brief, parasite cultures at the ring
stage were incubated at 1% parasitemia with 500 nM of the indicated compounds
(see Fig. 2) in a 96-well plate at 37°C for 48 h in an atmosphere of 90% N2, 5%
CO2, and 5% O2. Complete medium containing 0.1% Me2SO was used as the
negative control; at this concentration, the Me2SO did not affect parasite growth
in vitro. Wells containing NIRBCs in complete medium were used to obtain
background readings. We included the results only for assays in which the levels
of parasitemia of the Me2SO controls increased at least threefold from the initial
level of parasitemia. The levels of parasitemia were determined by fluorescent-
activated cell sorting (FACS) by counting 100,000 Sybr green-stained cells with
a FACScan instrument. The percent parasite growth inhibition was determined
by using the following formula: [(N ? B) ? (S ? B)]/(N ? B), where N is the
level of parasitemia in the Me2SO control well, B is the readout determined from
the wells containing NIRBCs, and S is the level of parasitemia of the sample. The
assays were run in triplicate and were repeated at least twice.
We treated several geographically representative P. falciparum strains at the
ring stage with graded concentrations of each compound and determined the
percent parasite growth inhibition. A plot of the percent parasite growth inhi-
bition versus drug concentration was made, and IC50s were calculated by using a
nonlinear regression curve fit. The specificity indices (SIs) were calculated as the
IC50of the drug for the mammalian cell/IC50of the drug for P. falciparum. The
antiproliferative activities of these compounds in mammalian cells have been
reported previously (14).
Quality control for GIA. Experiments were repeated by an independent sci-
entist by using the same assay to provide a minimum of six data points for each
drug concentration. The average and standard errors are reported for each drug
concentration. For selected compounds, the IC50s were also validated by inde-
pendent laboratories at Georgetown University and NIAID, NIH.
Assessment of reversibility of growth inhibition by HDAC inhibitor. To de-
termine if parasite growth resumes after HDAC inhibitor withdrawal, we treated
a P. falciparum 3D7 culture with YC-II-88 at 2.5 nM (the minimum concentra-
tion required to achieve complete parasite suppression in a 48-h GIA) for various
intervals. The drug (YC-II-88) was removed, and the levels of parasitemia were
measured every 12 h for 4 days to determine the percent parasite growth inhi-
Assessment of stage-specific effects of HDAC inhibitor. The erythrocytic cycle
of P. falciparum involves three stages of development: the ring, trophozoite, and
schizont stages. Various degrees of cell maturation and cell division occur at each
stage. Since HDAC inhibitors alter growth primarily by affecting these processes,
we investigated the differential toxicities of the drugs to the various parasite
stages (the ring, trophozoite, and schizont stages). Parasite rings (which appear
from 1 to 16 h postinvasion), trophozoites (which appear from 16 to 32 h after
merozoite invasion), and schizonts (which appear 32 to 48 h postinvasion) were
exposed to 1.25 nM YC-II-88 for 14 h. The levels of parasitemia were determined
at the end of drug treatment and 16 and 32 h later. All assays included positive
and negative controls (continuous exposure from 0 to 48 h with 1.25 nM YC-
II-88 and 0.1% Me2SO, respectively). The levels of parasitemia were compared
to those for the controls to assess the percent growth inhibition and growth
recovery. The assays were performed twice in triplicate.
In vivo toxicity and efficacy studies with mice. The animal protocols used here
were approved by the Animal Care and Use Committee of Georgetown Univer-
sity Medical Center. To determine the dose for use in vivo, we first determined
FIG. 1. Inhibition of PfHDAC activity. Trophozoite- and schizont-
containing crude cell lysates were the sources of PfHDAC activity.
Parasite lysates corresponding to 106parasites were mixed with the
indicated concentrations of YC-II-88 or SAHA and used to measure
the anti-PfHDAC activity by using a fluorimetric HDAC activity assay
kit from BioMol, and the manufacturer’s protocol was used, with slight
modifications (14). TSA (1 ?M) and Me2SO were included as negative
and positive controls, respectively. Assays were performed twice in
triplicate. The standard errors of the means are presented as bars.
1728AGBOR-ENOH ET AL.ANTIMICROB. AGENTS CHEMOTHER.
the minimum toxic dose of YC-II-88, the lead compound. Groups of nonimmune
ICR mice (five mice per group; Charles River Laboratories) were injected with
0, 0.5, 5, 50, or 100 mg/kg of body weight/day of YC-II- 88 divided into two doses
for 5 days and 14 days. The mice were observed for signs of distress (ruffled hair,
abdominal distention, porphyria, movement, and feeding). The mice were killed
on day 5 or 14, and the internal organs (brain, lungs, heart, spleen, liver, kidney,
adrenal, and bone marrow) were harvested for pathological examinations (gross
and microscopic). The only pathological change was an increase in the erythroid
mass, which was noted in a subset of drug-treated mice. This was scored by the
pathologist as mild, moderate, or severe. The data established a maximum dose
of YC-II-88 of 50 mg/kg/day for use in the efficacy studies.
In vivo efficacy studies were performed with cryopreserved P. berghei NK56
parasites injected intraperitoneally into ICR mice. The levels of parasitemia were
monitored every 2 days until they reached 10% or more. The blood was diluted
with phosphate-buffered saline, and 106parasites were injected intraperitoneally
into five groups of ICR mice (five mice per group). In our hands, 100% of the
mice infected in this way developed parasitemia by day 4 or earlier and died 8 to
14 days postinoculation. Each group of mice was simultaneously injected (intra-
peritoneally) with a graded concentration (0, 0.05, 0.5, 5, or 50 mg/kg/day) of
YC-II-88 divided into two doses for 4 days.
The mice were monitored daily for clinical signs of distress. On day 4, blood
was collected from a tail snip for two thin blood smears. The smears were stained
with Giemsa, and the levels of parasitemia were determined manually by count-
ing 1,500 cells from 10 random fields. The numbers of parasites on each slide
were counted by two experienced microscopists, providing a minimum of four
readings per time point per mouse. A reading from an independent, third
microscopist was obtained when the first two microscopists reported levels of
parasitemia that differed by 25% or greater. Slides with no observed parasites
were confirmed by retesting by staining with Sybr green. Sybr green is more
sensitive than Giemsa strain and detects very early rings that could otherwise be
We monitored the mice for a total of 6 weeks or until they died. During this
time, parasitemia and hematocrit levels were measured every 4 days. The rates of
survival were measured from the time of the initiation of treatment. Differences
in hematocrit levels, survival rates, levels of parasitemia, and other parameters
were compared by a paired Student t test with the assumption of equal variance.
TABLE 1. Effects of HDAC inhibitors on HDAC activities in
P. falciparum and HeLa cellsa
SAHA 127 800.63
aHADC inhibitors were mixed at the concentrations indicated in the text with trophozoite or schizont crude cell lysates or HeLa cell nuclear extracts (HeLa is a
human cervical cancer cell line) to determine the inhibition of Plasmodium and human HDAC activities. All experiments were performed in triplicate. The IC50
indicates the concentration of the HDAC inhibitor that conferred 50% HDAC enzyme activity inhibition.
bSpecificity index (SI) ? IC50HeLa cell extract/IC50Pf3D7.
VOL. 53, 2009 SELECTIVE AND POTENT HDAC INHIBITORS OF PLASMODIUM1729
Effects of HDAC inhibitors on PfHDAC activities. To deter-
mine the inhibitory activities of the HDAC inhibitors, P. fal-
ciparum crude extracts were assayed and were found to be
inhibited by known hydroxamic acid HDAC inhibitors (TSA
and SAHA) in a concentration-dependent manner. The
HDAC inhibitors showed no activity against NIRBC extracts
(data not shown). Among the compounds tested, YC-II-88
exhibited the highest level of inhibitory activity and had an IC50
of 0.23 nM, approximately 550-fold lower than the IC50of
SAHA (127 nM) (Fig. 1 and Table 1). YC-II-84, YC-II-90,
AG-THIA-01, AG-b, and K.2 also demonstrated PfHDAC-
inhibitory activities at lower levels. Furthermore, the mercap-
toacetamide W.2 (6) demonstrated low levels of PfHDAC-
inhibitory activity (IC50, 4,555 nM) (Table 1). Although
YC-II-88 also inhibited human HDACs in HeLa cells at the
highest level, its IC50for human HDACs (3.9 nM) was still 17
times greater than that for the PfHDAC (0.23 nM) (Table 1).
HDAC inhibitors selectively inhibit P. falciparum growth
and not human cell lines. Of the 22 different compounds
tested, 6 (YC-II-84, YC-II-88, YC-II-90, AG-THIA-01, AG-b,
and K.2) completely inhibited parasite growth at 500 nM (Fig.
2). These results were similar for the chloroquine-sensitive
strain (P. falciparum 3D7) (Fig. 2) and the multidrug-resistant
strain (P. falciparum FCB1) (data not shown). These six com-
pounds and SAHA, W.2, and I.2 were selected for determina-
tion of their IC50s. Consistent with its HDAC-inhibitory activ-
ity, YC-II-88 showed the greatest inhibition of parasite growth
and had an IC50of 1.25 nM for P. falciparum 3D7 (Table 2).
Other compounds (YC-II-90, AG-THIA-01, AG-b, YC-II-84,
and K.2) also had IC50s in the low nanomolar range (Table 2).
SAHA and YC-II-88 had IC50s for P. falciparum 3D7of 1.25 nM
and 900 nM, respectively, which indicates that YC-II-88 had an
estimated 600- to 2,350-fold greater potency than SAHA for the
various parasite strains tested. The antiplasmodial growth inhibi-
tion correlated with the inhibition of PfHDAC activity with a
correlation coefficient of 0.9.
The level of inhibition of the growth of the other P. falcip-
arum strains tested was similar to that observed for P. falcip-
arum 3D7 (Table 2). YC-II-88 consistently had greater growth-
inhibitory activity than any of the other compounds tested, with
IC50s ranging from 0.4 to 1.25 nM. AG-THIA-01 and YC-II-90
were also active, but their IC50s were about 1 log unit higher
than the IC50of YC-II-88. K.2, YC-II-84, and AG-b inhibited
all strains tested, and their IC50s were about 2 log units higher
than the IC50of YC-II-88. Similar potencies against chloro-
quine-sensitive strains (strains 3D7 and HB3) and chloro-
TABLE 3. YC-II-88 selectively inhibits P. falciparum growtha
Strain or cell lineYC-II-88 IC50(nM)
Human cell linesb
aThe IC50s for the human cell lines were compared to the IC50s for the P.
falciparum strains to calculate the SIs. The SIs ranged from 160 to 950.
bThe toxicity data for human cell lines have been published previously (14).
FIG. 2. Screening of HDAC inhibitors for their antimalarial activ-
ities. Antimalarial activities were screened by a GIA (24), with modi-
fications, by using P. falciparum strain 3D7. The levels of parasitemia
were determined by FACS and were used to calculate the percent
parasite growth inhibition. Assays were performed twice in triplicate,
and the averages and the standard errors of the means are presented.
Similar results were obtained with strain FCB1 (data not shown).
TABLE 2. HDAC inhibitors inhibit the growth of P. falciparum strainsa
IC50(nM) for the following P. falciparum strain (source):
7G8 (BRA)Dd2 (INDO) FCR3 (GAM)3D7 (UNK) HB3 (HON)V1/S (VIE) FCB1 (BRA)
aGeographically representative strains of P. falciparum were incubated with the indicated HDAC inhibitors, and the levels of parasitemia were determined by FACS
to determine IC50s. Abbreviations: BRA, Brazil; INDO, Indochina; GAM, The Gambia; UNK, unknown; HON, Honduras; VIE, Vietnam.
bThe structure of K.2 has been published elsewhere (14).
cND, not determined.
1730AGBOR-ENOH ET AL.ANTIMICROB. AGENTS CHEMOTHER.
quine-resistant strains (strains 7G8, Dd2, V1/S, FCB1, and
FCR3) of P. falciparum were also observed.
The HDAC inhibitors also inhibited the growth of several
human cell lines, but at concentrations much higher than those
required to inhibit P. falciparum strains. The preferential inhi-
bition of P. falciparum was determined by calculating the SI for
each compound. The lead compound, YC-II-88, inhibited the
P. falciparum strains with SIs of 160 to 950 (Table 3).
We also evaluated the reversibility of the antiplasmodial
activities of these compounds. Exposure to YC-II-88 for 48 h
resulted in the completely irreversible suppression of the par-
asites after YC-II-88 was withdrawn (data not shown). Shorter
treatments also resulted in irreversible parasite suppression if
the trophozoite window was included. The parasite levels did
not recover by 3 days after drug withdrawal (Fig. 3a). A similar
result was observed with compound K.2 (Fig. 3a).
The erythrocytic cycle of the malaria parasite requires ap-
proximately 48 h for development and maturation to transform
rings through the trophozoite and schizont stages to the infec-
tive merozoites stage, which is capable of reinvading RBCs to
restart the cycle. Since various developmental processes (cell
division, stage-specific gene expression, DNA synthesis) occur
at different stages of the cycle, we investigated the parasite
stages (rings [0 to 16 h], trophozoites [16 to 32 h], and schizonts
[32 to 48 h]) for their sensitivities to HDAC inhibitor-induced
parasite death. We treated the various parasite stages with 1.25
nM YC-II-88 for 14 h to determine parasite growth inhibition
(Fig. 3b). Analyses of the data indicate that trophozoites (16 to
32 h) were the most sensitive to HDAC inhibitor-induced
parasite death (25% and 35% growth inhibition at the end of
treatment and 16 after treatment, respectively) compared to
the sensitivities of the schizonts (20% and 25% growth inhibi-
tion at the end of treatment and 16 after treatment, respec-
tively) and rings (?5% and 15% at 16 and 32 h, respectively).
Toxicity of YC-II-88 in vivo. Both short (5-day) and long
(14-day) exposures to YC-II-88 produced no clinical signs of
distress or mortality in mice after the injection of 100 mg/kg/
day (Table 4). However, histological examinations of the har-
vested organs (lung, brain, adrenal, spleen, liver, bone marrow,
and kidney) revealed an increased erythroid mass in the
FIG. 3. HDAC inhibitor-induced parasite inhibition is irreversible and is most significant in mature parasite stages. (a) A synchronized P.
falciparum 3D7 culture was treated with the minimum concentrations of YC-II-88 (2.5 nM) and K.2 (1,000 nM) found to completely inhibit P.
falciparum 3D7 continuously over 4 days (solid lines) or from 10 to 34 h postinvasion (dashed line). Parasite growth was assessed every 12 h for
4 days by FACS, and the degree of growth inhibition was compared to that for the controls (parasites treated with Me2SO). The data are
representative of those from three separate experiments. (b) Rings (1 to 16 h postinvasion), trophozoites (16 to 30 h), and schizonts (32 to 48 h)
were treated with 1.25 nM YC-II-88 (the IC50for P. falciparum 3D7) or Me2SO for 14 h. The assay was performed twice in triplicate. The standard
errors of the means are presented as bars.
VOL. 53, 2009SELECTIVE AND POTENT HDAC INHIBITORS OF PLASMODIUM 1731
spleen, liver, and bone marrow, which was scored as mild,
moderate, or severe. Twenty percent (one of five) of the mice
injected with 100 mg/kg/day showed a mild increase in ery-
throid mass. However, 5-day exposures to doses of 0.5, 5, and
50 mg/kg/day of YC-II-88 resulted in no increase in erythroid
mass. Longer exposures (14 days) to YC-II-88 increased the
erythroid mass only with the two highest doses. It should be
noted that the erythroid cells from the drug-exposed and the
control mice showed no morphological differences.
HDAC inhibitors completely inhibit the growth of P. berghei
in mice and improve survival. Five groups of mice (five mice
per group) were injected with YC-II-88 intraperitoneally and
were simultaneously infected with P. berghei. The levels of
parasitemia were determined microscopically by the use of thin
blood smears stained with Giemsa or Sybr green on day 4.
None of the mice treated with 50 mg/kg/day of YC-II-88
showed detectable parasites or developed clinical signs of dis-
tress. Mice injected with split doses, 5 mg/kg/day and 0.5 mg/
kg/day, had detectable parasites on day 4, but the levels of
parasitemia were lower than those for the control mice (P ?
0.001 and P ? 0.02, respectively). Mice treated with 0.05 mg/
kg/day had levels of parasitemia comparable to those for the
control mice (P ? 0.08). The IC50of YC-II-88 for the control
P. berghei isolate was about 0.5 mg/kg/day (Fig. 4a).
Drug treatments were discontinued on day 4, and the levels
of parasitemia (Fig. 4b) and hematocrit (Fig. 4c) were assessed
for 6 weeks or until the mice died. On day 4 (the last day of
treatment), the mice in the control group and the group
treated with 0.05 mg/kg/day had comparable levels of para-
sitemia. By day 8, the levels of parasitemia in the group treated
with 0.5 mg/kg/day were comparable to those in the control
group. The levels of parasitemia in the group treated with 5
mg/kg/day reached the levels in the control group on day 20 of
the experiment. None of the mice in the group treated with 50
mg/kg/day developed parasitemia during the 6-week duration
of the experiment (Fig. 4b).
The hematocrit levels declined in the mice in all treatment
groups except the group receiving 50 mg/kg/day (Fig. 4c). The
hematocrit levels of the control mice dropped the fastest and
reached an average of about 18% on day 10 after infection.
The rate of decrease in the hematocrit level in the group
treated with 50 mg/kg/day were comparable to those observed
in the group treated with 0.05 mg/kg/day and the control group.
Mice treated with 0.5 and 5 mg/kg/day of YC-II-88 had slower
rates of decline in hematocrit levels. The hematocrits of the
mice receiving 50 mg/kg/day remained stable for the 6 weeks of
the experiment (Fig. 4c).
Even though the mice in the control group and the group
treated with 0.5 mg/kg/day had comparable levels of para-
sitemia starting on day 8 of the experiment, the control mice
lived an average of 11.7 days, while the mice in the group
treated with 0.5 mg/kg/day lived an average of 23.6 days (P ?
0.001) (Fig. 4d). The control mice also died as their levels of
parasitemia reached 50%, whereas the mice in the groups
treated with 0.5 and 5 mg/kg/day lived an average of about 10
days with levels of parasitemia of 50% or more before they
died. The mice in the group treated with 0.05 mg/kg/day group
lived an average of 3 days longer than the mice in the Me2SO-
treated control group (15.3 days), but this difference was not
significant (Fig. 4d).
HDACs are potential targets for the treatment of various
human diseases (10, 12, 21) and for antimalarial drug devel-
opment (2, 3, 8, 9, 20). These are particularly attractive targets
for antimalarial drug therapy because, unlike mammalian cells,
HDACs are more limited and potentially less redundant in
Plasmodium species. The P. falciparum genome reveals five
putative HDACs, two of which have been characterized (13,
23), and sequence analysis of PfHDAC1 (accession no.
PFI1260c) reveals 65% sequence homology with human class 1
HDAC but up to 96% sequence homology with HDAC1 of
other Plasmodium species.
Several HDAC inhibitors with antiprotozoal activity in vitro
and in vivo have been reported (2, 8, 20). For example, apici-
din, a fungal metabolite, inhibits plasmodial HDAC activity
and parasite growth with mean inhibitory concentrations in the
low-nanomolar range. However, apicidin failed to completely
suppress parasitemia in P. berghei-infected mice (8). Suberic
acid bisdimethylamide completely suppressed parasitemia in P.
berghei-infected mice, but the parasitemia recurred 8 to 12 days
posttreatment (3). In this investigation, we screened HDAC
inhibitors to find compounds that had high potencies and spec-
ificities for P. falciparum in vitro and in vivo and that could
irreversibly suppress parasites in vivo.
We evaluated these HDAC inhibitors showing high poten-
cies against a variety of geographically representative drug-
sensitive and multidrug-resistant strains of P. falciparum (Ta-
ble 2). In vitro, these drugs had mean inhibitory concentrations
as low as 0.4 nM for P. falciparum, a value that is several orders
of magnitude lower than the values for previously described
HDAC inhibitors. The growth-inhibitory activities were di-
rectly related to the enzyme-inhibitory activities. In our exper-
iments, the lead compound, YC-II-88, completely and irrevers-
ibly suppressed parasitemia in vitro (Fig. 3) and in vivo (Fig.
TABLE 4. Results of in vivo toxicity studiesa
Exposure time and
No. of mice that:
No. of mice with the
following increase in
erythroid mass/total no.
Had clinical signs
aGroups of ICR mice (five mice per group) were injected with YC-II-88 at the
indicated daily doses for 5 or 14 days. Control mice were injected with saline
containing 0.1% Me2SO. The mice were observed for death and clinical signs of
distress. The increase in erythroid mass (in the bone marrow, liver, and spleen)
was scored as mild, moderate, or severe. In case two organs from the same mouse
showed different scores, the higher score was assigned.
1732AGBOR-ENOH ET AL.ANTIMICROB. AGENTS CHEMOTHER.
4a). It is of note that increased concentrations of this drug
could not suppress HDAC activity in vitro below 25%. This
may be attributable to selective HDAC inhibition but is not
fully understood. The strain of P. berghei used in the in vivo
efficacy studies (strain NK56) is chloroquine sensitive; how-
ever, effects similar to those obtained in vitro are anticipated
with chloroquine-resistant strains (Fig. 2 and Table 2). Re-
cently, Dow and colleagues (9) reported the results of a chal-
lenge experiment with P. berghei in mice in which they failed to
effect a cure. The oral administration of YC-II-88 resulted in
no cures in P. berghei-infected mice at doses up to 640 mg/kg/
day for 3 days. They speculate that the failure of monotherapy
in mice may be due to the metabolic stability of YC-II-88,
which exhibits a short half-life after the administration of a
single oral dose of 50 mg/kg. However, we observed cures in P.
berghei-infected mice treated with YC-II-88 intraperitoneally
at doses of 50 mg/kg/day divided into two daily doses for 4 days.
We reason that the different routes of delivery may contribute
to the outcomes, since the solubility and stability of hydroxam-
ates are adversely affected by the low pH observed in the
stomach (16). Another possibility is related to the model sys-
tem used, since ours was a prophylactic model, whereas Dow et
al. used a curative mode (9).
HDACs regulate various cellular processes, including DNA
synthesis, cell division and differentiation, apoptosis, and oth-
ers. Inhibitors of HDACs arrest cell division and stimulate
apoptosis-related processes (22). For this reason, HDAC in-
hibitors show selective toxicity against growing malignant cells
and not against healthy cells. As predicted, among the three
stages of the erythrocytic cycle, trophozoites (in which DNA
and most macromolecular synthesis occur) and schizonts (in
which schizogony occurs) were more susceptible to HDAC
inhibitor-induced cell death than rings (Fig. 3).
Since HDACs show a high degree of sequence homology
among various species, HDAC inhibitors (like apicidin and
SAHA) show little selectivity among species (8). The lack of
selectivity raises the possibility of side effects, should these
drugs be used to treat human parasitic diseases. We observed
a relatively high degree of selective toxicity against P. falcipa-
rum compared to the toxicity observed against mammalian
cells (Table 3). The selectivity was greater for six of the com-
pounds (K.2, YC-II-84, YC-II-88, YC-II-90, AG-THIA-01,
and AG-b) than for SAHA. The lead compound, YC-II-88,
was up to 950 times more toxic to P. falciparum than to human
cells in vitro (Tables 2 and 3). Parasitemia was completely
suppressed in P. berghei-infected mice (Fig. 4a) treated with
YC-II-88 at 50 mg/kg/day, and there were no observable clin-
ical side effects (Table 4). We did not investigate higher infec-
tive doses of P. berghei or mice with already patent parasitemia.
The results of the toxicity studies indicated that mice in-
jected with 50 mg/kg/day of YC-II-88 for 4 days would not
show no any histological changes (Table 4), but longer expo-
sures or a higher dose (100 mg/kg/day) resulted in increases in
FIG. 4. HDAC inhibitors irreversibly inhibit Plasmodium in vivo
and increase the rate of survival. Five groups of mice (five mice per
group) were infected with 106P. berghei cells intraperitoneally and
were treated simultaneously with the indicated doses of YC-II-88
divided into two daily doses for 4 days. The percentage of parasite
growth inhibition was plotted against the drug concentration (a).
The mice were monitored for a total of 6 weeks or until they died; and
the levels of parasitemia (b), hematocrit levels (c), and rates of survival
(d) are shown. The standard errors of the means are presented as bars
in panels a, b, and c. CTRL, control.
VOL. 53, 2009 SELECTIVE AND POTENT HDAC INHIBITORS OF PLASMODIUM1733
erythroid mass (Table 4). The mechanism underlying the Download full-text
HDAC inhibitor-induced erythroid mass increase is not under-
stood; however, it has been reported that HDAC inhibitors
promote the differentiation of erythroid cells (17). Hematocrit
levels were comparable in control and drug-exposed mice in
the toxicity studies (data not shown). No histologic changes
were observed in the other organs examined (heart, kidney,
lungs, brains, and adrenals).
We thank Alan Kozikowski for providing the compounds; Carole
Long for providing P. falciparum 3D7 and performing growth inhibi-
tion assays; Paul Roepe (Georgetown University, Washington, DC) for
providing P. falciparum strains FCB1, Dd2, and HB3; Simon Metenou
(NIAID, NIH, Bethesda, MD) for reading the slides; the MR4 malaria
parasite depository resource for providing several P. falciparum and P.
berghei strains; and Manny Subramanian (Best Medical International,
Springfield, VA) and Geoffrey Dow (WRAIR, Silver Spring, MD) for
This work was supported by NIH/NCI grant P01CA074175 and a
fellowship from Krishnan Suthanthiran of Best Medical International,
1. Agbor-Enoh, S. T., R. N. Achur, M. Valiyaveettil, R. Leke, D. W. Taylor, and
D. C. Gowda. 2003. Chondroitin sulfate proteoglycan expression and binding
of Plasmodium falciparum-infected erythrocytes in the human placenta dur-
ing pregnancy. Infect. Immun. 71:2455–2461.
2. Andrews, K. T., T. N. Tran, A. J. Lucke, P. Kahnberg, G. T. Le, G. M. Boyle,
D. L. Gardiner, T. S. Skinner-Adams, and D. P. Fairlie. 2008. Potent anti-
malarial activity of histone deacetylase inhibitor analogues. Antimicrob.
Agents Chemother. 52:1454–1461.
3. Andrews, K. T., A. Walduck, M. J. Kelso, D. P. Fairlie, A. Saul, and P. G.
Parsons. 2000. Anti-malarial effect of histone deacetylation inhibitors and
mammalian tumor cytodifferentiating agents. Int. J. Parasitol. 30:761–768.
4. Bosman, A., and K. N. Mendis. 2007. A major transition in malaria treat-
ment: the adoption and deployment of artemisinin-based combination ther-
apies. Am. J. Trop. Med. Hyg. 77:193–197.
5. Butler, K. V., and A. P. Kozikowski. 2008. Chemical origins of isoform
selectivity in histone deacetylase inhibitors. Curr. Pharm. Des. 14:505–528.
6. Chen, B., P. A. Petukhov, M. Jung, E. Eliseeva, A. Velena, A. Dritschilo, and
A. P. Kozikowski. 2005. Chemistry and biology of mercaptoacetamindes as
novel histone deacetylase inhibitors. Bioorg. Med. Chem. Lett. 15:1389–
7. Cui, L., J. Miao, T. Furuya, Q. Fan, L. Xinyi, P. K. Rathod, X. Su, and L. Cui.
2008. Histone acetyltransferase inhibitor anacardic acid causes changes in
global gene expression during in vitro Plasmodium falciparum development.
Eukaryot. Cell 7:1200–1210.
8. Darkin-Rattray, S. J., A. M. Gurnett, R. W. Myers, P. M. Dulski, T. M.
Crumley, J. J. Allocco, C. Cannova, P. T. Meinke, S. L. Colletti, M. A.
Bednarek, S. B. Singh, M. A. Goetz, A. W. Dombrowski, J. D. Polishook, and
D. M. Schmatz. 1996. Apicidin: a novel antiprotozoal agent that inhibits
parasite histone deacetylase. Proc. Natl. Acad. Sci. USA 12:13143–13147.
9. Dow, D. S., Y. Chen, K. T. Andrews, D. Caridha1, L. Gerena1, M. Gettaya-
camin, J. Johnson, Q. Li, V. Melendez, N. Obaldia, T. N. Tran, and A. P.
Kozikowski. 2008. Antimalarial activity of phenylthiazolyl-bearing hydrox-
amate-based histone deacetylase inhibitors. Antimicrob. Agents Chemother.
10. Elaut, G., V. Rogiers, and T. Vanhaecke. 2007. The pharmaceutical potential
of histone deacetylase inhibitors. Curr. Pharm. Des. 13:2584–2620.
11. Fan, Q., L. An, and L. Cui. 2004. PfADA2, a Plasmodium falciparum homo-
logue of the transcriptional coactivator ADA2 and its in vivo association with
the histone acetyltransferase PfGCN5. Gene 336:251–261.
12. Griffiths, E. A., and S. D. Gore. 2008. DNA methyltransferase and histone
deacetylase inhibitors in the treatment of myelodysplastic syndromes. Semin.
13. Joshi, M. B., D. T. Lin, P. H. Chiang, N. D. Goldman, H. Fujioka, M. Aikawa,
and C. Syin. 1999. Molecular cloning and nuclear localization of a histone
deacetylase homologue in Plasmodium falciparum. Mol. Biochem. Parasitol.
14. Jung, M., A. Velena, B. Chen, P. A. Petukhov, A. P. Kozikowski, and A.
Dritschilo. 2005. Novel HDAC inhibitors with radiosensitizing properties.
Radiat. Res. 163:488–493.
15. Khan, N., M. Jeffers, S. Kumar, C. Hackett, F. Boldog, N. Khramtsov, X.
Qian, E. Mills, S. C. Berghs, N. Carey, P. Finn, L. S. Collins, A. Tumber,
J. W. Ritchie, P. B. Jensen, H. S. Lichenstein, and M. Sehested. 2000.
Determination of the class and isoform selectivity of small-molecule histone
deacetylase inhibitors. Biochem. J. 409:581–589.
16. Konsoula, R., and M. Jung 2008. In vitro plasma stability, permeability
and solubility of mercaptoacetamide histone deacetylase inhibitors. Int.
J. Pharm. 361:19–25.
17. Kosugi, H., M. Towatari, S. Hatano, K. Kitamura, H. Kiyoi, T. Kinoshita, M.
Tanimoto, T. Murate, K. Kawashima, H. Saito, and T. Naoe. 1999. Histone
deacetylase inhibitors are the potent inducer/enhancer of differentiation in
acute myeloid leukemia: a new approach to anti-leukemia therapy. Leuke-
18. Kutner, S., W. V. Breuer, H. Ginsburg, S. Aley, and Z. I. Cabantchik. 1985.
Characterization of permeation pathways in the plasma membrane of human
erythrocytes infected with early stages of Plasmodium falciparum: association
with parasite development. J. Cell. Physiol. 125:521–527.
19. Laufer, M. K., A. A. Djimde ´, and C. V. Plowe. 2007. Monitoring and deter-
ring drug-resistant malaria in the era of combination therapy. Am. J. Trop.
Med. Hyg. 77(6 Suppl.):160–169.
20. Mai, A., I. Cerbara, S. Valente, S. Massa, L. A. Walker, and B. L. Tekwani.
2004. Antimalarial and antileishmanial activities of aroyl-pyrrolyl- hydroxy-
amides, a new class of histone deacetylase inhibitors. Antimicrob. Agents
21. Marks, P. A., and M. Dokmanovic. 2005. Histone deacetylase inhibitors:
discovery and development as anticancer agents. Expert Opin. Investig.
22. Mehnert, J. M., and W. K. Kelly. 2007. Histone deacetylase inhibitors:
biology and mechanism of action. Cancer J. 13:23–29.
23. Merrick, C. J., and M. T. Duraisingh. 2007. Plasmodium falciparum Sir2: an
unusual sirtuin with dual histone deacetylase and ADP-ribosyltransferase
activity. Eukaryot. Cell 6:2081–2091.
24. Nikodem, D., and E. Davidson. 2000. Identification of a novel antigenic
domain of Plasmodium falciparum merozoite surface protein-1 that specifi-
cally binds to human erythrocytes and inhibits parasite invasion, in vitro. Mol.
Biochem. Parasitol. 30:79–91.
25. Trager, W. 1971. A new method for intraerythrocytic cultivation of malaria
parasites (P. coatneyi and P. falciparum). J. Protozool. 18:239–242.
1734AGBOR-ENOH ET AL.ANTIMICROB. AGENTS CHEMOTHER.