1. Identification of Potent Phosphodiesterase Inhibitors that
Demonstrate Cyclic Nucleotide-Dependent Functions in
Apicomplexan Parasites
Brittany L. Howard,†,#
Katherine L. Harvey,‡,∥,#
Rebecca J. Stewart,§,⊥,#
Mauro F. Azevedo,‡
Brendan S. Crabb,†,‡,∥
Ian G. Jennings,†
Paul R. Sanders,‡
David T. Manallack,†
Philip E. Thompson,*,†
Christopher J. Tonkin,*,§,⊥
and Paul R. Gilson*,†,‡
†
Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia
‡
Macfarlane Burnet Institute, Melbourne, Victoria, Australia
§
The Walter & Eliza Hall Institute, Melbourne, Victoria, Australia
∥
University of Melbourne, Melbourne, Victoria, Australia
⊥
Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia
*S Supporting Information
ABSTRACT: Apicomplexan parasites, including Plasmodium falciparum and
Toxoplasma gondii, the causative agents of severe malaria and toxoplasmosis,
respectively, undergo several critical developmental transitions during their
lifecycle. Most important for human pathogenesis is the asexual cycle, in which
parasites undergo rounds of host cell invasion, replication, and egress (exit),
destroying host cell tissue in the process. Previous work has identified important
roles for Protein Kinase G (PKG) and Protein Kinase A (PKA) in parasite egress
and invasion, yet little is understood about the regulation of cyclic nucleotides,
cGMP and cAMP, that activate these enzymes. To address this, we have focused
upon the development of inhibitors of 3′,5′-cyclic nucleotide phosphodiesterases
(PDEs) to block the breakdown of cyclic nucleotides. This was done by
repurposing human PDE inhibitors noting various similarities of the human and
apicomplexan PDE binding sites. The most potent inhibitors blocked the in vitro
proliferation of P. falciparum and T. gondii more potently than the benchmark compound zaprinast. 5-Benzyl-3-isopropyl-1H-
pyrazolo[4,3-d]pyrimidin-7(6H)-one (BIPPO) was found to be a potent inhibitor of recombinant P. falciparum Pf PDEα and
activated PKG-dependent egress of T. gondii and P. falciparum, likely by promoting the exocytosis of micronemes, an activity that
was reversed by a specific Protein Kinase G inhibitor. BIPPO also promotes cAMP-dependent phosphorylation of a P. falciparum
ligand critical for host cell invasion, suggesting that the compound inhibits single or multiple PDE isoforms that regulate both
cGMP and cAMP levels. BIPPO is therefore a useful tool for the dissection of signal transduction pathways in apicomplexan
parasites.
■ INTRODUCTION
The phylum Apicomplexa comprises a large group of obligate
intracellular parasites of wide social, economic, and medical
significance. Plasmodium falciparum, the causative agent of the
most severe form of malaria, remains one of the biggest
scourges to human health, infecting 207 million people a year
and killing 627 000.1,2
Toxoplasma gondii on the other hand is
chronically carried by 30−80% of the human populations, and
while it causes few problems in healthy individuals, acute
infection in a developing fetus or people with compromised
immune systems (HIV, transplant recipients) can cause massive
tissue destruction. This can lead to a range of disease
manifestations including hydrocephalus, epilepsy, mental
retardation when passed to the unborn fetus, or blindness
and neurological deficits in the immuno-compromised.
Both P. falciparum and T. gondii are obligate intracellular
pathogens and thus rely on the invasion of host cells for survival
and proliferation. During the asexual amplification stage, P.
falciparum exclusively targets human erythrocytes, whereas T.
gondii can infect almost any cell in most vertebrates. After
replication inside a host cell, both T. gondii and P. falciparum
must activate a cellular program, which elicits egress (exit) from
one cell and reinvasion of the next. Molecular processes that are
required for invasion and egress are of significant interest given
their potential as vaccine and drug targets for the treatment and
prevention of toxoplasmosis and malaria.
Received: May 16, 2014
Accepted: January 2, 2015
Articles
pubs.acs.org/acschemicalbiology
© XXXX American Chemical Society A DOI: 10.1021/cb501004q
ACS Chem. Biol. XXXX, XXX, XXX−XXX

2. Recently, it has been determined that three second
messenger signaling pathways control parasite egress and
reinvasion of host cells.3−6
An increase in cytoplasmic
concentration of the secondary messengers cyclic-AMP
(cAMP), cyclic-GMP (cGMP), and calcium ions (Ca2+
),
typically causes changes in the cellular program by activating
kinases through their direct or indirect binding. This results in
phosphorylation of specific substrates, which ultimately leads to
a change in cellular function. cAMP, for example, is a known
activator of Protein Kinase A (PKA), and this enzyme has been
implicated in the phosphorylation of the cytoplasmic tail of the
parasite adhesin Apical Membrane Antigen 1 (AMA1), which is
essential for P. falciparum erythrocyte invasion.5
Protein Kinase
G (PKG) on the other hand is directly activated upon a rise in
cGMP concentration, and in apicomplexan parasites this
enzyme appears to occupy an important nexus during multiple
transition stages within P. falciparum4,7−9
and during egress and
motility of Toxoplasma.6,10,11
Given that PKA and PKG require
a rise in intracellular concentration of cAMP and cGMP,
enzymes that control the production and degradation of these
cyclic nucleotides likely play a critical role in the activation of
egress and invasion. 3′-5′-Cyclic nucleotide phosphodiesterases
(PDEs) hydrolyze cAMP and cGMP into AMP and GMP,
respectively, thus attenuating their cellular accumulation.
Indeed, physiological PDE inhibition (eg, by regulatory domain
phosphorylation) has been described as a mechanism to
increase cAMP and cGMP levels, consequently activating
downstream events.12
Small molecule inhibitors of parasite
PDEs could therefore assist in the determination of events that
activate egress and invasion in apicomplexan parasites.
The human PDE5 (hPDE5) inhibitor, zaprinast (1; Figure
1), was the first compound identified as an inhibitor of parasite
PDEs.13
Specifically, it was shown to inhibit P. falciparum
proliferation (IC50 35 μM), inhibit recombinant P. falciparum
PDEα activity, and cause elevation of intracellular cGMP
levels.14
Zaprinast has also been used in the study of parasite
egress where it was shown to induce premature egress from
erythrocytes, which can be blocked by the PKG inhibitor,
Compound 1 (Cmpd1).4
Indeed, zaprinast can also induce
PKG-dependent egress in T. gondii.2
While zaprinast has been useful to determine the role of
PDEs in infection, and as a tool for activating parasite egress, it
is only a moderately potent inhibitor and therefore has limited
use. Several other PDE inhibitors were evaluated in the 2009
National Institute of Health (NIH) malaria screen,15
including
the nonisoform selective PDE inhibitor, Dipyridamole, and the
hPDE3 inhibitor Cilostazol with EC50 values against the 3D7
strain of approximately 10 μM. In addition, Beghyn and co-
workers originally explored the repurposing of the hPDE5
inhibitor, tadalafil (2; Figure 1) as a potential antiplasmodium
compound. The most potent tadalafil analogue (3; Figure 1)
had an EC50 of 0.5 μM.16
Recently, we reasoned that more potent parasite PDE
inhibitors might be identified by examining the homology at the
catalytic site of P. falciparum PDEs (PfPDE) as compared to
human PDEs,17
and using this we hypothesized that hPDE9
and hPDE1 isoforms were most like Pf PDE, thus providing a
mechanism to identify new apicomplexan PDE inhibitors. Here,
we have extended our examination of this topic now to the
apicomplexan parasite T. gondii PDEs (TgPDE). Furthermore,
we show that a series of compounds that inhibit hPDE1/9 elicit
much more potent cell responses than zaprinast on both P.
falciparum and T. gondii. We provide evidence as to the mode-
of-action of these new inhibitors, showing that they potently
inhibit recombinant Pf PDEα and induce host cell egress in
both P. falciparum and T. gondii, which can be blocked by
inhibiting PKG, and this is likely due to their ability to potently
activate microneme secretion. Interestingly, we also show that
our new PDE inhibitors act to induce an accumulation of
cAMP during AMA1 tail phosphorylation, thus suggesting that
the PDE target(s) of these compounds breaks down both
cAMP and cGMP during egress, motility, and invasion in
apicomplexan parasites. Together, these data describe potent
new compounds that can be used to study cyclic nucleotide
signaling across the Apicomplexa, which in time could offer new
insights for future disease control therapies.
■ RESULTS AND DISCUSSION
Homology of Pf PDEs and TgPDE. Previous work
demonstrating that zaprinast and other compounds are active
against P. falciparum and T. gondii suggested that these parasites
rely on one or more PDEs for critical cellular processes.2,4
The
P. falciparum genome encodes four highly similar PfPDEs, of
which only two, Pf PDEα and Pf PDEβ, appear to be expressed
in asexual blood stages. It is this stage that produces
symptomatic disease and is therefore the stage that most
PDE inhibition studies have targeted. Collins et al. suggested
Pf PDEβ as a good candidate for zaprinast inhibition,4
but the
Pf PDEs are highly homologous to one another at the active
site, and it is conceivable that it inhibits multiple isoforms. The
Pf PDEs are also homologous to the human targets of zaprinast
hPDE1, -5, and -9.17
Yuasa et al. showed that zaprinast inhibits
Pf PDEα with an IC50 of 3.8 μM, and in their studies of
Pf PDEδ knockout gametocytes, Taylor et al. showed that
Pf PDEδ is relatively insensitive to zaprinast whereas the
residual activity is zaprinast-sensitive.13,18
To extend this work, we wished to determine the PDE
target(s) of zaprinast in T. gondii. We retrieved 18 putative PDE
sequences from ToxoDB, the T. gondii genome database, and
reviewed the homology to human and Plasmodium sequences.
When aligned to the Pf- and hPDE sequences,
TgME49_202540 showed good homology to Pf PDEβ (27%)
and hPDE1B (25%) and in particular showed 72% homology at
the cyclic nucleotide binding site with Pf PDEβ (Table 1) and
matched hPDE1/9 nucleotide-binding residues as well at a
number of conserved amino acid positions. Other T. gondii
sequences also showed sufficient homology to the hPDE and
Pf PDE sequences to be considered candidate PDEs and
include TgME49_266920 [Pf PDEβ (26%) and hPDE1 (28%)]
and TgME49_228500 [Pf PDEβ (29%) and hPDE1 (33%)]
(data not shown).
In developing homology models of the PfPDE isoforms,17
we identified some key features that we believed would dictate
Figure 1. Structures zaprinast (1), tadalafil (2), and tadalafil analogue
(3).
ACS Chemical Biology Articles
DOI: 10.1021/cb501004q
ACS Chem. Biol. XXXX, XXX, XXX−XXX
B

3. the potency of inhibitors of those PDEs, and these were found
to be conserved in the T. gondii orthologues (Table 1). In
particular, the residue adjacent to the conserved “purine-
scanning” glutamine is small in hPDE9 (A452
), hPDE1, Pf PDEβ
(S283
, Figure 2), and Tg49_202540, compared to a bulky M in
most human isoforms (Table 1 and Figure 2). We had also
identified a residue in the core pocket (N405
in hPDE9) as H or
N in the various parasite sequences (H235
in PfPDEβ, Figure 2)
as well as hPDE1, -4, -7, -8, and -9, but A in hPDE5. The larger
H residue in PfPDEs may explain why the hPDE5 inhibitor
sildenafil is a much less potent inhibitor in P. falciparum
proliferation assays relative to zaprinast. Finally we identified a
common hydrophobic pocket formed by three residues (L421
,
Y424
, and F441
of hPDE9 ; L251
, F254
, and L271
in Pf PDEβ, Figure
2). Overall, the similarity of key nucleotide binding residues of
hPDE1 or hPDE9 with the parasite PDEs supported inhibitors
of human hPDE1 and/or -9 as potential starting points from
which to develop parasite PDE inhibitors. We speculated that a
series of reported 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one
hPDE1/9 inhibitors described by De Ninno et al. displayed
the requisite binding pharmacophore (Figure 2).19
Subse-
quently, an analogous 1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
compound in cocrystal with hPDE9 was reported adopting the
pose as modeled by us.19,20
Discovery of 5-Benzyl-3-isopropyl-1H-pyrazolo[4,3-d]-
pyrimidin-7(6H)-one (6). Given that both P. falciparum and
T. gondii most closely resemble hPDE1/9 we prepared a
focused library of compounds based upon a series of already
reported inhibitors of this enzyme.19
The synthesis of all the
compounds derive from the key precursor 4 prepared by
adaptation of literature methods (Scheme 1). Condensation of
4 with various carboxylic acids using PyBroP under microwave
conditions yielded intermediate amides 5, which underwent
base catalyzed cyclization to yield the desired pyrazolopyr-
imidinones (6−23).21
An N-methyl derivative 24 was prepared
by direct methylation of 6 (see Supporting Information).
The compounds were first tested for their growth inhibitory
activity in cultured asexual blood stage P. falciparum parasites
Table 1. Alignment of Human, Pf, and Tg PDE Catalytic Site Residues Based upon Full Sequence Alignment
residuea
regionb
hPDE1 hPDE5 hPDE9a
PfPDEβa
TgME49_202540
292 M H H H292
H119
H
293 M D D D293
D120
D
296 M H H H296
H123
H
322 M E E E322
E149
E
325 M H H H325
H152
H
402 M D D D402
D232
D
251 Q Y Y F251
Y78
Y
405 Q H A N405
H235
N
413 Q H Q A413
H243
H
420 Q + H L V L420
V250
L
423 Q E E E423
E253
E
453 Q Q Q Q453
Q284
Q
456 Q F F F456
F287
F
490 Q W W Y490
W322
W
421 H M A L421
L251
S
424 H F F Y424
F254
F
441 H L L F441
L271
L
301 L N N N301
N128
N
302 L N S T302
L129
A
303 L F Y Y303
F130
L
452 L S M A452
S283
S
455 L G G G455
T286
G
459 L F A F459
F290
F
406 P I E406
G236
C
417 T A V417
C247
C
a
Numbering based upon hPDE9 X-ray structure PDB code 3DYN and Pf PDEβ model based on ref 17. b
Region guide M = metal binding, Q = core
pocket, H = hydrophobic pocket, L = Lid region (see ref 35). Shaded residue positions are discussed in the text.
Figure 2. Homology model of PfPDEβ catalytic site modeled with
BIPPO (6) by analogy to hPDE9 cocrystal (PDB: 3JSI). Residues are
highlighted which may dictate selective interactions between inhibitors
and binding site including Q284 conserved glutamine (Q453 in
hPDE9), H243 (A413), H235 (N405), S283 (A452), L251 (L421),
F254 (Y424), L271 (F441), and H277 (V447).
ACS Chemical Biology Articles
DOI: 10.1021/cb501004q
ACS Chem. Biol. XXXX, XXX, XXX−XXX
C

4. by measuring the activity of the parasite enzyme lactate
dehydrogenase, after 72 h of parasite cultivation with the
inhibitors (Scheme 1 and Table 2). Compounds 6−11 showed
the most potent growth inhibitory activity in that order.
Compound 6, or 5-benzyl-3-isopropyl-1H-pyrazolo[4,3-d]-
pyrimidin-7(6H)-one, which we abbreviated to “BIPPO” and
with no substitution on the phenyl ring, was the most effective
compound with an IC50 of 0.4 μM in P. falciparum. The
potency of BIPPO was followed by compounds 7 and 8,
indicating that the respective para-chloro and para-fluoro
substitutions were tolerated (Scheme 1 and Table 2). The
presence of other substituents, shorter or longer links to the
aryl substituent, reduced potency as did, most notably,
methylation at the 7-positon (24) consistent with the proposed
pharmacophore.
Proliferation inhibitory studies were performed also with T.
gondii tachyzoites growing in human fibroblasts. Growth
potential was qualitatively measured by the number and size
of zones of host cell clearance (plaques) created by the lytic
growth of T. gondii. The three best compounds, as determined
by P. falciparum growth inhibition studies (above), had their
ability to block T. gondii growth determined. BIPPO showed
effective inhibition at concentrations as low as 2 μM and was at
least 30 times more effective than zaprinast at reducing plaque
formation (Figure 3). Just as for P. falciparum, compounds 7
and 8 were the next most potent at reducing tachyzoite
proliferation, and all were much more effective than zaprinast
(Figure 3). Note that the IC50 cannot be calculated using this
plaque assay as a readout.
BIPPO, 6, with its robust activity in both growth inhibition
assays appeared to be the compound to investigate further to
establish parasite PDE inhibition as the mechanism of action.
We first attempted to confirm that BIPPO was a potent
inhibitor of Pf PDEs by testing it against recombinant enzymes.
We expressed PfPDEα with a glutathione S-transferase (GST)
tag in Escherichia coli and obtained a partially purified protein
(Supporting Information Figure s1) that showed robust
hydrolysis of cGMP. While full characterization of the enzyme
and inhibitors was not possible due to its poor stability, it was
found that BIPPO inhibited this activity in a dose dependent
manner, with an IC50 of approximately 150 nM, while zaprinast
was much less potent at 5 μM affording only approximately
40% inhibition, (consistent with the report of Yuasa et al.;13
Supporting Information Figure s2). We also observed that the
enzyme showed modest hydrolytic activity against cAMP
(Supporting Information Figure s3), and when both cGMP
and cAMP were in the same reaction, hydrolysis of cGMP was
virtually complete while significant cAMP remained (Support-
ing Information Figure s4). We also attempted to prepare
recombinant Pf PDEβ, and while observing some activity
against cGMP the enzyme was even less stable than PfPDEα,
making reproducible measurements problematic, and the data
Scheme 1. Synthesis of Compounds 6−24 (Top) and
Structures of R Groups (Bottom)
Table 2. Antiproliferative Activity of the
Pyrazolopyrimidinones against the Asexual Blood Stage of
Plasmodium falciparum 3D7 Strain
compound IC50 (μM)
1, zaprinast 35 ± 4.2a
6 (BIPPO) 0.40 ± 0.14b
7 0.64 ± 0.21
8 0.73 ± 0.30
9 0.74 ± 0.23
10 1.2 ± 0.24
11 2.4 ± 0.6
12 2.9 ± 0.6
13 3.6 ± 0.85
14 4.7 ± 1.9
15 7.8c
16 5.6
17 10
18 11.5 ± 3.7
19 25
20 35 ± 6.7
21 40
22 >100
23 >100
24 70
a
Reference 13. b
IC50 ± SEM (n = 3 or 4). c
IC50 (n = 2).
Figure 3. PDE inhibitors preventing T. gondii growth. Parasites
incubated with confluent HFF cells for 7 days with indicated
concentrations (μM) of PDE inhibitors. Each zone of clearance
(white plaques) represents one tachyzoite undergoing repeated rounds
of invasion, replication, and egress. T. gondii is more sensitive to new
PDE inhibitors than to zaprinast with plaques prevented at
concentrations upward of 55 μM of PDE inhibitors tested. Scale bar
= 5 mm.
ACS Chemical Biology Articles
DOI: 10.1021/cb501004q
ACS Chem. Biol. XXXX, XXX, XXX−XXX
D

5. will not be presented here. BIPPO was consequently also
screened against recombinant hPDE isoforms and confirmed
potent hPDE9 activity (IC50 = 30 nM), with significant
inhibition at 1 μM against hPDE1 (42%), hPDE5 (66%), and
hPDE6 (64%). Attempts to directly measure the increase in
levels of these cyclic nucleotides in cultured blood stage P.
falciparum, using commercial microplate kits, were unsuccessful
since nucleotide levels were too low to reliably measure (data
not shown).
BIPPO Potently Activates Parasite Egress in P.
falciparum and T. gondii. We assessed the activity of
BIPPO in comparison to zaprinast in a number of cyclic
nucleotide dependent functional assays in both P. falciparum
and T. gondii. To quantitatively measure egress in P. falciparum
cultures, we created an egress reporter parasite line by
transfecting the 3D7 strain with a highly active luciferase called
NanoLuc fused to a signal peptide sequence at its N-
terminus.22,23
The signal peptide promotes secretion of
NanoLuc into the parasitophorous vacuole (PV) space that
surrounds the intracellular parasite. Just prior to egress, the
parasite breaks down the PV membrane and the plasma
membrane of the erythrocyte host, and the extent to which this
occurs can be ascertained by measuring the levels of luciferase
released into the supernatant. After 20 min of incubation,
BIPPO induced greater egress at substantially lower concen-
trations than zaprinast and induced a 2.5 fold higher egress
peak at saturating levels of the compounds (Figure 4a).
To determine if the egress response seen in P. falciparum due
to PDE inhibition was also seen in T. gondii, we monitored
egress response to a titration of our panel of compounds and
compared their effectiveness to zaprinast. Egress was measured
as a function of host cell lactate dehydrogenase (LDH) release,
which occurs due to damage created by egressing tachyzoites.2
In comparison to zaprinast, T. gondii egress was more sensitive
to five compounds (BIPPO, 7, 8, 10, and 12, Scheme 1 and
Table 2); one compound (18) showed a similar response to
zaprinast (1), and one compound was ineffective at inducing
tachyzoite egress (15; Figure 4b−h). The data largely correlates
with the activity shown by the compounds in the T. gondii
proliferation (plaque) assay (Figure 3).
To understand further the effect that PDE inhibition has on
parasite egress, we undertook live cell imaging of both P.
falciparum and T. gondii. Zaprinast has been recently shown to
induce premature egress in asexual, late stage blood cultures of
P. falciparum with an EC50 of 25 μM4
and to also activate
tachyzoite egress from host cells in T. gondii.2
To confirm that
BIPPO was actually inducing the breakdown of the PV
membrane and the plasma membrane of infected erythrocytes,
time-lapse imaging was performed on late stage parasites. Over
a 30 min period, 2 μM BIPPO appeared to induce the
breakdown of the erythrocyte and vacuole membranes around
fully formed merozoites, but they could not invade the
surrounding erythrocytes presumably because either they
were not developmentally mature or because BIPPO also
inhibited the invasion process (Figure 5A, left; Movie 1).
Immature parasite forms undergoing cytokinesis were also
released, highlighting the fact that BIPPO was accelerating
breakdown of the host before the merozoites were mature,
presumably through an increase in cGMP levels (Figure 5a,
right; Movie 1). Zaprinast induced egress was not undertaken
since it has been performed previously.4
To investigate the response of T. gondii to these new
inhibitors, BIPPO and zaprinast were compared for egress
induction via live microscopy (Figure 5B and Movie 2).
Tachyzoites were allowed to invade and replicate for 30 h to
produce vacuoles with >16 parasites. Tachyzoite egress was
seen rapidly after the addition of the compounds (Cf =55 μM)
Figure 4. BIPPO (compound 6) inducing parasite egress. (a) In P.
falciparum blood stages, BIPPO more efficiently induces egress than
zaprinast through being more active at lower concentration and
achieving higher peak activity after 20 min of incubation. Egress was
quantified by measuring luciferase activity as relative light units (RLU)
in the growth media of parasites induced to breakdown their
enveloping vacuole and erythrocyte membranes, thereby releasing
the luciferase. The data points represent mean ± SD, n = 3. (b−f)
Tachyzoite egress following 5 min treatment with different
concentrations of PDE inhibitors was measured as a function of
lactate dehydrogenase release from host cells, and normalized to
maximal egress. Each plot displays indicated compound treatment
compared with zaprinast. Mean ± S.E.M, n = 3. Plots indicate that
compounds BIPPO (b), C7 (c), C8 (d), C12 (e), and C10 (f)
outperform zaprinast as an egress stimulant, while C18 (g) is
comparable to zaprinast and C15 (h) is ineffective.
ACS Chemical Biology Articles
DOI: 10.1021/cb501004q
ACS Chem. Biol. XXXX, XXX, XXX−XXX
E

6. with BIPPO stimulating egress at 1:30 min faster than zaprinast
(5:30). Compounds 7 and 8 were also tested, and they too
induced egress with rapidity consistent with the compound
curves (Supporting Information Figure s5). It is also worth
noting that T. gondii and P. falciparum undergo intracellular
replication in a different manner. T. gondii undergoes binary
division and is always ready to egress; thus premature egress is
not possible.
PKG Inhibitor Can Block BIPPO Indicating Specificity
for a cGMP-Dependent PDE. In other systems, cGMP can
function by activating ion channels and promoting phosphor-
ylation via PKG. If BIPPO and the other PDE inhibitors exert
their egress-promoting effects in a PKG-dependent processes,
then inhibition of this kinase should block the egress-promoting
effects of BIPPO. Compound 1 (Cmpd1) is a specific inhibitor
of apicomplexan PKG,11
and it has been shown that Cmpd1
blocks the effects of zaprinast in both P. falciparum and T.
gondii.2,4,24
To determine if Cmpd1 could therefore block the
pro-egress effects of BIPPO, the P. falciparum NanoLuc
reporter parasites were incubated ±2.5 μM of Cmpd1 along
with the approximate IC50 levels of BIPPO (0.7 μM) and
zaprinast (40 μM) for 0, 10, 20, and 40 min (Figure 6A). Under
these conditions, Cmpd1 efficiently blocked the egress-
promoting effects of zaprinast and BIPPO, confirming the
latter is also a cGMP PDE inhibitor (Figure 6A).
We performed parallel experiments in T. gondii, which is
susceptible to zaprinast in a Cmpd1-dependent manner.2
After
pretreatment of intracellular tachyzoites with a titration of
Cmpd1, parasites were stimulated to egress using a fixed
concentration of our compounds (BIPPO, 7, 8, and zaprinast)
(Figure 6b−d), while increasing the concentration of Cmpd1.
The most potent PDE inhibitor, BIPPO, required greatest
concentrations of Cmpd1 to inhibit egress than 7, 8, and
zaprinast. Furthermore, as visualized by live video microscopy,
Cmpd1 pretreatment completely ablated the tachyzoite
response to the C7, C8 and BIPPO compounds, showing a
Figure 5. Movie stills showing rapid egress of Plasmodium falciparum and Toxoplasma gondii induced by BIPPO (added at black arrow). (a, left)
Sequential video images showing P. falciparum merozoite egress (red box) beginning 10:30 after the addition of 2 μM BIPPO. (Right) After 15:30,
BIPPO induces breakdown of membranes surrounding the doubly infected erythrocyte before the schizonts have undergone complete cytokinesis.
Time stamp is minutes:seconds. Scale bar = 5 μm. (b) Intracellular T. gondii tachyzoite vacuoles (outlined by dashed white line) were exposed to
either zaprinast (500 μM) or BIPPO (55 μM) at 30 s (arrow). BIPPO-treated T. gondii respond much more rapidly and at a lower concentration
than zaprinast treatment. Scale bar = 20 μm.
ACS Chemical Biology Articles
DOI: 10.1021/cb501004q
ACS Chem. Biol. XXXX, XXX, XXX−XXX
F

7. complete lack of response even after 10 min of stimulation
(Supporting Information Figure s5, Movie 3).
PDE Inhibitors Stimulate the Release of Secretory
Organelles in Toxoplasma. Host cell egress and motility in
apicomplexan parasites relies on the secretion of adhesins and
perforin-like molecules from the microneme organelles, which
is controlled by intracellular calcium and PKG signaling
events.6,11,25
Given that our new PDE inhibitors rapidly
stimulate host cell egress in both T. gondii and P. falciparum,
we hypothesized that this was due to the activation of
microneme secretion. To test this, we applied the inhibitors
to a well-established semiquantitative microneme secretion
assay in T. gondii6,25
(this assay is not well developed for P.
falciparum and hence could not be used here). Here, the
amount of proteolytically cleaved Mic2 (a major Toxoplasma
adhesin) is measured in the supernatant of extracellular
tachyzoites after stimulation and is used as a surrogate for
total microneme release. To do this, we treated extracellular
tachyzoites for 20 min with 500 μM of zaprinast or 55 μM of
BIPPO, 7, and 8. The supernatant (containing any excreted and
cleaved MIC2 in response to stimulation) and pellet fraction
were collected by centrifugation and then fractionated by SDS-
PAGE. Western blot was then used to determine the amount of
MIC2 present in the supernatant. The three PDE inhibitors
induced the secretion of MIC2 into the supernatant and appear
more potent over the given time frame than zaprinast (Figure
7). Overall, this provides further evidence that the new
compounds are much more potent PDE inhibitors than
zaprinast and act, at least in part, to induce parasite egress
through the stimulation of microneme secretion.
BIPPO Enhances PKA/cAMP-Dependent AMA1 Tail
Phosphorylation. Given the restricted expression of PDE
isoforms through the blood stage of the parasite in P.
falciparum, we hypothesized that the target(s) of BIPPO may
also break down cAMP. We have shown previously in P.
falciparum (not yet studied in T. gondii) that the cytoplasmic
tail of the merozoite invasion ligand apical membrane antigen 1
(AMA1) is phosphorylated by the cAMP-dependent protein
kinase A, at serine 610.5
To measure phosphorylation here, an
in vitro reaction was performed on the recombinant GST-
AMA1 tail in the presence of parasite lysate, [γ‑32
P] ATP, and 2
μM cAMP. To determine if BIPPO could block cAMP
hydrolysis, we added 6 μM to a series of in vitro
phosphorylation reactions containing 2, 1, 0.5, 0.25, and 0
μM cAMP (Figure 8 and Supporting Information). Without
BIPPO, the levels of AMA1 tail phosphorylation declined very
rapidly with decreasing cAMP concentrations, suggesting the
nucleotide was being rapidly hydrolyzed. The rate of
phosphorylation decline, however, could be substantially
inhibited by BIPPO, suggesting it acts against a cAMP-
degrading Pf PDE (Figure 8).
Given their greater potency and rapid action, BIPPO and its
analogues have great potential as tools to understand signaling
pathways mediating egress and invasion in Apicomplexan
parasites. Previously, use of calcium ionophores (ionomycin or
A23187) was standard for assessing active or defective
biological responses such as egress, motility, microneme
secretion, and invasion.3,6,25−27
However, this approach is
problematic as calcium ionophores induce a crude, global Ca2+
change in the intracellular environment by indiscriminately
releasing calcium from intracellular stores and allowing Ca2+
to
enter from external sources.28
This completely ablates the
intricacies and importance of calcium dynamics, such as calcium
flux, amplitude, and periodicity. Furthermore, signal pathway
induction by ionophores prohibits analysis of any events
Figure 6. cGMP-specific protein kinase G (PKG) inhibitor compound
1 (Cmpd1) blocking egress-promoting effects of BIPPO and other
putative PDE inhibitors confirming the inhibitors are targeting parasite
cGMP-specific PDEs. (a) Cmpd1 at 2.5 μM was able to block the pro-
egress effects of 0.7 μM BIPPO and 40 μM zaprinast upon NanoLuc
expressing P. falciparum blood stage parasites. Egress was measured via
luciferase present in the supernatant after host cell lysis. RLU was
plotted as a function of time for the various treatments (mean ± SD, n
= 3). (b−d) Inhibition of tachyzoite egress following 20 min
preincubation with different concentrations of Cmpd1 (Ctop 18 μM)
and 5 min treatment with a fixed concentration of PDE inhibitors (Cf
55 μM). Egress was measured as a function of lactate dehydrogenase
release from host cells and normalized to maximal egress. Each plot
displays indicated drug treatment compared with zaprinast. Mean ±
S.E.M., n = 3. Plots demonstrate egress response was inhibited by
Cmpd1 at a comparable rate to zaprinast.
Figure 7. PDE inhibitors stimulate microneme secretion. Extracellular
tachyzoites treated with zaprinast (Cf 500 μM) or PDE inhibitors (Cf
55 μM). Microneme secretion was determined by Western Blot
detection of secreted and cleaved micronemal protein MIC2 (cMic2)
relative to nonsecreted, full length MIC2 (fMIC2). Supn (super-
natant).
ACS Chemical Biology Articles
DOI: 10.1021/cb501004q
ACS Chem. Biol. XXXX, XXX, XXX−XXX
G

8. preceding calcium release. Indeed, cGMP signaling has been
suggested to act before intracellular Ca2+
release and therefore
argues that the use of BIPPO provides a superior alternative to
current stimuli in a more biologically relevant environment.9
The combination of the use of BIPPO, Cmpd1, and Ca2+
ionophore may indeed help tease out the order of events that is
required for Apicomplexan host cell egress and help understand
how a change in extracellular K+
activates egress or how the
engagement of host cell receptors promotes invasion.
The consequences of inducing rapid and premature parasite
egress in an infected host are not absolutely clear, and so the
therapeutic potential of PDE inhibition remains to be
examined. On the other hand, dysregulation of key events in
host cell invasion such as AMA1 phosphorylation, that rely
upon cAMP dependent Pf PKA phosphorylation for efficiency,
may provide one mechanistic pathway to a therapeutically
useful application of PDE inhibition or render parasites more
susceptible to other drugs.
In conclusion, the broad homology between the mammalian
and parasitic PDE active site residues14
suggested that it should
be possible to repurpose hPDE inhibitors for use in studying
parasite signaling. Our approach to using homology modeling
to select the most homologous of the human enzymes for
parasite orthologues led us to develop BIPPO, which is much
more potent than the previously utilized tool compound
zaprinast, at inhibiting growth and inducing premature parasite
egress in a PKG-dependent manner.
While BIPPO shows potent inhibition of Pf PDEα consistent
with its observed cell-based activity, the specific Pf PDE and
TgPDE targets of BIPPO in parasites are still unknown, and the
effect on both cGMP and cAMP pathways suggests they may
emerge as being a single dual specificity PDE, multiple PDEs,
or a cGMP or cAMP-PDE involved in PDE cross-talk, and this
is a key goal of future work. The greater activity of PfPDEα
against cGMP over cAMP reflects the current order in which
we think these nucleotides function; i.e., cGMP stimulates
egress followed by cAMP dependent phosphorylation of
AMA1, which enables invasion. Optimizing the pharmaco-
logical inhibitors of parasite PDE functions may provide novel
compounds for the study of signal transduction processes
governing apicomplexan host cell egress and invasion and
identify new intervention points for therapy in these diseases.
■ METHODS
Plasmodium falciparum Strains and Transfections. P.
falciparum (strain 3D7) asexual blood stage parasites were
cultured as per ref 29 in RPMI-HEPES media supplemented
with L-glutamine (Sigma) and Albumax II (Invitrogen). To
express a luciferase enzyme that could be secreted into the
parasitophorous vacuole of infected red blood cells, DNA
sequence corresponding to 23 amino acid endoplasmic
reticulum signal sequence of merozoite surface protein 1 was
appended onto the 5′ end of the Promega NanoLuc sequence.
3D7 parasites were transfected with the NanoLuc construct by
culturing in erythrocytes that had been electroporated with 100
μg of the DNA30,31
and selected on 5 μg/mL blasticidin S.
Toxoplasma Culture. Toxoplasma was grown in Human
Foreskin Fibroblasts (HFFs) and passaged as required. Here,
HFFs were grown to confluency in DME supplemented with
10% Cosmic Calf Serum (Hyclone) and just before inoculation
with T. gondii media was refreshed and serum levels dropped to
1% fetal calf serum. All culture and assay conditions performed
at 37 °C, 10% CO2.
Plasmodium Growth Assay. Asexual blood stage parasites
12−24 h post infection (hpi) were grown for 72 h in
compounds serially diluted in DMSO (0.2% v/v in culture
media). Parasite growth was assessed by measuring the activity
of parasite lactate dehydrogenase using the Malstat assay.32
Toxoplasma Plaque Assay. Growth was determined by
the ability of tachyzoites to form zones of clearance (plaques)
in host cells through repeated cycles of invasion, replication,
and egress. This was done by adding tachyzoites to confluent
HFFs (Cf 100cells/ml) and treated with indicated concen-
trations of the test compound for the entirety of the assay.
Plates were left undisturbed for 7 days at 37 °C, fixed with 70%
methanol, stained with Crystal Violet, and imaged.
Plasmodium Egress Assays. 3D7_NanoLuc parasites23
were cultured until 44 h postinvasion at 5% parasitemia. The
culture was washed in RPMI medium to remove any NanoLuc
from the supernatant and resuspended at 2% hematocrit in
RPMI medium. Cmpd1 was added to half of the culture at 2.5
μM and 0.2 μL of zaprinast or BIPPO dilutions in DMSO were
added to 100 μL of Cmpd1-treated and -untreated culture in
duplicate. Samples were allowed to warm to 37 °C for 5 min
and then placed on ice after a 0, 10, 20, or 40 min incubation
period. Cells were pelleted at 3000g for 5 min; then 10 μL of
culture supernatant was mixed with 10 μL of NanoLuc assay
buffer (2× Promega cell lysis buffer and 1 μL Nano Glo
substrate per mL) in a luminometer plate, and the activity was
measured with a FluoStar Optima instrument (BMG Labtech).
Figure 8. BIPPO blocks the decline of cAMP-dependent phosphor-
ylation by inhibiting degradation of cAMP by PDEs in P. falciparum
extracts. (a) Diagram of recombinant GST-AMA1S610only tail fusion
protein that has had all its known phosphorylation sites mutated to
alanine (red) except for the S610 PKA site (blue). (b) An
autoradiograph of [γ32
P] labeled GST-AMA1S610only tail proteins that
have been phosphorylated by Pf PKA in lysates from P. falciparum
blood stage parasites. At lower levels of cAMP, phosphorylation
declines rapidly, presumably due to degradation by native PDEs, which
could be reversed by BIPPO. (c) Densitometry plots as a function of
cAMP concentration (mean ± SD, n = 3).
ACS Chemical Biology Articles
DOI: 10.1021/cb501004q
ACS Chem. Biol. XXXX, XXX, XXX−XXX
H

9. Relative light units (RLU) were plotted as a function of
inhibitor concentration or time in Prism (GraphPad), using
nonlinear regression analysis as a sigmoidal dose−response
curve with variable slope. Samples were assayed in triplicate.
Live cell imaging of P. falciparum parasites was performed as
per ref 33 after the addition of 2 μM BIPPO.
Toxoplasma LDH Egress Assay. Egress was determined as
a function of lactate dehydrogenase (LDH) released from the
host cell as parasites egress.2
Tachyzoites (Cf 5 × 105
cells/ml)
were added to confluent HFFs in a 96 well plate format and
grown for 32 h. Wells were washed once with Ringers/5%FCS
before 1:3 titration of PDE inhibitors and zaprinast for 5 min at
37 °C (Ctop 500 μM). For Cmpd1 inhibition, cells were
pretreated with a titration of Cmpd1 (Ctop 18 μM) for 20 min
at 37 °C; then stimulated with PDE inhibitors (55 μM) or
zaprinast (500 μM) for 5 min at 37 °C. Supernatants were
taken and LDH detected using Promega CytoTox LDH assay
kit according to manufacturer’s instructions. Data were
normalized to 100% egress response in each assay. IC50 and
EC50 were determined using Prism (GraphPad) plotting
normalized, log transformed (x axis), nonlinear regression
analysis as a sigmoidal dose−response curve with variable slope.
Samples were assayed in triplicate for each assay, n = 3.
Treatments did not induced significant LDH release in the
absence of parasites (data not shown).
Toxoplasma Live Egress. Tachyzoites were added to
confluent HFFs in imaging chambers at 1 × 104
cells/mL and
grown for 30 h. In relevant instances, cells were pretreated with
2 μM Cmpd1 for 20 min at 37 °C. Wells were rinsed, and
media was replaced with Ringers/5%FCS. Cells were imaged
using a heated chamber at 37 °C with PDE inhibitors (55 μM)
and zaprinast (500 μM) added at 00:30 time-points. Images
were recorded for 10 min.
Toxoplasma Microneme Secretion Assay. Fresh extrac-
ellular cells (2 × 108
cells/mL) were treated for 20 min at 37
°C with PDE inhibitors (Cf 55 μM) and zaprinast (Cf 500 μM)
in 3%FCS DME. Microneme secretion was detected using
Western blot, probing for micronemal protein MIC2.34
■ ASSOCIATED CONTENT
*S Supporting Information
Movie 1: Treatment of Plasmodium falciparum blood stage
parasites with 2 μM BIPPO results in premature egress. Movie
2: Treatment of Toxoplasma gondii infected human fibroblasts
with 55 μM BIPPO triggers rapid parasite egress from their
host cells. Movie 3: Pretreatment of Toxoplasma gondii infected
human fibroblasts with 2 μM Cmpd1 blocks the egress
normally triggered by BIPPO. This material is available free of
charge via the Internet at http://pubs.acs.org.
■ AUTHOR INFORMATION
Corresponding Authors
*E-mail: Philip.Thompson@monash.edu.
*E-mail: tonkin@wehi.edu.au.
*E-mail: gilson@burnet.edu.au.
Author Contributions
#
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
B.L.H., K.L.H., and R.J.S. are recipients of Australian
Postgraduate Awards, and C.J.T is recipient of an Australian
Future Fellowship (FT1200100164). This work was supported
by NHMRC Project grants 1025598 and 603720 as well as the
Victorian State Government Operational Infrastructure Support
Program and NHMRC IRIISS. We are grateful to the
Australian Red Cross for the supply of red blood cells. We
also thank K. Rogers for help with microscopy and T. Luc for
technical assistance.
■ REFERENCES
(1) World_Health_Organisation. (2013) World Malaria Report.
(2) Lourido, S., Tang, K., and Sibley, L. D. (2012) Distinct signalling
pathways control Toxoplasma egress and host-cell invasion. EMBO J.
31, 4524−4534.
(3) Billker, O., Lourido, S., and Sibley, L. D. (2009) Calcium-
dependent signaling and kinases in apicomplexan parasites. Cell Host
Microbe 5, 612−622.
(4) Collins, C. R., Hackett, F., Strath, M., Penzo, M., Withers-
Martinez, C., Baker, D. A., and Blackman, M. J. (2013) Malaria parasite
cGMP-dependent protein kinase regulates blood stage merozoite
secretory organelle discharge and egress. PLoS Pathog. 9, e1003344.
(5) Leykauf, K., Treeck, M., Gilson, P. R., Nebl, T., Braulke, T.,
Cowman, A. F., Gilberger, T. W., and Crabb, B. S. (2010) Protein
kinase a dependent phosphorylation of apical membrane antigen 1
plays an important role in erythrocyte invasion by the malaria parasite.
PLoS Pathog. 6, e1000941.
(6) Lourido, S., Shuman, J., Zhang, C., Shokat, K. M., Hui, R., and
Sibley, L. D. (2010) Calcium-dependent protein kinase 1 is an
essential regulator of exocytosis in Toxoplasma. Nature 465, 359−362.
(7) Dvorin, J. D., Martyn, D. C., Patel, S. D., Grimley, J. S., Collins, C.
R., Hopp, C. S., Bright, A. T., Westenberger, S., Winzeler, E.,
Blackman, M. J., Baker, D. A., Wandless, T. J., and Duraisingh, M. T.
(2010) A plant-like kinase in Plasmodium falciparum regulates parasite
egress from erythrocytes. Science 328, 910−912.
(8) McRobert, L., Taylor, C. J., Deng, W., Fivelman, Q. L.,
Cummings, R. M., Polley, S. D., Billker, O., and Baker, D. A. (2008)
Gametogenesis in malaria parasites is mediated by the cGMP-
dependent protein kinase. PLoS Biol. 6, e139.
(9) Brochet, M., Collins, M. O., Smith, T. K., Thompson, E.,
Sebastian, S., Volkmann, K., Schwach, F., Chappell, L., Gomes, A. R.,
Berriman, M., Rayner, J. C., Baker, D. A., Choudhary, J., and Billker, O.
(2014) Phosphoinositide metabolism links cGMP-dependent protein
kinase G to essential Ca(2)(+) signals at key decision points in the life
cycle of malaria parasites. PLoS Biol. 12, e1001806.
(10) Wiersma, H. I., Galuska, S. E., Tomley, F. M., Sibley, L. D.,
Liberator, P. A., and Donald, R. G. (2004) A role for coccidian cGMP-
dependent protein kinase in motility and invasion. Int. J. Parasitol. 34,
369−380.
(11) Donald, R. G., Allocco, J., Singh, S. B., Nare, B., Salowe, S. P.,
Wiltsie, J., and Liberator, P. A. (2002) Toxoplasma gondii cyclic GMP-
dependent kinase: chemotherapeutic targeting of an essential parasite
protein kinase. Eukaryotic Cell 1, 317−328.
(12) Francis, S. H., Blount, M. A., and Corbin, J. D. (2011)
Mammalian cyclic nucleotide phosphodiesterases: molecular mecha-
nisms and physiological functions. Physiol. Rev. 91, 651−690.
(13) Yuasa, K., Mi-Ichi, F., Kobayashi, T., Yamanouchi, M., Kotera, J.,
Kita, K., and Omori, K. (2005) PfPDE1, a novel cGMP-specific
phosphodiesterase from the human malaria parasite Plasmodium
falciparum. Biochem. J. 392, 221−229.
(14) Wentzinger, L., Bopp, S., Tenor, H., Klar, J., Brun, R., Beck, H.
P., and Seebeck, T. (2008) Cyclic nucleotide-specific phosphodies-
terases of Plasmodium falciparum: PfPDEalpha, a non-essential
cGMP-specific PDE that is an integral membrane protein. Int. J.
Parasitol. 38, 1625−1637.
ACS Chemical Biology Articles
DOI: 10.1021/cb501004q
ACS Chem. Biol. XXXX, XXX, XXX−XXX
I

10. (15) Yuan, J., Johnson, R. L., Huang, R. L., Wichterman, J., Jiang, H.
Y., Hayton, K., Fidock, D. A., Wellems, T. E., Inglese, J., Austin, C. P.,
and Su, X. Z. (2009) Genetic mapping of targets mediating differential
chemical phenotypes in Plasmodium falciparum. Nat. Chem. Biol. 5,
765−771.
(16) Beghyn, T. B., Charton, J., Leroux, F., Laconde, G., Bourin, A.,
Cos, P., Maes, L., and Deprez, B. (2011) Drug to Genome to Drug:
Discovery of New Antiplasmodial Compounds. J. Med. Chem. 54,
3222−3240.
(17) Howard, B. L., Thompson, P. E., and Manallack, D. T. (2011)
Active site similarity between human and Plasmodium falciparum
phosphodiesterases: considerations for antimalarial drug design. J.
Comput. Aided Mol. Des 25, 753−762.
(18) Taylor, C. J., McRobert, L., and Baker, D. A. (2008) Disruption
of a Plasmodium falciparum cyclic nucleotide phosphodiesterase gene
causes aberrant gametogenesis. Mol. Microbiol. 69, 110−118.
(19) Deninno, M. P., Andrews, M., Bell, A. S., Chen, Y., Eller-Zarbo,
C., Eshelby, N., Etienne, J. B., Moore, D. E., Palmer, M. J., Visser, M.
S., Yu, L. J., Zavadoski, W. J., and Michael Gibbs, E. (2009) The
discovery of potent, selective, and orally bioavailable PDE9 inhibitors
as potential hypoglycemic agents. Bioorg. Med. Chem. Lett. 19, 2537−
2541.
(20) Verhoest, P. R., Proulx-Lafrance, C., Corman, M., Chenard, L.,
Helal, C. J., Hou, X., Kleiman, R., Liu, S., Marr, E., Menniti, F. S.,
Schmidt, C. J., Vanase-Frawley, M., Schmidt, A. W., Williams, R. D.,
Nelson, F. R., Fonseca, K. R., and Liras, S. (2009) Identification of a
brain penetrant PDE9A inhibitor utilizing prospective design and
chemical enablement as a rapid lead optimization strategy. J. Med.
Chem. 52, 7946−7949.
(21) Wang, C., Ashton, T. D., Gustafson, A., Bland, N. D., Ochiana,
S. O., Campbell, R. K., and Pollastri, M. P. (2012) Synthesis and
evaluation of human phosphodiesterases (PDE) 5 inhibitor analogs as
trypanosomal PDE inhibitors. Part 1. Sildenafil analogs. Bioorg. Med.
Chem. Letters 22, 2579−2581.
(22) Hall, M. P., Unch, J., Binkowski, B. F., Valley, M. P., Butler, B.
L., Wood, M. G., Otto, P., Zimmerman, K., Vidugiris, G., Machleidt,
T., Robers, M. B., Benink, H. A., Eggers, C. T., Slater, M. R.,
Meisenheimer, P. L., Klaubert, D. H., Fan, F., Encell, L. P., and Wood,
K. V. (2012) Engineered luciferase reporter from a deep sea shrimp
utilizing a novel imidazopyrazinone substrate. ACS Chem. Biol. 7,
1848−1857.
(23) Azevedo, M. F., Nie, C. Q., Elsworth, B., Charnaud, S. C.,
Sanders, P. R., Crabb, B. S., and Gilson, P. R. (2014) Plasmodium
falciparum transfected with ultra bright NanoLuc luciferase offers high
sensitivity detection for the screening of growth and cellular trafficking
inhibitors. PLoS One 9, e112571.
(24) Diaz, C. A., Allocco, J., Powles, M. A., Yeung, L., Donald, R. G.,
Anderson, J. W., and Liberator, P. A. (2006) Characterization of
Plasmodium falciparum cGMP-dependent protein kinase (PfPKG):
antiparasitic activity of a PKG inhibitor. Mol. Biochem. Parasitol. 146,
78−88.
(25) Carruthers, V. B., and Sibley, L. D. (1999) Mobilization of
intracellular calcium stimulates microneme discharge in Toxoplasma
gondii. Mol. Microbiol. 31, 421−428.
(26) McCoy, J. M., Whitehead, L., van Dooren, G. G., and Tonkin, C.
J. (2012) TgCDPK3 regulates calcium-dependent egress of
Toxoplasma gondii from host cells. PLoS Pathog. 8, e1003066.
(27) Nebl, T., Prieto, J. H., Kapp, E., Smith, B. J., Williams, M. J.,
Yates, J. R., 3rd, Cowman, A. F., and Tonkin, C. J. (2011) Quantitative
in vivo analyses reveal calcium-dependent phosphorylation sites and
identifies a novel component of the Toxoplasma invasion motor
complex. PLoS Pathog. 7, e1002222.
(28) Pressman, B. C. (1973) Properties of ionophores with broad
range cation selectivity. Fed Proc. 32, 1698−1703.
(29) Trager, W., and Jensen, J. B. (1976) Human malaria parasites in
continuous culture. Science 193, 673−675.
(30) Deitsch, K. W., Driskill, C. L., and Wellems, T. E. (2001)
Transformation of malaria parasites by the spontaneous uptake and
expression of DNA from human erythrocytes. Nucleic Acids Res. 29,
850−853.
(31) Hasenkamp, S., Russell, K., and Horrocks, P. (2012)
Comparison of the absolute and relative efficiencies of electro-
poration-based transfection protocols for Plasmodium falciparum.
Malaria J. 11, 210.
(32) Makler, M. T., and Hinrichs, D. J. (1993) Measurement of the
lactate dehydrogenase activity of Plasmodium falciparum as an
assessment of parasitemia. Am. J. Trop Med. Hyg. 48, 205−210.
(33) Gilson, P. R., and Crabb, B. S. (2009) Morphology and kinetics
of the three distinct phases of red blood cell invasion by Plasmodium
falciparum merozoites. Int. J. Parasitol. 39, 91−96.
(34) Wan, K. L., Carruthers, V. B., Sibley, L. D., and Ajioka, J. W.
(1997) Molecular characterisation of an expressed sequence tag locus
of Toxoplasma gondii encoding the micronemal protein MIC2. Mol.
Biochem. Parasitol. 84, 203−214.
(35) Sung, B. J., Hwang, K. Y., Jeon, Y. H., Lee, J. I., Heo, Y. S., Kim,
J. H., Moon, J., Yoon, J. M., Hyun, Y. L., Kim, E., Eum, S. J., Park, S. Y.,
Lee, J. O., Lee, T. G., Ro, S., and Cho, J. M. (2003) Structure of the
catalytic domain of human phosphodiesterase 5 with bound drug
molecules. Nature 425, 98−102.
ACS Chemical Biology Articles
DOI: 10.1021/cb501004q
ACS Chem. Biol. XXXX, XXX, XXX−XXX
J