Caged garcinia xanthones, a novel chemical scaffold with potent antimalarial activity

Article abstract of DOI:10.1128/AAC.01220-16

Caged Garcinia xanthones (CGXs) constitute a family of natural products that are produced by tropical/subtropical trees of the genus Garcinia. CGXs have a unique chemical architecture, defined by the presence of a caged scaffold at the C ring of a xanthone moiety, and exhibit a broad range of biological activities. Here we show that synthetic CGXs exhibit antimalarial activity against Plasmodium falciparum, the causative parasite of human malaria, at the intraerythrocytic stages. Their activity can be substantially improved by attaching a triphenylphosphonium group at the A ring of the caged xanthone. Specifically, CR135 and CR142 were found to be highly effective antimalarial inhibitors, with 50% effective concentrations as low as 10 nM. CGXs affect malaria parasites at multiple intraerythrocytic stages, with mature stages (trophozoites and schizonts) being more vulnerable than immature rings. Within hours of CGX treatment, malaria parasites display distinct morphological changes, significant reduction of parasitemia (the percentage of infected red blood cells), and aberrant mitochondrial fragmentation. CGXs do not, however, target the mitochondrial electron transport chain, the target of the drug atovaquone and several preclinical candidates. CGXs are cytotoxic to human HEK293 cells at the low micromolar level, which results in a therapeutic window of around 150-fold for the lead compounds. In summary, we show that CGXs are potent antimalarial compounds with structures distinct from those of previously reported antimalarial inhibitors. Our results highlight the potential to further develop Garcinia natural product derivatives as novel antimalarial agents.

Full text of DOI:10.1128/AAC.01220-16

AAC Accepted Manuscript Posted Online 31 October 2016
Antimicrob. Agents Chemother. doi:10.1128/AAC.01220-16
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Caged Garcinia xanthones: a novel chemical scaffold with potent antimalarial activity
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Hangjun Ke,a,# Joanne M. Morrisey,a Shiwei Qu,b Oraphin Chantarasriwong,c Michael W.
Mather, a Emmanuel A. Theodorakis,b Akhil B. Vaidyaa
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Center for Molecular Parasitology, Department of Microbiology and Immunology, Drexel
University College of Medicine, Philadelphia, Pennsylvania, USAa;
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla,
California, USAb;
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Department of Chemistry, Faculty of Science, King Mongkut’s University of Technology
Thonburi, Bangmod, Thungkru, Bangkok, Thailandc
Running Head: Caged Garcinia Xanthones as novel antimalarial agents
# Address correspondence to Hangjun Ke, hangjun.ke@drexelmed.edu

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Abstract
Caged Garcinia xanthones (CGXs) constitute a family of natural products that are produced by
tropical/sub-tropical trees of the genus Garcinia. CGXs have a unique chemical architecture,
defined by the presence of a caged scaffold at the C ring of a xanthone moiety and exhibit a
broad range of biological activities. Here we show that synthetic CGXs exhibit antimalarial
activity against Plasmodium falciparum, the causative parasite of human malaria, at the intra-
erythrocytic stages. The activity can be substantially improved by attaching a
triphenylphosphonium group at the A ring of the caged xanthone. Specifically, CR135 and
CR142 were found to be highly effective antimalarial inhibitors with EC50s (effective
concentration that inhibits growth by 50%) as low as ~10 nM. CGXs affect malaria parasites at
multiple intra-erythrocytic stages, with mature stages (trophozoites and schizonts) being more
vulnerable than immature rings. Within hours of CGX treatment, malaria parasites display
distinct morphological changes, significant reduction of parasitemia (the percentage of infected
red blood cells) and aberrant mitochondrial fragmentation. CGXs, however, do not target the
mitochondrial electron transport chain (mtETC), the target of the drug atovaquone and several
preclinical candidates. The cytotoxicity of CGXs in human HEK293 cells is at the low µM level,
which results in a therapeutic window around 150 fold for the lead compounds. In summary, we
show that CGXs are potent antimalarial compounds with structures distinct from previously
reported antimalarial inhibitors. Our results highlight the potential to further develop Garcinia
natural product derivatives as novel antimalarial agents.

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Introduction
Malaria remains a leading infectious disease in the tropical and subtropical regions of the world.
In the past 15 years, the morbidity and mortality of malaria has decreased dramatically due to
the wide use of artemisinin-combined chemotherapy (ACT), indoor residual spraying, the
distribution of insecticide-treated bed nets, and other malaria prevention and research efforts (1-
3). However, malaria parasites have evolved mechanisms to adapt to various immunological
and chemical pressures. They display genomic plasticity, readily accumulating mutations and
rearrangements to overcome antimalarial drugs (4). Clinical isolates that are resistant to the
majority of antimalarial drugs available have spread widely in malaria endemic areas (5,6). The
facile development and spread of parasite drug resistance clearly threatens the achievements
made so far, as well as impeding the goal of eradicating malaria in the near future. The recent
appearance and spread of artemisinin tolerance underscores the need for continued urgent
efforts to develop new antimalarial reagents (7-9).
Plants of the genus Garcinia produce an intriguing family of caged xanthone-derived natural
products that have a documented value in traditional Eastern medicine (10). Collectively
referred to as caged Garcinia xanthones (CGXs), these compounds are structurally defined by
an unusual motif in which the C-ring of an allylated xanthone has been converted into a tricyclic
cage (Figure 1). This motif is further decorated via A-ring substitutions and peripheral oxidations
to produce a variety of natural products with a broad range of bioactivities (11,12). Gambogic
acid (GBA), the archetype of this family, potently inhibits cancer cell proliferation in solid tumors
(13-17) and hematological malignancies (18), and has entered clinical trials in China for patients
with non-small cell lung, colon and renal cancers (19). In addition, the ability of several CGXs to
exhibit potent cytotoxicity at low µM concentrations has been well documented (20-23). Efforts
to unveil the minimum structural motif of CGXs that is accountable for the observed anti-cancer
activity led to the identification of cluvenone (CLV) (24,25). This compound was found to have

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similar potency to GBA in inhibiting cancer cell growth against the NCI60 cell panel and a
promising window of selectivity against non-tumor cells (26). Although the detailed mechanism
of action of GBA, CLV and related compounds has not yet been delineated, several studies
indicate that they localize to mitochondria and exhibit their bioactivity by affecting mitochondrial
structure and function (27,28). Along these lines, the hydroxylated cluvenones MAD28 and
MAD44 were recently found to bind to the mitoNEET family of iron-sulfur containing proteins
that are located at the outer mitochondrial membrane (29).
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The mitochondrion of malaria parasites is an essential organelle and has been validated as an
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antimalarial drug target (30,31). Compounds that disrupt essential mitochondrial functions within
the parasite are either in clinical use or in clinical trials as potential antimalarial agents (32) . For
instance, atovaquone (a component of Malarone®) is a clinically approved drug that selectively
inhibits the parasite mitochondrial electron transport chain (mtETC) at the cytochrome bc1
complex, leading to collapse of the mitochondrial membrane potential (33). Moreover, the
dihydroorotate dehydrogenase (DHODH) inhibitor DSM265 is currently undergoing Phase II
clinical trials (34). Inhibition of DHODH blocks pyrimidine biosynthesis, which is an essential
pathway in malaria parasites (35). Since malaria parasites require mitochondrial functions for
survival, we speculated that GBA and derivative compounds might exhibit antimalarial activity.
In this study, we examine the antimalarial activities of GBA and synthetic CGXs against the
human malaria parasite Plasmodium falciparum.
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Materials and Methods
1. Parasite lines and parasite culture. P. falciparum strains Dd2 (resistant to chloroquine,
mefloquine and pyrimethamine) and 3D7 (drug sensitive) are the wild type lines used in this
study. P. falciparum 3D7attB-yDHODH is a transgenic line bearing a copy of the yeast
dihydroorotate dehydrogenase gene (yDHODH) in the genome, rendering the parasites

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resistant to inhibitors targeting the mitochondrial electron transport chain (mtETC) (35,36).
Parasites were cultured in human O+ erythrocytes (Interstate Blood Bank) in complete RPMI
1640 medium supplemented with 0.5% AlbuMAX (Invitrogen), 15 mM HEPES, 10 mg/L
hypoxanthine, 25 mM NaHCO3 and 50 µg/L gentamicin. Cultures were incubated at 37 °C in an
incubator filled with a low oxygen gas mixture (89% N2, 5% CO2, and 6% O2).
2. Growth inhibition assay via 3H-hypoxanthine incorporation. The antimalarial activity was
determined by measuring 3H-hypoxanthine incorporation in parasites exposed to compounds in
96-well plates. Compounds tested were initially dissolved in DMSO at 10 mM concentrations.
Parasite cultures at 1% parasitemia and 1.5% hematocrit were exposed to serial dilutions of
each compound or no compound media for 24 h. After 24 h, each well was pulsed with 0.5 µCi
of 3H-hypoxanthine and incubated for another 24 h. Then parasites were frozen at -80 °C
overnight. Parasites were lysed by thawing and nucleic acids were collected onto filters using a
cell harvester (PerkinElmer Life Sciences). Filters were dried by air, and 30 µl MicroScint O
(PerkinElmer Life Sciences) was added to each well. Incorporation of 3H-hypoxanthine was
quantified by a TopCount scintillation counter (PerkinElmer Life Sciences).
3. Flow cytometry assessment of parasitemia. Dd2 parasites were tightly synchronized by
multiple rounds of alanine treatment (0.5 M alanine/10 mM HEPES, pH 7.6) of ring stage
cultures (37). Upon reaching the mid-trophozoite stage, synchronized parasites were inoculated
into a 24-well plate with each well harboring a 2 ml culture with 2.5% hematocrit. Parasites were
exposed to DMSO (0.5 µl/ml) or compounds at 10x EC50 concentration. Specifically, the
concentrations were 0.1 µM, 0.15 µM, 2.6 µM and 1.1 µM for CR135, CR142, MAD28, SQ129,
respectively. At specified time points post treatment (2 h, 4 h, 8 h, 24 h and 48 h), a small
aliquot was taken from each well and used to prepare a Giemsa-stained thin smear for
morphological examination. At the same time points, another aliquot of each parasite culture (5-

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10 µl of pellet) was fixed with 4% formaldehyde at 37 °C for 1 h or 4 °C overnight on a rotator.
The samples were then washed three times with PBS and stained with SYBR Green
(ThermoFisher Scientific) for 1h at room temperature on a rotator. They were then washed three
times and ~1 µl of the pellet was resuspended in 1 ml of sterile H2O. The samples were
analyzed by a BD Accuri C6 flow cytometer. Uninfected RBCs, unstained and stained with
SYBR Green, and unstained infected trophozoites were used as controls. For each sample,
1,000,000 events were collected and cell debris was removed by proper gating strategy. The
percentage of SYBR Green positive events was taken to be the parasitemia.
4. Microscopy. Thin blood smears were fixed with 100% methanol, air dried and stained with
Giemsa dye solution for 10 min. Morphologies of infected red blood cells were examined under
a Leica microscope and pictures were taken with a 16-megapixel camera. When parasitemia
was determined microscopically, at least 1000 red blood cells were counted. For mitochondrial
morphologies, parasites were stained with MitoTracker Red (Invitrogen) at 60 nM for 30 min,
washed three times with PBS, and then incubated with a test compound. After compound
treatment, parasites were lightly fixed with 1% formaldehyde for 10 min and observed under an
Olympus fluorescence microscope.
5. Immunofluorescence Assay (IFA). Dd2 parasites were tightly synchronized with several
rounds of alanine treatment. At the early trophozoite stage, parasites were exposed to DMSO or
a 10xEC50 concentration of selected CGX compounds for 8 h. 30 min before sample harvesting,
60 nM MitoTracker Red CMXRos (Invitrogen) was added to each culture. Parasites were then
washed 3 times with PBS. Thin blood smears were made for each condition. The remaining
cultures were fixed with 4% formaldehyde/0.0075% glutaraldehyde at 4 °C overnight. The
samples were then permeabilized with 0.1%Triton X-100, reduced with 0.1 mg/ml sodium
borohydride, and blocked with 5% BSA-PBS, according to our standard IFA procedure (38). To

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monitor the morphological changes of the food vacuole upon drug treatment, an anti
Plasmepsin II polyclonal antibody (Rabbit antiserum MRA-66) was obtained from BEI
Resources (NIAID and NIH) (BEIResources.org). The antibody was diluted 1:1000 and
incubated with the samples at 4 °C overnight. Then an AlexaFluor 488 conjugated anti-rabbit
secondary antibody (Molecular Probes) was added at a dilution of 1:350 and incubated at 4 °C
overnight. All other steps followed the standard protocol (38). The parasites were then
visualized under the Olympus fluorescence microscope.
6. MTT cell growth assay. HEK293 is a non-carcinoma cell line stably transformed with
adenovirus DNA and can grow indefinitely in vitro (39). The mammalian cells were cultured with
complete Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine
serum. The cytotoxicity of CGX compounds against HEK293 cells was determined by an MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in 96 well plates following
the manufacture’s protocol (EMD Millipore Corporation). Briefly, 10, 000 cells in a volume of 50
µl DMEM were inoculated into each well. The plates were incubated in a 37 °C incubator (5%
CO2) for 2 h to allow the cells to attach. Compounds were then serially diluted in another 96 well
plate and 50 µl aliquots with various concentrations were added into wells previously seeded
with cells. The final volume of medium in each well equaled 0.1 ml. After exposure to
compounds for 24 h and 48 h, 10 µl of MTT solution (5 mg/ml) was added to each well and the
plates were incubated at 37 °C for 4 h to allow MTT to be reduced to purple formazan in live
cells. At the end of 4 h incubation with MTT, 100 µl isopropanol containing 0.04 N HCl was
added to each well. The isopropanol dissolves formazan, yielding homogenous blue solution
that can be measured colorimetrically. Absorbance was measured with an ELISA plate reader
(Tecan US) at 570 nm versus a reference wavelength of 630 nm.

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7. Compound synthesis. GBA was isolated from gamboge resin via its pyridine salt (27). CLV
and the hydroxylated cluvenones MAD28 and MAD44 were synthesized as previously reported
(25,40). The synthesis of the triphenylphosphonium salt conjugates is shown in Supplementary
Material. CR135 and CR142 were synthesized from MAD28 and MAD44, respectively, by
treating them with 1,4-dibromobutane (5 equiv) and potassium carbonate (2 equiv) in
dimethylformamide (DMF) (41). The resulting bromide was then converted to the
triphenylphosphonium salt upon treatment with Ph3P (5 equiv) in acetonitrile at 150 °C under
microwave irradiation. Preparation of SQ129 proceeded in three steps that included: (a)
protection of the catechol functionality of trihydroxylated xanthone (42,43) with diiodomethane
and sodium bicarbonate; (b) bromination of the C6 phenol with 1,4-dibromobutane and (c)
treatment of the resulting bromide with triphenylphosphine. Detailed experimental procedures
and spectroscopic and analytical data are shown in the Supplemental Material.
8. Data analysis. For 3H-hypoxanthine incorporation and MTT assays, triplicate wells were set
for each condition tested. The mean values of measurements made with parasites or HEK293
cells treated with DMSO alone were set as 100%. All other measurements were compared to
DMSO controls. The dose-response data was then analyzed by GraphPad Prism Version 4 to
obtain curves fitted by nonlinear regression and corresponding EC50 values. The
mean±standard error of all biological replicates for each condition is reported in the Results.
Results
Effect of CGXs on the growth of the human malaria parasite P. falciparum. The
erythrocytic stage of malaria parasites causes all clinical symptoms associated with malaria and
is the target for most antimalarial drugs. P. falciparum strains isolated from various geographical
regions harbor different sensitivities to antimalarial drugs. Dd2 is a multi-drug resistant clone
selected from an Indochina isolate using mefloquine pressure (44). To test if GBA and related

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CGXs (Figure 1) have activities against drug resistant parasites, we performed growth inhibition
assays based on 3H-hypoxanthine incorporation by Dd2 parasites (Figure 2). Artemisinin was
included as a control in this assay, which yielded an EC50 value of 12.4±1.5 nM, similar to
previous reports (45). As shown in Figure 2, GBA and CLV exhibited moderate antimalarial
activities with EC50 values of 0.28±0.03 µM and 0.75±0.03 µM, respectively. A similar
antimalarial activity was observed with MAD28, the C6 hydroxylated CLV, which exhibited an
EC50 of 0.26±0.02 µM. However, MAD44, the C18 hydroxylated CLV, was less effective, with an
EC50 of 4.1±0.3 µM. CR135 and CR142 were synthesized from MAD28 and MAD44,
respectively, by conjugating a triphenylphosphonium group at C6 of MAD28 and C18 of MAD44
(41). Importantly, CR135 and CR142 exhibited remarkable antimalarial activities, with EC50s as
low as 7.9 nM and 11.1 nM, respectively. Thus, conjugating the A ring of the caged xanthone
structure with a triphenylphosphonium group drastically improves the antimalarial activity.
Specifically, adding this group to the C6-hydroxyl group of MAD28 decreased the EC50 by about
30 fold from 267 nM (MAD28) to 7.9 nM (CR135). The same modification at the C18-hydroxyl
group decreased the EC50 about 370 fold from 4100 nM (MAD44) to 11.1 nM (CR142). To test if
a caged xanthone was required for the robust activity of CR135, we replaced the caged
xanthone structure of CR135 with a planar xanthone, yielding the compound SQ129. The
antimalarial activity of SQ129 (EC50, 106.5±13.1 nM) was much weaker than that of CR135
(Figure 2), suggesting that a caged xanthone moiety is also needed for optimal antimalarial
activity.
We repeated the growth inhibition assays several times with selected CGX compounds in drug
sensitive (3D7) and drug resistant (Dd2) P. falciparum parasites and the average EC50 values
are presented in Table 1. Collectively, these data show that GBA and related CGXs have
moderate antimalarial potency, but their efficacy increases dramatically upon conjugation of the
caged xanthone motif with a triphenylphosphonium group.

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Effect of CGXs on the intra-erythrocytic development cycle of P. falciparum. Within the red
blood cells, P. falciparum undergoes a 48 h intra-erythrocytic development cycle, during which
the maturation process can be subdivided into ring, trophozoite, and schizont stages (46).
Compounds CR135, CR142 and MAD28, which exhibited the best EC50 values, were selected
for this study while DMSO and SQ129 served as controls. We tightly synchronized Dd2
parasites and subsequently treated them at the trophozoite stage with CGX compounds at 10x
EC50 concentrations (Materials and Methods) and monitored them from 2 h to 48 h post addition.
At each time point, thin blood smears were prepared for morphological studies, and parasite
aliquots were fixed and stained with SYBR Green for determination of parasitemia by flow
cytometry.
Parasite morphological changes and parasitemia during the treatment time course are
presented in Figure 3. In the DMSO control, parasites progressed normally throughout the 48 h
life cycle (Figure 3A, top panel). However, trophozoites treated with CR135 or CR142 displayed
a characteristic morphological change, the appearance of a large Giemsa stain resistant area
(Figure 3A, red arrows), giving the appearance of an empty volume that fills much of the
parasite, as soon as 2 h after treatment. For future references, we named this morphologically
aberrant structure as Giemsa-stain negative body (GNB). The proportion of these GNB
parasites containing a large stain-negative structure increased from ~20% (2 h drug treatment)
to ~30% after 4 h drug treatment (Figure 3B). In samples treated with CR135 or CR142 for 8 h,
the proportion of empty-looking parasites decreased to ~ 5%, due to the progression of the
parasite growth cycle producing a large population of newly invaded erythrocytes containing
young ring parasites (Figure 3B). These newly formed ring stage parasites seemed to be
morphologically normal (Figure 3A). During an additional 16 h exposure to CR135 or CR142
(24 h total), these new rings progressed to late rings without any observable morphological
defects (Figure 3A). However, in the next 24 h (48 h total post addition, Figure 3A), growth

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appeared to become blocked at the early trophozoite stage, with the blocked parasites regaining
the characteristic empty-looking appearance.
On the other hand, MAD28 seemed to kill parasites by different mechanism(s). As shown in
Figure 3A, MAD28 did not cause the formation of a large empty area inside parasites during the
treatment time course. Nevertheless, at the concentration tested (2.6 µM), MAD28 significantly
delayed progression of the parasites. After 4 h treatment, fewer trophozoites progressed to
multi-nucleated schizonts. After 8 h MAD28 treatment, there were very few new rings in the
sample. SQ129 behaved distinctly from CR135/CR142 or MAD28 (Figure 3A). It did not induce
a large GNB in the parasites or inhibit parasite growth as dramatically as MAD28. In all, these
data suggest that CR135 works similarly to CR142; however, CR135 and CR142 kill malaria
parasites through mechanism(s) distinct from those of MAD28.
Quantitative parasitemia data for the same 48 h treatment time course, determined by flow
cytometry, is presented in Figure 3C. CR135 and CR142 knocked down parasitemia
significantly after drug treatment for 24 h. Even though there were some residual “parasites”
present after drug treatment for 48 h with CR135 or CR142, these parasites were dead and did
not progress (Figure 3A). After a 96 h treatment with CR135 or CR142, the parasitemia dropped
down to an undetectable level, and there were just a few dead remnant parasites or purple dots
within the host cells observed after staining (data not shown). MAD28, on the other hand,
seemed to be a faster killer than CR135 or CR142. It reduced the parasitemia significantly after
8 h of drug treatment (Figure 3B). The kinetics of these parasite killing data suggests that
CR135 and CR142 eliminate parasites at a slower pace than does MAD28 and, again, it
appears that CR135 and CR142 work similarly, while MAD28 kills parasites through different
mechanism(s).

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As shown in Figure 3A, CR135 and CR142 caused the formation of a large Giemsa stain
resistant area (GNB) inside the parasite. It was noticeable that this large empty area was
adjacent to the hemozoin crystal. The intimate localization of this structure to hemozoin
prompted us to investigate the integrity of the food vacuole under drug treatment with CR135
and CR142 by IFA using a food vacuole marker. Dd2 parasites were synchoronized and treated
with CR135 and CR142 (10x EC50 concentration), respectively, for 8 h from early-trophozoite
stage to mid-trophozoite stage. Compared to a 4 h treatment starting with mid trophozoites, we
noticed that the percentage of GNB parasites increased significantly (up to 50%) when
treatment started at a younger trophozoite stage and lasted longer (8 h) (data not shown). Post
treatment, the parasites were then fixed with formaldehyde/glutaraldehyde. An anti Plasmepsin
II polyclonal antibody was used to visualize the structure of food vacuole; Plasmepsin II, an
aspartic protease for hemoglobin digestion, is a marker for this organelle (47,48). As shown in
Figure 4, in the control parasite, one, relatively large globular structure containing the hemozoin
crystal was clearly visible, and a few small spots next to the main body were also present. This
staining pattern represents the normal food vacuole architecture (47,48). Under treatment with
CR135 and CR142, however, the intactness of the food vacuole appeared to be lost. As shown
in Figure 4, the main globular staining disappeared; instead, there were many small granular
fluorescing particles scattering throughout the cytosol, suggesting that the food vacuole integrity
was damaged by treatment with the compounds. A correlation between the large vacant space
in the GNB parasites (Figure 3A) and the IFA images (Figure 4) is not evident; thus, the nature
of this feature remains unclear at present (see Discussion).
To further characterize the effects of these CGX compounds on parasites, we also treated P.
falciparum with 10x EC50 concentrations starting with synchronized schizonts and with
synchronized rings. As shown in the Supplementary Figure 1A, when treatment was begun at
the schizont stage, these compounds significantly inhibited parasite development. At 10x EC50

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concentration, MAD28 was the most effective compound in this regard. As shown in
Supplementary Figure 1B, when a parasite culture at 5% schizont parasitemia was treated with
MAD28, the parasitemia only declined to 3% after 8 h treatment, indicating that ~60% of the
schizonts still remained inside the host cells. In control parasites treated with DMSO in parallel,
only ~10% of the schizonts had not egressed after 8 h incubation. CR135 and CR142 also
inhibited parasite growth, but to a lesser extent than MAD28. Due to the strong inhibitory effect
of MAD28, the parasitemia after 24 h treatment dropped to a very low level (Supplementary
Figure 1C). Again, CR135 and CR142 were slow killers, and parasitemias after 24 h drug
treatment remained much higher than that of MAD28 (Supplementary Figure 1C). Collectively,
these data suggest that CGX compounds inhibit parasite growth at the schizont stage, possibly
via blocking schizont maturation and/or parasite egress. MAD28 was a strong inhibitor at the
concentration used (2.6 µM). However, due to its narrow therapeutic window, MAD28 is quite
toxic to mammalian cells at 10x EC50 concentration (see Table 1).
Different phenomena were observed when treatments were initiated at the ring stage. As shown
in Supplementary Figure 2A, the compounds did not inhibit the progression of young rings to
mature rings significantly, as 8 h drug treatments did not cause dramatic morphological changes.
For all the compounds tested, the parasitemia of an 8 h drug treatment was comparable to that
of the control DMSO treatment (Supplementary Figure 2B). The antimalarial effects of these
compounds became much more evident after 24 h of drug treatment: CR135 and CR142
arrested the parasites at early trophozoite stages and formed a large GNB inside the parasites;
MAD28 blocked the parasites at an earlier stage, but did not cause the formation of a large
empty-looking structure. The parasitemias after 24 h of treatment with each of the compounds
are presented in Supplementary Figure 2C.

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Mitochondrial morphology of P. falciparum parasites treated with CGXs. GBA and CGXs
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are pleiotropic compounds that kill cancer cells via multiple mechanisms (11). One important
target is the mitochondrion, which undergoes apoptosis under drug treatment (27). MAD28 has
been recently shown to inhibit an iron-sulfur cluster binding protein, mitoNEET, on the outer
membrane of mitochondria in breast cancer cells (29). MitoNEET is involved in cellular iron
homeostasis and various mitochondrial functions (49).
To determine the effects of these compounds on parasite mitochondria, we preloaded
synchronized parasites at the early trophozoite stage with MitoTracker (Materials and Methods),
treated them with compounds at 10x EC50 concentrations, and observed their mitochondrial
morphologies over time. In parasites treated with CR135, CR142, or MAD28 for 4 h, their
mitochondria were morphologically indistinguishable from those treated with DMSO vehicle
(data not shown). However, in samples treated with compounds for 8 h, we observed significant
morphological changes in the mitochondria (Figure 5). As shown in Figure 5A, in the DMSO
control parasite, the mitochondrion formed a continuous tubular filament structure, indicating a
healthy mitochondrion. However, in parasites treated with CR135, CR142, or MAD28, the
mitochondria appeared fragmented and punctate. Each parasite contained one or several
MitoTracker stained dots, but no long tubular structures. To further quantify this phenomenon,
we examined 200 parasites from each treatment (drug or vehicle) and classified them as having
either tubular mitochondria or punctate mitochondria. As shown in Figure 5B, in the DMSO
control, >95% of the parasites had tubular structures; however, the percentage of tubular
mitochondria decreased to <5% in drug treated parasites. These data strongly suggest that
these CGX compounds have a strong detrimental effect on the parasite mitochondria.
Effect of CGXs on the mitochondrial electron transport chain (mtETC). During the asexual
blood stages, the mtETC of malaria parasites is required to recycle ubiquinol to ubiquinone,

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which serves as the electron acceptor for DHODH, an essential enzyme in the pyrimidine
biosynthesis pathway (35). Providing the parasite with a yeast DHODH enzyme that utilizes a
different electron acceptor establishes an alternate pathway for pyrimidine biosynthesis and
renders the parasite resistant to all bc1 complex inhibitors (35). Thus, yDHODH transgenic lines
have become a convenient tool to determine if a compound targets the mtETC. Here, we
utilized the 3D7attB-yDHODH line, which has the yDHODH gene integrated into the genome at
a non-essential locus (36). As shown in Figure 6, this transgenic line was fully resistant to
atovaquone, as expected, but was still susceptible to all CGX compounds tested, suggesting
that the target(s) of the CGXs do not reside in the parasite mtETC. Consequently, these drugs
likely target other essential function(s), either inside or outside of the mitochondrion. We note
that the EC50 values of these compounds against the 3D7attB-yDHODH parasites (Figure 65)
were slightly higher than those found with the 3D7 parasites (Table 1). The reasons for the
variation are undetermined at present, but possibly may arise during the genetic transfections
and lengthy selection procedures required to generate the 3D7attB-yDHODH line.
Toxicity of CGXs to human cells. To test the toxicity of these CGXs to human cells, we
performed the standard MTT assay on HEK293 cells. The cells were exposed to a range of
concentrations of each selected CGX compound for 24 h and 48 h, as described in Materials
and Methods. As shown in Figure 7, after 24 h drug exposure, we found that the EC50s of
CR135, CR142 and MAD28 fell in the range of low µM concentrations. SQ129 was less toxic to
HEK293 cells with a significantly higher EC50. The average EC50 values from 3 independent
experiments are shown in Table 1. The EC50 values for the 48 h drug exposure were quite
similar to those of the 24 h treatment (data not shown). These data suggest that these CGX
compounds are toxic to human cells at low µM concentrations. Accordingly, the therapeutic
window is around 150 fold for CR135 or CR142, while it is much narrower for MAD28 (~7 fold).

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Discussion
In this study, we tested the antimalarial activities of CGX compounds against P. falciparum
malaria parasites at the intra-erythrocytic stages. GBA and synthetic derivatives have been of
great interest to medicinal chemistry due to their potent activities against cancer cells. For the
first time, this study shows that these compounds also possess antimalarial activity both against
drug sensitive and drug resistant parasite lines. Our data extend the biological functions of GBA
and CGXs beyond their known anticancer (11), antibacterial (50), and antiviral (51) effects to
include antiparasitic activity.
GBA and its derivatives showed moderate antimalarial activity with EC50s at sub micromolar
concentrations. This activity was greatly enhanced for compounds CR135 or CR142, in which
the caged xanthone motif was conjugated with a triphenylphosphonium group, resulting in EC50s
at low nM concentrations. It has been proposed that delocalized lipophilic cations, such as the
triphenylphosphonium group, can specifically and efficiently drive their cargo to active
mitochondria, which maintain a negative-inside transmembrane gradient (52,53). Such a
delivery strategy has led to the development of MitoQ, a ubiquinone-triphenylphosphonium
conjugate that has entered clinical trials against neurodegenerative diseases (54,55). Moreover,
improvement of antimalarial potency has been reported for a series of 1,4-naphthoquinones (the
chemical class that include atovaquone) conjugated with a triphenylphosphonium group (56,57).
In the infected red blood cell, the parasite’s mitochondrion is the organelle with the most
negative membrane potential. Therefore, we reason that CR135 and CR142 accumulate in or at
the parasite’s mitochondrion in higher concentrations compared to the rest of the parasite and
the host. In turn, the parasite mitochondrion is likely a major action site for these compounds. In
support of this hypothesis, we also observed that CR135 and CR142 caused mitochondrial
fragmentation in the parasite 8 h post drug treatment (Figure 4), indicating that the parasite
mitochondrion contains, at least, one target of CR135 and CR142. Interestingly, in cancer cells,

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both CR135 and CR142 showed decreased toxicity compared to their parental compounds,
MAD28 and MAD44 (41), suggesting that structural modifications of the CGX motif could
improve both potency and selectivity against malaria parasites. Besides the
triphenylphosphonium group, the caged xanthone moiety of CR135 and CR142 is also critical
for function since SQ129, a synthetic derivative that contains a triphenylphosphonium group but
lacks the caged xanthone, showed only moderate antimalarial activity.
We found that CGXs kill malaria parasites at multiple asexual stages. The metabolically active
trophozoite and schizont stages are more sensitive to these compounds compared to the ring
stage parasites (Figure 3). Our data also show that treatment with different CGXs caused
distinct morphological changes to the parasites, suggesting that the various individual
modifications to these compounds may result in differing modes of action. Particularly, CR135
and CR142 exhibited antimalarial activities at low nanomolar concentrations (Figure 2). These
compounds caused the formation of a large Giemsa-stain negative structure (GNB) inside the
parasite that occurred during trophozoite development (Figure 3). The appearance of GNB
seemed to be specific for CR135 or CR142, not for MAD28 (Figure 3). Since this enlarged area
was close to the hemozoin particle (Figure 3), we thought that it might be an enlargement of the
food vacuole. Indeed, the integrity of food vacuole was damaged by CR135 and CR142
treatment as determined by IFA with a food vacuole marker (Figure 4). Under drug treatment
(CR135 or CR142), the globular structure of the food vacuole of a normal parasite was absent,
and the vacuole marker Plasmepsin II distributed among many punctate particles scattered
throughout the parasite cytosol (Figure 4). These small particles, however, did not appear to
outline a large structure resembling the empty area seen in Giemsa stained parasites after drug
treatment (compare Figure 3 and Figure 4). Therefore, at present, the nature of GNB seen in
thin blood smears remains unknown.

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At present, the mechanisms of action of CGXs against malaria parasites remain unknown. In
cancer cells, GBA and other CGXs are known to cause mitochondrial damage and apoptosis
(11). The mtETC is an essential process in malaria parasites and a known antimalarial drug
target. We have shown that in the asexual blood stages, the critical function of the mtETC is to
sustain the activity of the parasite dihydroorotate dehydrogenase (DHODH) for pyrimidine
biosynthesis (35,36). Provision of the parasites with the yeast DHODH, which does not rely on
the mtETC, makes the parasites resistant to all mtETC inhibitors (35,36). Importantly however,
yeast DHODH transgenic parasites were found to be sensitive to CGXs (Figure 5). This result
rules out the possibility that CGXs target the mtETC. It is likely that CR135 and CR142 target
other as yet unknown essential mitochondrial function(s) or other pathways beyond the
mitochondrion.
CGX compounds seem to target multiple pathways. The integrity of food vacuole is lost after 8 h
of treatment with CR135 and CR142. We attempted to generate resistant clones by culturing
10^8 Dd2 parasites under 3x EC50 and 5x EC50 of CR135, CR142, and MAD28, individually.
However, none of these conditions yielded any resistant parasites over a two month period
(data not shown), consistent with the assumption that CGX compounds have various targets.
Targeting multiple pathways can be beneficial to prevent or delay the appearance of drug
resistance. Indeed, the standard antimalarial chemotherapy involves the use of several agents
in a combination, since monotherapy can often select resistant parasites rapidly. Therefore, it
could be an advantage for CGX compounds to target multiple pathways. On the other hand,
action against multiple targets can make it challenging to optimize parasite specific inhibitors via
a rational program of chemical modifications. CGX compounds kill mammalian cells at low
micromolar concentrations (1.5-2.2 µM; Figure 7), somewhat below the common concentration
(>10 µM) for other antimalarial compounds under development. While the initial GCX
compounds had a narrow selective window, addition of a triphenylphosphonium group to the

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xanthone moiety expanded the therapeutic window dramatically from 7 fold (MAD28) to 150 fold
(CR135/CR142) (Table 1). Thus, it is possible to develop better CGX derivatives through
medicinal chemistry. As the most efficient antimalarial compound of the series, CR135 could be
a lead for future chemical optimization.
From a drug development point of view, GBA and related CGXs clearly have potential for
further development as antimalarial drugs. Importantly, conjugation of the CGX motif with
selective transporters and delivery systems can substantially improve the potency and target
selectivity. This was shown by CR135 and CR142, which exhibit strong antimalarial activity at
low nanomolar concentrations. It is thus reasonable to predict that further chemical optimization
of the caged xanthone backbone would yield compounds with more potent and selective
antimalarial activities. Interestingly, CGX compounds were not included in the “malaria box”, a
set of 400 antimalarial compounds made available to the research community by Medicines for
Malaria Venture and partner companies and organizations (58). The CGX compounds appear to
represent a new and unexplored class of antimalarial agents with a totally different chemical
backbone from those of other chemical scaffolds discovered by high through-put screenings or
other methods.
GBA and gamboge have been used in Eastern medicine for hundreds of years. GBA has
entered a Phase II clinical trial in China as an anti-cancer agent in patients with non-small cell
lung, colon and renal cancers (19).Toxicity studies on GBA in mice, dogs, and rats indicated
that GBA has decent therapeutic windows for cancer therapies (59,60) without inducing any
toxic symptoms on blood pressure, heart rate and respiratory frequency at pharmacologically
relevant doses (61). An innocuous dose of GBA in rats was established to be 60 mg/kg after
administration for a total of 13 weeks at a frequency of one administration every other day. This
dose was more than 10 times higher than that used in human clinical trials (58, 59). In addition,

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bioavailability studies (40 and 80 mg/Kg) in rats showed that GBA was rapidly accumulated in
liver where it can be metabolized by various routes, including oxidation, hydration,
glutathionylation and glucosidation, and was mainly excreted in bile from 0-24 h post-dosing
(62,63). These studies attest to the pharmacological potential of GBA and the CGX motif.
Moreover, the developed synthetic strategies allow rapid and high yielding access to designed
CGX analogs thereby potentially paving the way for the development of CGX-based antimalarial
agents.
In conclusion, we evaluated the effect of gambogic acid and related synthetic analogues of
caged Garcinia xanthones as antimalarial agents. We found that these compounds induce
cytotoxicity in P. falciparum malaria parasites at submicromolar concentrations. Importantly,
conjugating these compounds with a phosphonium salt improved the efficacy by about two
orders of magnitude, resulting in lead compounds with a promising therapeutic window. Further
modification of the caged xanthone motif and/or the delivery subunit could further increase the
selective cytotoxicity of the compound and lead to the development of a promising lead
candidate.
Acknowledgement
The Plasmepsin II antibody was obtained through BEI Resources (NIAID and NIH). The
antibody was originally contributed by Daniel E. Goldberg from Washington University at St.
Louis. We thank Dr. James M. Burns Jr. from Drexel University College of Medicine for help in
arranging the delivery of antibody. Human embryonic kidney cells (HEK293) were kindly
provided by Dr. Sandhya Kortagere from Drexel University College of Medicine. We thank Dr.
Wei Xu for technical assistance in culturing HEK293 cells and Sihem Sassi-Gaha for help with
MTT assays. We also thank Dr. Anthony Mrse (UCSD NMR Facility) and Dr. Yongxuan Su
(UCSD MS Facility) for assistance with the spectroscopic characterizations.

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Funding information
This work was support by grants AI028398 from NIH to Dr. Akhil B. Vaidya, CRC-15-380737
from Cancer Research Coordinating Committee to Dr. Emmanuel A. Theodorakis, and
TRG5780085 from Thailand Research Fund to Dr. Oraphin Chantarasriwong.
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731
732
733
734
735
Figure Legend
Figure 1. Chemical structures of gambogic acid (GBA) and related caged Garcinia
xanthones (CGXs). Cluvenone (CLV) defines the structure of the common CGX motif.
Hydroxylation at the C6 and C18 centers of CLV produces MAD28 and MAD44, respectively.

26
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
Attachment of a triphenylphosphonium salt at these hydroxylated sites produces CR135 and
CR142, respectively.
Figure 2. The antimalarial effect of CGX compounds in P. falciparum parasites. The
antimalarial efficiency of CGXs in Dd2 parasites was measured by the 3H-hypoxanthine
incorporation assay (Materials and Methods). The x axis indicates the concentrations of a tested
compound, and the y axis indicates the percentage of 3H-hypoxanthine incorporation compared
to that in no-drug controls. The assays were set up using triplicate wells for each concentration
of each tested compound. The averaged data of five independent experiments are shown (n=5).
Figure 3. Viability P. falciparum parasites treated with CGX derivatives. Dd2 parasites were
tightly synchronized and treated with compounds at 10x EC50 concentrations starting at mid-
trophozoite stage. Giemsa smears were made for morphological studies with representative
images shown in (A). The red arrows in the panel indicate the specific morphological structures
not stained by Giemsa. In (B), the percentage of GNB parasites in control and drug treated
cultures (CR135 and CR142) is quantified. In (C), the parasitemia at each time point is plotted,
as determined by SYBR Green staining and flow cytometry. For each sample, 1,000,000 events
were collected and analyzed. The time-course was repeated three times and error bars in (B)
and (C) indicate the standard error of three biological replicates.
Figure 4. The food vacuole integrity of P. falciparum parasites treated with CR135 and
CR142. Representative IFA images show food vacuole morphology in parasites treated with
vehicle, CR135, and CR142, individually. Anti-Plasmepsin II polyclonal antibody (rabbit) was
diluted 1:1000 and an AlexaFluor 488 conjugated anti-rabbit secondary antibody was diluted
1:350. MitoTracker Red stains active mitochondria.

27
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765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
Figure 5. The mitochondrial morphologies of P. falciparum parasites treated with CGX
derivatives. (A) Representative images showing mitochondrial morphology in parasites treated
with vehicle or compounds for 8 h. (B) Quantitation of the relative fraction of healthy tubular
mitochondria in parasites treated with vehicle or compounds for 8 h. For each treatment,
mitochondrial morphology was assessed in 200 parasites. This experiment was repeated three
times. Error bars indicate the standard error of three biological replicates.
Figure 6. CGX compounds do not target the mitochondrial electron transport chain of P.
falciparum. The potency of CGX compounds against the mtETC-independent strain 3D7attB-
yDHODH was determined with the 3H-hypoxanthine incorporation assay. Calculated EC50s of
compounds [nM] against this strain from three independent experiments: CR135 (58.0±5.7),
CR142 (61.8±6.4), MAD28 (383.6±19.5), and SQ129 (101.3±8.1).
Figure 7. Cytotoxicity of CGX derivatives in HEK293 cells. The cytotoxicity of CGX
compounds in human HEK293 cells treated for 24 h was determined by an MTT assay
(Materials and Methods). For each concentration of each compound tested, triplicate wells were
set up. The y axis (% Growth) indicates the percentage of MTT signal in a drug treated sample
compared to that in a no-drug control. The average of three biological replicates is shown.
Calculated EC50s are listed in Table 1.
781
782
783
784
Table 1. EC50 values of CR135, CR142, MAD28 and SQ129 in malaria parasites and
human cells.
785
786
787

28
788
Calculated EC50 values and standard errors resulting from 3-5 independent experiments. Please
789
790
791
note that data are shown in nanomolar for P. falciparum lines Dd2 and 3D7, but in micromolar
for HEK293.