Antiplasmodial Mode of Action of Pantothenamides: Pantothenate Kinase Serves as a Metabolic Activator Not as a Target

Article abstract of DOI:10.1021/acsinfecdis.7b00024

N-Substituted pantothenamides (PanAms) are pantothenate analogues with up to nanomolar potency against blood-stage Plasmodium falciparum (the most virulent species responsible for malaria). Although these compounds are known to target coenzyme A (CoA) bio

Full text of DOI:10.1021/acsinfecdis.7b00024

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Article
Antiplasmodial mode of action of pantothenamides: pantothenate
kinase serves as a metabolic activator, not as a target
Marianne de Villiers, Christina Spry, Cristiano Joao Macuamule,
Leanne Barnard, Gordon Wells, Kevin J Saliba, and Erick Strauss
ACS Infect. Dis., Just Accepted Manuscript • Publication Date (Web): 24 Apr 2017
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Antiplasmodial mode of action of pantothenamides:
pantothenate kinase serves as a metabolic activator, not
as a target
†
‡
†
†
Marianne de Villiers *, Christina Spry , Cristiano J. Macuamule , Leanne Barnard , Gordon
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†
‡,§
†
Wells , Kevin J. Saliba and Erick Strauss *
†
Department of Biochemistry, Stellenbosch University, Stellenbosch, 7600, South Africa.
‡
§
Research School of Biology, and Medical School, College of Medicine, Biology and Environment, The
Australian National University, Canberra, Australian Capital Territory, 2601, Australia.
*To whom correspondence should be addressed: mdevilliers@sun.ac.za; estrauss@sun.ac.za
De Villiers et al.
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ABSTRACT
Nꢀsubstituted pantothenamides (PanAms) are pantothenate analogues with up to nanomolar
potency against bloodꢀstage Plasmodium falciparum (the most virulent species responsible for
malaria). Although these compounds are known to target coenzyme A (CoA) biosynthesis and/or
utilization, their exact mode of action (MoA) is still unknown. Importantly, PanAms that retain
the natural βꢀalanine moiety are more potent than other variants, consistent with the involvement
of processes that are selective for pantothenate (the precursor of CoA) or its derivatives. The
transport of pantothenate and its phosphorylation by P. falciparum pantothenate kinase (PfPanK,
the first enzyme of CoA biosynthesis) are two such processes previously highlighted as potential
targets for the PanAms’ antiplasmodial action. In this study, we investigated the effect of
PanAms on these processes using their radiolabeled versions (synthesized here for the first time),
which made possible the direct measurement of PanAm uptake by isolated bloodꢀstage parasites,
and PanAm phosphorylation by PfPanK present in parasites lysates. We found that the MoA of
PanAms does not involve interference with pantothenate transport, and that inhibition of PfPanKꢀ
mediated pantothenate phosphorylation does not correlate with PanAm antiplasmodial activity.
Instead, PanAms that retain the βꢀalanine moiety were found to be metabolically activated by
PfPanK in a selective manner, forming phosphorylated products that likely inhibit other steps in
CoA biosynthesis, or are transformed into CoA antimetabolites that can interfere with CoA
utilization. These findings provide direction for the ongoing development of CoAꢀtargeted
inhibitors as antiplasmodial agents with clinical potential.
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Keywords
Coenzyme A biosynthesis, antimicrobial, antimetabolite, pantothenamide, pantothenate uptake.
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INTRODUCTION
N-substituted pantothenamides (PanAms) are analogues of pantothenate (Pan), the
precursor of the essential metabolic cofactor coenzyme A (CoA) (Figure 1a). These compounds
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inhibit the proliferation of bloodꢀstage Plasmodium falciparum, the parasite responsible for
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most of the deaths attributed to malaria. In fact, the most potent antiplasmodial member of the
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class identified to date—Nꢀphenethylꢀpantothenamide (N-PEꢀPanAm)—has a potency
comparable to that of chloroquine (IC , i.e. the concentration that gives halfꢀmaximal inhibition,
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of ~20 nM). However, PanAms are sensitive to degradation by pantetheinase, a serum enzyme
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that belongs to the Vanin protein family, and their potency is therefore considerably reduced
4,9
unless this enzyme is inactivated. We previously demonstrated that exchanging the βꢀalanine
moiety of the PanAm structure with either glycine or γꢀaminobutyric acid (yielding αꢀPanAms
and HoPanAms respectively; the “normal”, unmodified structures are referred to as nꢀPanAms)
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reduces degradation significantly (Figure 1b). However, this change also reduces the potency of
the best of the molecules, suggesting that the βꢀalanine moiety is required for the PanAms’ mode
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of action (MoA).
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,4ꢀ5
The finding that addition of Pan reverses the antiplasmodial effect of the PanAms,
indicates that these compounds target processes involved in Pan utilization (i.e. its uptake and
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biosynthetic conversion to CoA) and/or processes that require CoA or its thioesters. However,
the exact antiplasmodial MoA of the PanAms remains unknown. Four possible MoAs have been
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proposed, as shown in Figure 1a. Among these possibilities, PfPanK has long been considered
a strong candidate for the target of PanAmꢀmediated antiplasmodial activity. Indeed, Pan is the
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only vitamin that is required for survival of bloodꢀstage P. falciparum (thiamine becomes
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essential only after extensive depletion from erythrocytes ), and its phosphorylation by PfPanK
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allows the parasite to compete for the vitamin with its human host. However, the activity of
PfPanK has only been studied in lysates (due to a failure to date to express the enzyme
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heterologously ) using an assay that tracks the phosphorylation of radiolabeled substrates.
These reports indicated that PfPanK has a submicromolar K for Pan, and that its ability to
M
phosphorylate Pan is potently inhibited by certain PanAms (and other Pan analogues), especially
those with hydrophobic substituents. However, the relevance of these data to the MoA of the
PanAms has thus far remained unclear.
In contrast, the importance of PanK to the MoA of the PanAms in bacteria has been
21ꢀ25
explored in more detail,
partly because prokaryotes have been shown to have three different
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types of PanK. These types differ in regards to structure, activity profiles and the level of
substrate specificity, providing an ideal tool for the study of PanK’s involvement in PanAm
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inhibition in these organisms. The studies demonstrated that in organisms (such as Escherichia
coli) that have nonꢀselective type I PanKs, the enzyme acts as a metabolic activator that initiates
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the transformation of the PanAms (including its αꢀPanAm and HoPanAm variants ) into CoA
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antimetabolites,
lowering CoA levels and inhibiting CoAꢀdependent processes
—
including fatty acid biosynthesis, which is dependent on CoA for activation of the apoꢀacyl
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carrier protein (ACP) of the type II fatty acid synthase to its active holoꢀform. In contrast,
organisms that have highly selective type III PanKs are completely resistant to inhibition by the
PanAms, as these enzymes do not recognize them as substrates and therefore they are not
metabolically activated. However, between these extremes is at least one example—
Staphylococcus aureus—in which PanAmꢀmediated growth inhibition occurs at least partially
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through direct inhibition of the target organism’s PanK. The S. aureus PanK (SaPanK) is an
atypical type II PanK that shows high specificity for Pan and the nꢀPanAms, accepting the latter
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as substrates. Recently, it was shown that the phosphorylated products get trapped on the
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enzyme, leading to its inhibition in vitro. Due to the enzyme’s specificity, the αꢀPanAms and
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HoPanAms do not inhibit SaPanK and show no inhibition of S. aureus growth.
Taken together, these previous studies indicate that to gain a better understanding of
PfPanK’s role in PanAm inhibition of P. falciparum—including the requirement of the βꢀalanine
moiety for the maintenance of these molecules’ potency—a more inꢀdepth study of the kinetics
and specificity of the parasite enzyme is required. Furthermore, since P. falciparum is highly
dependent on Pan, the selective inhibition of the uptake of the vitamin by the parasite’s Pan
transporter presents an additional potential vulnerability that could also introduce the observed
structural selectivity filter. Yet no studies have been performed on the susceptibility of Pan
uptake to inhibition by the PanAms to date.
In this study we set out to investigate the basis for the PanAms’ specificity profile in P.
falciparum, and shed light on their antiplasmodial MoA. This was achieved through the use of
various PanAm variants and their analogues (including radiolabeled versions) in uptake studies
and in phosphorylation assays using parasite lysates. Our results show that the PanAm MoA does
not depend on the inhibition of Pan uptake or PfPanK activity. Instead, PfPanK acts as a highly
specific metabolic activator of the PanAms. This study provides clear direction for the future
development of different variants of the PanAms as antiplasmodial agents based on a suggested
MoA that depends on the inhibition of a target beyond PanK.
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Figure 1. a) Biosynthesis of CoA in P. falciparum. Uptake of pantothenate (Pan) is followed by its
biosynthetic conversion to CoA in a fiveꢀstep pathway catalyzed by pantothenate kinase (PanK),
phosphopantothenoylcysteine synthetase (PPCS), phosphopantothenoylcysteine decarboxylase (PPCDC),
phosphopantetheine adenylyltransferase (PPAT), and finally dephosphoꢀCoA kinase (DPCK), that
produce
4'ꢀphosphopantothenate
(PꢀPan),
4'ꢀphosphopantothenoylcysteine
(PꢀPanCys),
4'ꢀ
phosphopantetheine (PꢀPantSH), 3'ꢀdephosphoꢀCoA (dPCoA) and CoA, respectively. The accumulation of
Pan can be represented by the combined effects of the transport of the vitamin into the parasite and its
metabolism by the CoA pathway starting with PanKꢀmediated phosphorylation. This ultimately leads to
trapping of Pan within the parasite. Pantothenamides (shown as PanAm–R in the figure, with R denoting
the amide substituent) can also be taken up by the parasite to exert their effect on CoA biosynthesis and/or
utilization. PanAms are proposed to exert an antiplasmodial effect via one of four possible processes, or a
combination thereof: 1) by inhibiting Pan transport, 2) by inhibiting PanKꢀmediated Pan phosphorylation,
3) by lowering CoA levels due to formation of CoA antimetabolites (antiꢀCoA) rather than CoA, and 4) by
CoA antimetabolites interfering with CoAꢀdependent processes. b) PanAms that contain a βꢀalanine
moiety (i.e. nꢀPanAms, middle) are susceptible to degradation by pantetheinase. Modification of the βꢀ
alanine moiety that displaces the scissile bond produces the αꢀPanAm (left) or HoPanAm (right) variants
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that are resistant to pantetheinase.
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RESULTS AND DISCUSSION
Investigating the impact of PanAm variants on Pan accumulation in isolated P. falciparum
parasites. We first determined whether the PanAms have an impact on the accumulation of Pan
(i.e. the combination of transport of extracellular Pan across the parasite plasma membrane and
its metabolism by the enzymes of the CoA biosynthesis pathway, see Figure 1a) in intact P.
falciparum parasites. This was achieved by measuring the uptake of radiolabeled Pan by isolated
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parasites in the presence of two different n-PanAms—Nꢀpentyl pantothenamide (N5ꢀPan) and
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NꢀPEꢀPanAm—that showed good antiplasmodial activity in a previous study. The αꢀPanAm and
HoPanAm counterparts of these compounds were also tested to determine if the observed
selectivity for the βꢀalanine moiety of the nꢀPanAms could be related to the accumulation
process.
Intact trophozoiteꢀstage P. falciparum parasites were isolated by saponinꢀmediated
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permeabilization of the erythrocytic membranes and accumulation of [ C]Pan was measured in
the presence of increasing concentrations of the various PanAms after 20 minutes (i.e. within the
linear phase of accumulation). The accumulation was found to be inhibited by the PanAms in a
doseꢀdependent manner (Figure 2a and b). The IC50 values calculated from the corresponding
dose response curves indicated that while all the PanAms tested showed inhibition, the nꢀPanAm
and HoPan variants (with either of the tested substituents) are more potent than their αꢀPanAm
counterparts (Table 1, left). This finding correlates with the antiplasmodial potency of these
compounds, suggesting that the observed selectivity is based on one of the processes involved in
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accumulation (Figure 2c). In addition, we found that the [ C]Pan distribution ratio (i.e. the ratio
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of [ C]Pan inside the cell to that in the extracellular solution) reached a value of approximately
one as the PanAm concentrations were increased. This value indicates that in the presence of the
PanAms the compound continues to equilibrate across the parasite membrane but that little to no
metabolism takes place. These results are consistent with the PanAms having a negative impact
on the metabolism, rather than the uptake, of the vitamin (Figure 2a and b).
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Figure 2. Effect of αꢀ, nꢀ and HoPanAms (N5 series in panel a and NꢀPE series in panel b, respectively)
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on [ C]Pan accumulation (the combined effect of transport and metabolism) as measured by saponinꢀ
isolated P. falciparum trophozoites after 20 minutes. Accumulation decreases with increasing
concentrations of the PanAms and reaches a distribution ratio of approximately one (indicated by a black
dashed line). Accumulation data are an average of two separate experiments, each performed in duplicate.
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All error bars denote range/2. c) Correlation of the potency of inhibition of [ C]Pan accumulation in
isolated P. falciparum trophozoites by PanAms and the antiplasmodial potency obtained for PanAms
against cultured bloodꢀstage P. falciparum parasites. Antiplasmodial IC50 values were obtained from
4
ꢀ5
previous studies. Error bars denote SEM or range/2 as appropriate for the particular data set.
Table 1. Inhibition of the accumulation of radiolabeled Pan or PanAms in isolated P.
falciparum parasites by non-labeled substrates.
Inhibition of accumulation of
Inhibition of accumulation of
indicated [ C]PanAms by Pan
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[
C]Pan by indicated PanAms
a
a
Non-labeled
IC50 (µM)
Radiolabeled
substrate variant
IC50 (µM)
N6 series N-Bn series
0.87 ± 0.01 0.64 ± 0.09
inhibitor variant
N5 series
N-PE series
αꢀPanAm
nꢀPanAm
HoPanAm
27 ± 1
16 ± 8
αꢀPanAm
nꢀPanAm
HoPanAm
1.1 ± 0.8
1.7 ± 0.1
0.11 ± 0.06
0.76 ± 0.07
44 ± 21
18 ± 2
6.8 ± 2.6
7.0 ± 0.7
a
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The IC50 of PanAmꢀmediated inhibition of [ C]Pan accumulation (on the left) and Panꢀmediated inhibition of
1
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[
C]PanAm accumulation (on the right) in intact isolated trophozoiteꢀstage parasites after a 20 minute incubation at
7 ºC. The reported values represent the averages of two independent experiments each performed in duplicate; the
3
errors indicate the range/2.
Synthesis of two sets of radiolabeled PanAms provides suitable tool and control compounds
for studying their uptake and metabolism in P. falciparum. More detailed investigations into the
uptake and metabolism of PanAms by saponinꢀisolated parasites required the synthesis of
radiolabeled PanAms. Consequently, we performed the first synthesis of radiolabeled PanAms
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described to date by modification of a previously developed method.
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Two sets of PanAm variants—one based on N6ꢀPanAm that shows good antiplasmodial
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activity , and another based on NꢀBnꢀPanAm that is inactive —were prepared for this study using
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[ C]hexylamine and [ C]benzylamine, two radiolabeled amines that are commercially available.
We included the inactive compound specifically to act as control, i.e. with the expectation that
they should show behavior distinct from their antiplasmodial counterparts. With the compounds
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in hand, we turned to performing accumulation experiments as described for [ C]Pan above.
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The accumulation of PanAms in isolated P. falciparum parasites points to an effect related to
metabolism, not uptake. The accumulation of the synthesized radiolabeled PanAm variants by
saponinꢀisolated trophozoiteꢀstage parasites was monitored over time and compared to
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accumulation of [ C]Pan (Figure 3a). The PanAms were accumulated to higher concentrations
inside the parasites than in the extracellular medium (i.e. distribution ratio > 1), consistent with
the PanAms not just being taken up by the parasite, but also metabolised (Figure 3b and c).
Importantly, no correlation was found between the accumulation profiles of N6ꢀPanAm and
BnꢀPanAm and their antiplasmodial activities.
Nꢀ
Interestingly,
NꢀBnꢀHoPanAm was accumulated to a much higher distribution ratio than
its
n
ꢀPanAm and ꢀPanAm counterparts and that of the N6ꢀseries; however, this increase does
α
not correlate with it having improved antiplasmodial potency, as N6ꢀPanAm—which is
accumulated to a much lower distribution ratio—still has a lower antiplasmodial IC50 value (0.55
5
±
0.1 µM compared to 4.7 ± 0.1 µM for
N
ꢀBnꢀHoPanAm ). Nonetheless, Pan still accumulates to
a distribution ratio ~2ꢀfold higher than that of
N
ꢀBnꢀHoPanAm, indicating a strong preference for
the transport and/or metabolism of Pan compared to the PanAms (Figure 3a). To investigate this
selectivity further, we studied the effect of increasing Pan concentrations on the accumulation of
the radiolabeled PanAms. The IC values determined from the obtained activity profiles (Figure
50
3d and e) indicated that Pan had the most potent effect on the accumulation of the two
PanAms, whereas HoPanAm and ꢀPanAm accumulation were inhibited to a lesser extent (Table
, right).
To ascertain whether the compounds are being transported by the H ꢀdependent Pan
transporter, the distribution ratio was monitored after 30 minutes in the absence and presence of
αꢀ
n
1
+
14
phloretin, which is a known Pan transport inhibitor. Addition of phloretin blocked the
accumulation of radiolabeled ꢀPanAms, but surprisingly the accumulation of the ꢀPanAms and
HoPanAms variants was apparently stimulated by it (Figure 3f). This suggests that the ꢀPanAms
enter the parasite through the Pan transporter, but that the ꢀPanAms and HoPanAms use
alternative means to gain entry. The stimulation of the accumulation of the latter two variants—
α
n
α
n
which is statistically significant in the case of the N6 derivatives (Figure 3f,
p = 0.0024 for N6ꢀ
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PanAm;
p
= 0.04 for N6ꢀHoPanAm)—is in agreement with previous published work that showed
that phloretin stimulates the uptake of pantothenol, another pantothenate analogue that exhibits
1
8,35
antiplasmodial activity and targets CoA metabolism/utilization.
The reason for this
stimulatory effect is still unclear, however it might be due to alleviation of feedback regulation of
PanK by CoA.
+
To establish if the PanAms are transported by an alternative process dependent on the H
1
1
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0
gradient across the parasite plasma membrane, the experiments were repeated in the presence of
the ionophore carbonyl cyanide
m
ꢀchlorophenyl hydrazine (CCCP), which is known to cause
+
14
rapid dissipation of the H gradient. A similar trend was observed with accumulation of the
αꢀ
PanAms being blocked by the ionophore, while the
nꢀPanAms and HoPanAms still accumulated
in the parasite in amounts comparable to the controls with no ionophore present (Figure 3f).
Taken together, the data are consistent with the
n
ꢀPanAms and HoPanAms being transported by
+
an alternative process that is not dependent on the H gradient—perhaps diffusion through the
3
6
membrane, as has been shown to be the case for pantetheine in certain bacteria and pantothenol
18
in the parasite. In contrast, uptake of the
αꢀPanAms seems to occur by means of the
14
Plasmodium Pan transporter.
These results are consistent with the difference in the antiplasmodial activity of the three
PanAm variants not being linked to the method of their uptake, but instead on a process related to
Pan metabolism. This directed our attention to the first step of CoA biosynthesis, the
phosphorylation of Pan to 4
'
ꢀphosphopantothenate as mediated by PfPanK (Figure 1a).
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Figure 3. Accumulation of [ C]Pan and [ C]PanAm in saponinꢀisolated P. falciparum trophozoites. a)
14
Accumulation of [ C]Pan over 2 hours. Data represent an average of three separate experiments, each
1
4
performed in duplicate and error bars denote SEM. b) Accumulation of the Cꢀlabelled N6ꢀseries of
1
4
PanAm variants over 2 hours. c) As for b, but for the Cꢀlabelled NꢀBnꢀseries of PanAm variants. The
data in panels b and c represent an average of two separate experiments, each performed in duplicate and
1
4
error bars denote range/2. d) and e) Effect of increasing Pan concentration on [ C]PanAm accumulation
(panel d and e for the N6 and NꢀBn series respectively) by saponinꢀisolated P. falciparum trophozoites
after 60 minutes. Accumulation is plotted as percentage of a control with no Pan present. Both d and e
represent an average of two separate experiments, each performed in duplicate and error bars denote
1
4
range/2. f) Distribution ratio of both series of [ C]PanAm variants after accumulation in isolated P.
falciparum parasites for 30 minutes (indicated in black bars). Accumulation was also measured in the
presence of 400 ꢁM phloretin (light grey bars) and 10 ꢁM CCCP (dark grey bars) for each compound
under investigation. The data are an average of 2ꢀ4 separate experiments, each performed in duplicate and
error bars denote SEM or range/2. Statistical analysis was done by performing an unpaired student’s tꢀtest
(where * is p < 0.05 and ** is p < 0.01).
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PanAm inhibition of Pan phosphorylation by PfPanK in parasite lysates confirms an effect on
metabolism, not uptake. The ability of the PanAms to interfere with the phosphorylation of
14
[
C]Pan by PfPanK (either by direct inhibition or by acting as alternative substrates that compete
with Pan for the enzyme’s active site) was investigated using lysates prepared from isolated
parasites. For these tests, unlabeled versions of the same PanAms (i.e. the N5 and ꢀPEꢀ
substituted series) that were used in the accumulation studies were investigated. PfPanK activity
N
1
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
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9
0
1
2
3
4
5
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9
0
14
was determined by measuring the amount of [ C]Pan phosphorylated after 30 minutes (a time
period during which Pan phosphorylation increased linearly under control conditions) in the
presence or absence of the PanAm of interest. Concentration response profiles generated for each
14
compound allowed the determination of IC50 values for the inhibition of [ C]Pan
phosphorylation (Figure 4a and b; Table 2). The results showed that for both sets of PanAms, the
HoPanAm and
nꢀPanAm variants were much more potent inhibitors of Pan phosphorylation than
their ꢀPanAm counterparts, exhibiting IC50 values in the subꢀmicromolar range (0.2–0.4 ꢁM)
α
compared to values in the 7–9 ꢁM range for the latter. These trends correlate directly with those
observed for the inhibition of Pan accumulation (Figure 2a and 2b; Table 1, left), providing
further evidence that the PanAms interfere with Pan metabolism, rather than its transport.
Table 2. Inhibition of PfPanK-catalyzed phosphorylation of radiolabeled Pan by non-
labeled PanAms.
14
Inhibition of phosphorylation of [ C]Pan
by indicated PanAm
a
Non-labeled
variant
IC50 (µM)
N5 series
N-PE series
αꢀPanAm
nꢀPanAm
7.6 ± 0.1
9.8 ± 0.9
0.21 ± 0.01
0.31 ± 0.01
11 ± 2
0.21 ± 0.02
0.43 ± 0.03
>100
HoPanAm
DeoxyꢀPanAm
b
4'ꢀphosphoꢀPanAm
ND
1.3 ± 0.2
a
14
The IC50 of PanAmꢀmediated inhibition of [ C]Pan phosphorylation by parasite lysates after a 45–60 minute
incubation at 37 ºC. The reported values represent the average of two independent experiments, each performed in
duplicate; the errors indicate the range/2.
b
ND, not determined.
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Figure 4. Inhibition of phosphorylation of 2 µM [ C]Pan by PfPanK present in parasite lysate by (a) N5ꢀ
and (b) NꢀPEꢀsubstituted series of PanAm variants. Data were calculated as percentage of control
reactions in which no PanAm was added (100% phosphorylation). For both panels a and b the data are an
average of two separate experiments, each performed in duplicate; the error bars denote range/2.
PfPanK phosphorylates the PanAms, showing kinetic distinction between the different
variants and N-substituents. Previous studies of the interaction of PanAms with SaPanK showed
similar results to those observed in the experiment described above: poor inhibition of Pan
phosphorylation by the αꢀPanAm series, while members of both the nꢀPanAm and HoPanAm
2
7
series potently inhibit Pan phosphorylation. To establish if the observed inhibition of PfPanK
shows a similar distinction, we investigated whether the enzyme accepts PanAms as substrates
14
using the synthesized radiolabeled N6ꢀseries, as well as [ C]NꢀBnꢀPanAm (insufficient amounts
of the radiolabeled NꢀBnꢀαꢀPanAm and NꢀBnꢀHoPanAm variants were available to allow for
their inclusion in these experiments).
Phosphorylation reactions performed with these compounds showed that they all act as
substrates of PfPanK, however, they are phosphorylated more slowly than Pan (Figure 5a).
Activity profiles were next obtained for each compound from the initial rates measured over 30
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minutes (the linear portion of the progress curves) using a range of concentrations (Figure 5b).
From these profiles kinetic parameters were determined for each compound by fitting the data to
either the MichaelisꢀMenten or Hill equation by nonꢀlinear regression (Table 3). The Hill
equation was considered as a model for the kinetic data in light of the recent report of the
37
allosteric behavior of the human PanK3 isoform (HsPanK3). Instead, we found that for PfPanK
the data for both Pan and N6ꢀαꢀPanAm were still best described by the MichaelisꢀMenten
equation. However, the Hill equation gave a better fit to the data obtained for N6ꢀPanAm and
HoPanAm, with Hill numbers greater than 1.5 in both cases, while the data for NꢀBnꢀPanAm
were described equally well by either equation. A Hill number >1.5 is consistent with PfPanK
positive cooperativity in certain cases, depending on the structure of the substrate and its
interactions with the enzyme’s active site (see below).
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The determined kinetic parameters highlighted several trends (Table 3). Specifically, N6ꢀ
HoPanAm showed K0.5 and Vmax values similar to the K and V obtained for Pan, while the
M
max
parameters determined for N6ꢀαꢀPanAm are much higher (K
M
and Vmax are ~13ꢀ and ~2.7ꢀfold
higher than the corresponding parameters for Pan, respectively). For the two nꢀPanAms the
analysis is more complex. Both N6ꢀPanAm and NꢀBnꢀPanAm have K0.5 values that are an order
of magnitude lower than the K for Pan, indicating high affinity interactions with the enzyme.
M
Moreover, their Vmax values are also smaller than that of Pan, although the reduction is much
larger in magnitude for NꢀBnꢀPanAm than for its N6 counterpart.
Table 3. Kinetic parameters determined for the phosphorylation of Pan and PanAm
a
variants by PfPanK present in P. falciparum lysate.
V
max
-1
Radiolabeled
substrate
K
M
or K0.5
Hill
number
Equation
fitted
(µmol.h per
10 parasites)
(µM)
12
Pan
0.397 ± 0.092
5.14 ± 1.97
13.2 ± 1.8
36.7 ± 1.7
8.58 ± 1.35
11.3 ± 2.2
2.24 ± 0.11
ꢀ
MichaelisꢀMenten
N6ꢀαꢀPanAm
N6ꢀPanAm
N6ꢀHoPanAm
NꢀBnꢀPanAm
ꢀ
MichaelisꢀMenten
0.054 ± 0.012
0.180 ± 0.004
0.013 ± 0.003
2.11 ± 0.01
1.57 ± 0.17
1.17 ± 0.59
Hill
Hill
Hill
a
The reported values represent the average parameters determined from of two independent experiments, each done
in duplicate; the errors indicate the range/2.
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Figure 5. a) [ C]PanAm phosphorylation by parasite lysate over 2 hours, shown as % conversion of total
substrate added. Dashed lines indicate the linear portion of each curve, which is maintained over at least
3
0 min. The data are from one experiment performed in duplicate and error bars denote SD. b) Activity
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14
14
profiles of PfPanK present in parasite lysate acting on [ C]Pan, [ C]N6ꢀ and [ C]NꢀBnꢀPanAm as
substrates. c) Activity profiles of PfPanK present in parasite lysate when supplied with increasing
14
14
concentrations of [ C]N6ꢀαꢀPanAm or [ C]N6ꢀHoPanAm as substrates. The dashed line shows the Pan
activity profile for reference. For both panels b and c the solid lines are the curves obtained from fitting
either the MichaelisꢀMenten (for Pan and N6ꢀαꢀPanAm) or Hill (for the nꢀPanAms and N6ꢀHoPanAm)
equations to the data. The data are an average of two separate experiments, each performed in duplicate
and error bars denote range/2. d) Structure of HsPanK3 with N7ꢀPan (stick structure with Cꢀatoms in
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white) bound in the active site (PDB id: 3SMS) showing the hydrogen bonding interactions with Arg207,
which bridges the carbonyl oxygen atoms of the ligand’s amide groups, as well as the hydrogen bonds
between the backbone carbonyls of Val268' and Ala269' and the ligand’s amide hydrogens. The four
residues (N299', S303', R306' and W341') that are substituted for Tyr in PfPanK are also indicated. e)
Model of PfPanK with NꢀPEꢀPanAm (stick structure with Cꢀatoms in blue) bound showing the same
hydrogen bonding interactions between the ligand and Arg295, Leu357' and Ala358'. The four Tyr
residues (Tyr395', Tyr399', Tyr402' and Tyr437') that form the hydrophobic pocket that accommodates the
phenyl group of the ligand are indicated. f) The same model of PfPanK showing the αꢀPanAm (stick
structure with Cꢀatoms in orange) and HoPanAm (stick structure with Cꢀatoms in magenta) variants of Nꢀ
PEꢀPanAm bound. In the case of NꢀPEꢀHoPanAm, only the interactions between Arg295 and the ligand’s
proximal amide group, and between the same amide N–H and the backbone carbonyl of Ala358' are
maintained (indicated with magenta dashes), while the distance between Arg295 and its distal amide
increases, leading to a weakened or lost interaction. In contrast, NꢀPEꢀαꢀPanAm maintains interactions
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(
shown in orange dashes) between its distal amide and Arg295 and Leu357' respectively, as the proximal
amide is rotated away. The rotation also leads to a very different placement of the geminal dimethyl
groups as well as the 4'ꢀOH group that is to be phosphorylated. The amide substituent is still
accommodated in the pocket formed by the Tyr residues shown in panel e, but these were omitted for
clarity.
From this data set—the first to be obtained for PfPanK with PanAms as substrates—we
conclude that the core moiety of the PanAm dictates the affinity of the interaction of the substrate
with PfPanK, with the βꢀalanine moiety being preferred, while the amide substituent apparently
affects the rate of turnover, at least in the nꢀPanAm series. Importantly, the data show that
PfPanK kinetically differentiates between the three PanAm variants in a manner similar to that
described for SaPanK above. However, the data obtained here show that the reduction in the Vmax
values for PfPanK is not as extensive as was found for SaPanK. Moreover, the difference in the
V
max values determined for N6ꢀPanAm and NꢀBnꢀPanAm does not correlate with their
antiplasmodial activities, as would be expected if reduced PfPanK turnover was a factor in the
PanAm’s antiplasmodial MoA. Since the cost and difficulty in synthesizing an extended set of
radiolabeled PanAms made an expanded structure activity relationship (SAR) analysis difficult,
we decided to turn to the use of other tools to further investigate the interactions of the PanAms
with PfPanK.
The modeled PfPanK active site interactions with the PanAms support the analysis drawn
from the kinetic data. We first set out to investigate if we could gain information from the
architecture of the PfPanK active site. While the molecular identity of the PfPanK is still to be
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confirmed, and as a consequence there is no available crystal structure to investigate the
interactions of the PanAms in the active site of the enzyme, the most likely PfPanK candidate
does show a high level of sequence similarity (apart from some parasite specific insertions) with
15
the human PanK isoforms PanK1α and PanK1β, PanK2 and PanK3. Among these, the coꢀ
crystal structure of the homodimeric HsPanK3 with Nꢀheptylꢀnꢀpantothenamide (N7ꢀPan) and
ADP bound in both active sites has been determined (PDB id: 3SMS). This structure shows that
key substrateꢀrecognizing interactions are the two hydrogen bonds made between the side chain
of Arg207 and the two amide carbonyls of the PanAm, and the hydrogen bonds between the
0
1
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0
PanAm’s amide N–H groups and the backbone carbonyls of Val268' and Ala269' (in the adjacent
monomer) (Figure 5d). Since these hydrogen bonds frame the substrate’s βꢀalanine moiety, we
considered that these interactions could also be responsible for PfPanK’s selectivity for the
PanAm variants. To investigate if this is indeed the case, the HsPanK3 coꢀstructure was used as a
15
template to construct a model structure of PfPanK (in which its parasiteꢀspecific inserts are
excluded) that could be used to interrogate its active site interactions. The structure of N7ꢀPan
was modified in situ to reflect the structures of NꢀPEꢀPanAm (the most potent antiplasmodial nꢀ
PanAm discovered to date) and its αꢀPanAm and HoPanAm counterparts. All structures were
subsequently reꢀdocked into the active site of the PfPanK model; to investigate the binding
interactions between the enzyme and the PanAms further, molecular dynamics simulations were
performed to compare the extent to which the observed binding interactions are maintained.
Based on the resulting model structures, we predict that PfPanK forms the same key
hydrogen bonding interactions with the two amide groups flanking the βꢀalanine moiety of NꢀPEꢀ
PanAm (Figure 5e). In addition, the molecular dynamics simulations indicated that these
interactions are maintained in both active sites for the course of the 1.2 ns simulation. In the case
of NꢀPEꢀHoPanAm introduction of the extra methylene group results in greater flexibility and
loss of the hydrogen bond with the Leu357' backbone carbonyl, while the distance between its
distal amide and Arg295 increases, leading to a potentially weaker interaction (Figure 5f).
Simulation with NꢀPEꢀαꢀPanAm indicated the formation of alternate interactions with its amide
groups (Figure 5f). The shortening of the distance between the amides due to the exchange of βꢀ
alanine for glycine apparently prevents both amide bonds from simultaneously forming the
expected hydrogen bonds, resulting in fewer interactions with PfPanK compared to the other two
substrates. These differences correlate with the differences in PfPanK’s kinetic parameters for the
three PanAm variants, and provide an explanation for the enzyme’s preference for substrates in
which the βꢀalanine moiety is maintained.
In addition to the hydrogen bonding interactions that are necessary for binding of the
substrate, comparison of the PfPanK model and the HsPanK3 coꢀstructure also revealed several
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9
substitutions of polar residues for Tyr that result in the formation of a hydrophobic pocket in
PfPanK in the vicinity of the PanAm’s amide substituent (Figure 5e). This hydrophobic pocket (a
cage lined by Tyr395', Tyr399', Tyr402' and Tyr437') is likely to afford more favorable binding
for PanAms substituted with alkyl, alkyl thioether and aromatic groups, compared to the natural
substrate Pan. Furthermore, occupation of the hydrophobic pocket causes both amide bonds of
the PanAms to lock into a favorable conformation for hydrogen bonding with the protein
backbone, whereas the carboxylic acid group of Pan would remain free to rotate. Finally, the
model with NꢀPEꢀPanAm bound highlights the formation of potential πꢀstacking interactions
between the aromatic substituent and Tyr399' and Tyr437'. Such interactions would also explain
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the lower K
M
determined for NꢀBnꢀPanAm compared to N6ꢀPanAm, as well as the former’s
lower Vmax (due to slower release of its phosphorylated product). Overall, these observations
correlate with PfPanK having a preference for binding nꢀPanAms with hydrophobic, and
specifically aromatic, substituents.
Poor inhibition of PfPanK and P. falciparum by DL-4'-deoxy-PanAms point to PfPanK not being
the target of PanAm inhibition. The results of the modeling studies mirror the findings of the
study on the interaction of antistaphylococcal PanAms with SaPanK, which also showed
27,38
preferential binding of nꢀPanAms with specific amide substituents.
Moreover, in the SaPanK
case a linear correlation was observed between an individual nꢀPanAm’s IC50 value for inhibition
of Pan phosphorylation and its kcat value; subsequent experiments showed this to be due to the
PanAms being trapped in the active site after being phosphorylated, strongly suggesting that
inhibition of SaPanK is an important contributor to the MoA of the PanAms in the inhibition of S.
33
aureus.
While it would be tempting to extrapolate the findings of the S. aureus study to P.
falciparum and to conclude that the PanAms act against the parasite by inhibiting Pan
phosphorylation, the observed trends in the antiplasmodial potency of the PanAm variants could
also be explained by their selective metabolic activation (phosphorylation). Since the
experimental limitations of performing extensive kinetic analysis of PfPanK activity and
inhibition in parasite lysates prevented us from performing a similar IC50/Vmax correlation
analysis, we decided to turn to studying a different set of PanAm analogues to establish these
compounds’ antiplasmodial MoA. The n-PanAm analogues DLꢀ4
ꢀdeoxyꢀPEꢀPanAm (dNꢀPEꢀPanAm) were therefore prepared; these compounds have the
same amide substitutions as N5ꢀPan and NꢀPEꢀPanAm respectively, but lack the 4 ꢀhydroxyl
'ꢀdeoxyꢀN5ꢀPan (dN5ꢀPan) and
DLꢀ4
'
'
27
group that is phosphorylated by PanK enzymes (Figure 6a). While this exchange makes it
impossible for the compound to be metabolically activated through phosphorylation, and
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therefore to have any inhibitory effects on processes and enzymes other than PfPanK, they are
expected to maintain all the other important binding interactions with the enzyme. Consequently,
one would expect such compounds to largely retain their antiplasmodial activity if inhibition of
PfPanK formed part of the MoA of the PanAms.
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Figure 6. a) Chemical structure of DLꢀ4'-deoxyꢀPanAms in which the 4'-OHꢀgroup of the parent compound
14
is removed. b) Inhibition of phosphorylation of 2 µM [ C]Pan by PfPanK present in parasite lysate by
dN5ꢀPan and dNꢀPEꢀPanAm. The data are an average of two separate experiments, each performed in
duplicate; error bars denote range/2. c) and d) Antiplasmodial activity of N5ꢀPan (panel c) and NꢀPEꢀ
PanAm (panel d) in comparison to the corresponding deoxy variants of each compound in culture medium
in which pantetheinase has been inactivated. Data for parent compounds are from previous published
4
ꢀ5
work. Data for the deoxyꢀvariants are an average of two separate experiments, each performed in
triplicate and error bars denote range/2.
The interaction of dN5ꢀPan and dNꢀPEꢀPanAm with PfPanK was first determined by
14
measuring the phosphorylation of [ C]Pan by parasite lysates in the presence of increasing
concentrations of the two deoxyꢀcompounds (Figure 6b). The IC50 values obtained from the
resulting dose response profiles showed that dNꢀPEꢀPanAm has a ~500ꢀfold reduction in
inhibition potency compared to NꢀPEꢀPanAm, with an additional order of magnitude difference
in the case of the N5 series compounds (Table 2). These results stand in contrast to those obtained
for a similar experiment performed with SaPanK and dN5ꢀPan and N5ꢀPan, which exhibited the
2
7
same inhibitory effect on this enzyme. The poor inhibition of PfPanKꢀmediated Pan
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phosphorylation by the DLꢀ4
'
ꢀdeoxyꢀPanAms indicates that the 4'ꢀOH group forms crucial
binding interactions with the enzyme, and suggests that PfPanK is predisposed to binding
PanAms that can act as PanK substrates, i.e. compounds that can be metabolically activated
through phosphorylation.
The poor interaction of dN5ꢀPan and dNꢀPEꢀPanAm with PfPanK also suggested that
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these compounds would show poor antiplasmodial activity. Indeed, both DLꢀ4'ꢀdeoxyꢀPanAms
have antiplasmodial IC50 values >200 ꢁM, while the two parent compounds showed potent
antiplasmodial activities with IC50 values of 7.5 ± 6.2 ꢁM and 0.020 ± 0.002 ꢁM, respectively
4
ꢀ5
(Figure 6c and d). Again, these findings do not correlate with results found in S. aureus, in
which the dN5ꢀPan was only ~30ꢀfold less potent than N5ꢀPan. Moreover, dNꢀPEꢀPanAm’s ~10ꢀ
fold higher inhibition of Pan phosphorylation compared to dN5ꢀPan did not translate to it
showing a similarly higher parasite killing activity. Taken together, these results strongly indicate
that the MoA of the PanAms is different in S. aureus and in P. falciparum, and that the inhibition
of PanK does not play an important role in PanAmꢀmediated inhibition of proliferation of the
latter.
4’-Phospho-NꢀPE-PanAm is a poor inhibitor of PfPanK-mediated Pan phosphorylation, but still
inhibits P. falciparum proliferation. The results obtained with the deoxy variants are consistent
with the PanAms having to be metabolically activated through phosphorylation by PfPanK to be
able to exert an antiplasmodial effect. Consequently, phosphorylated PanAms can be expected to
show antiplasmodial effects if they are stable under the growth assay conditions, and can cross
the parasite membranes. Recently it was shown that 4'ꢀphosphoꢀpantetheine (PꢀPantSH), an
intermediate in the CoA biosynthetic pathway and CoA degradation product that is structurally
very similar to a phosphorylated PanAm, is stable in serum and can cross the membranes of
3
9ꢀ40
several eukaryotic cell models.
We therefore prepared the phosphorylated version of NꢀPEꢀ
PanAm to determine if it has any effect on PfPanK activity and on P. falciparum bloodꢀstage
proliferation.
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Figure 7. a) Chemical structure of 4'ꢀphosphoꢀNꢀPEꢀPanAm in which the 4'ꢀOHꢀgroup of the parent
compound is phosphorylated. b) 4'ꢀPhosphoꢀNꢀPEꢀPanAmꢀmediated inhibition of phosphorylation of 2
1
4
µM [ C]Pan by PfPanK present in parasite lysate. The data are an average of two separate experiments,
each performed in duplicate and error bars denote range/2. The dotted line shows the fit for NꢀPEꢀPanAmꢀ
mediated inhibition (see Figure 4b) for comparison. c) Antiplasmodial activity of 4'ꢀphosphoꢀNꢀPEꢀ
PanAm in culture medium containing 1 ꢀM Pan with and without pantetheinase activity. Activity was also
measured in medium containing 100 ꢀM Pan without pantetheinase activity. All data are an average of 3–
4 separate experiments, each performed in triplicate; error bars denote SEM. The dotted line shows the fit
for NꢀPEꢀPanAmꢀmediated inhibition (see Figure 6d) for comparison.
4
'ꢀPhosphoꢀNꢀPEꢀPanAm (Figure 7a) was tested for its ability to inhibit the
14
phosphorylation of [ C]Pan by PfPanK present in parasite lysate. While inhibition was observed,
the determined IC50 of 1.3 ± 0.2 ꢁM is 6.5 times higher than the value obtained for NꢀPEꢀPanAm
(Figure 7b). This result provides evidence against a mechanism of inhibition similar to that seen
3
3
for SaPanK, where the phosphorylated PanAm is trapped in the enzyme. However, 4'ꢀphosphoꢀ
NꢀPEꢀPanAm was found to still inhibit bloodꢀstage parasite proliferation in culture media either
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with or without pantetheinase, with IC values of 2.00 ± 0.25 ꢁM and 1.84 ± 0.25 ꢁM (SEM,
50
n=4) respectively (Figure 7c). These values are relatively high compared to the value of ~20 nM
4
determined previously for the parent compound (NꢀPEꢀPanAm) in media without pantetheinase ;
this can likely be ascribed to the two compounds having different rates of uptake, although this
remains to be tested. More importantly though, the observed antiplasmodial activity could not be
antagonized by the addition of 100 ꢁM Pan to the medium (IC50 2.6 ± 0.5 ꢁM; SEM, n=3) as
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could be done for the parent compound in a previous study, indicating that the 4'ꢀphospho
version does not exert its antiplasmodial effect by inhibiting a process related to Pan
accumulation (such as its phosphorylation by PfPanK). Taken together, these results point to the
PanAms exerting their antiplasmodial effects only after being metabolically activated into their
phosphorylated forms by PfPanK, and that PfPanK itself is not the target of their inhibition.
Consequently, the antagonism of the PanAm’s antiplasmodial activity by increased amounts of
Pan is best explained by the latter interfering with PanAm activation, and not through
competition for the actual molecular target.
CONCLUSION
Although the antiplasmodial potential of the PanAms has been highlighted in several
studies, little information was known regarding their MoA and the basis for the apparent
requirement of the βꢀalanine moiety to retain activity against intraerythrocytic stage parasites. In
this study, we demonstrated that differentiation between the PanAm variants is already
+
established during uptake, with αꢀPanAms being transported by the H ꢀmediated Pan transporter
whereas nꢀ and HoPanAm variants enter the parasite by a different route(s). However, we could
find no evidence that inhibition of Pan uptake forms part of the PanAm’s antiplasmodial MoA.
Instead, our results show that upon uptake, PfPanK phosphorylates the PanAms, and that the
kinetic efficiency of this phosphorylation correlates with the antiplasmodial potency of the
compounds. Since we found that PfPanK prefers the PanAms as substrates over Pan, it is possible
that PanAm treatment could initially lead to a temporary reduction in CoA levels as the PanAms
outcompetes Pan for access to the enzyme. While such a process could be likened to the action of
a competitive inhibitor acting on PfPanK, the finding that the PanAms are also substrates of the
enzyme means that the analysis of their inhibitory activity is more complex. In fact, our data
indicate that PfPanK’s main role in the PanAms’ MoA is to contribute to their metabolic
activation through phosphorylation. Recently it was shown that the murine malaria parasites
Plasmodium yoelii and Plasmodium berghei undergo normal asexual stage development even
41ꢀ42
when lacking the genes encoding their putative PanK enzymes.
This could be explained by a
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preference by these parasites to infect immature red blood cells (reticulocytes) that provide
complex reservoirs of metabolites.
While we demonstrated that a phosphorylated PanAm has antiplasmodial activity, it is
unclear whether it directly acts on a specific target, or is further activated by PPAT and DPCK
into a CoA antimetabolite that act on CoAꢀdependent processes. The CoA biosynthetic enzymes
PPCS and PPAT are both excellent candidates to act as the molecular targets of 4'ꢀphosphoꢀ
PanAms, especially since the former has been identified as the target of other phosphorylated Pan
analogues (the fungal natural product CJꢀ15,801, and pantothenol) that show both antibacterial
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35,43ꢀ45
and antiplasmodial activity.
Whether it is a CoAꢀdependent process or a CoA biosynthetic
enzyme, identification of the molecular target(s) of the activated PanAms remains an important
goal considering that the citric acid cycle and fatty acid biosynthesis—both processes in which
CoA plays a central role, and which otherwise would be considered to contain ideal target
46ꢀ48
candidates—are not essential for survival of the bloodꢀstage parasite.
Achieving this goal will
therefore not only advance our search for new antiplasmodial agents, but will also make a
significant contribution to our understanding of P. falciparum biology and its vulnerability
related to its essential requirement for Pan.
METHODS
General materials and methods. Nonꢀradiolabeled Nꢀsubstituted PanAms and precursors
1
13
previously published were prepared as previously described and their purity confirmed by H, C
2
,5,27,34
NMR and HRMS analysis.
The PanAms were dissolved in dimethylsulfoxide (DMSO) to
yield stock solutions at a concentration of 200 mM and in all assays the final DMSO
concentrations never exceeded 0.1% (v/v). General chemicals, reagents and media were
purchased from SigmaꢀAldrich (Aldrich, Sigma or Fluka), Merck Chemicals or BDH and were of
the highest purity available. All solvents used were CHROMASOLV HPLC grade. Gentamycin
and Albumax II for cell culture were obtained from Invitrogen. Scintillation fluid (MicroScintꢀO
and MicroScint40), Whatman UNIFILTER 350 white opaque 96ꢀwell plates, and TopSealꢀA
Pressꢀon Adhesive Sealing Film were purchased from PerkinꢀElmer.
Analyses of all concentrationꢀresponse curve data and MichaelisꢀMenten curves were
done using Sigmaplot 12 (Systat software). For concentrationꢀresponse curve data the percentage
was plotted against the logarithm of increasing compound concentration and curve fitting was
b
done by nonlinear regression (using the equation y = a/(1 + (x/x
0
) ) to yield the compound
concentration required for 50% inhibition of the process evaluated.
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Synthesis of radiolabeled PanAms. Sodium
D
ꢀ[1ꢀ C]pantothenate ([ C]pantothenate) with
specific activity of 50 mCi/mmol was purchased from American Radiolabeled Chemicals, Inc. (S.
Louis, Mo, USA) and was used in initial experiments. Radiolabeled versions of the N6ꢀ and Nꢀ
Bnꢀseries of PanAms were synthesized by modification of a method previously used to prepare
34
14
nonꢀradiolabeled PanAms, using [1ꢀ C]Nꢀhexylamine hydrochloride (55 mCi/mmol) and [7ꢀ
1
4
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C]Nꢀbenzylamine hydrochloride (55 mCi/mmol) (both obtained from American Radiolabeled
Chemicals) as radioactive starting materials. Briefly, a 250 ꢁL reaction mixture was constituted
by combining the phenyl thioester of αꢀpantothenic acid, Pan, or homopantothenic acid (75 ꢁL of
a 20 mM solution in acetonitrile; 1.5 ꢁmol), triethylamine (50 ꢁL of a 20 mM solution in water; 1
ꢁ
mol), the radiolabeled amine hydrochloride (50 ꢁL of a 18.2 mM solution in water; 0.91 ꢁmol;
5
0 ꢁCi) and 75 ꢁL acetonitrile. The mixture was incubated at 40 °C for 4 hours, followed by the
addition of 1,4ꢀdiaminobutane (75 ꢁL of a 20 mM solution in acetonitrile; 1.5 ꢁmol) and
incubation at 40 °C for a further hour. The mixture was then loaded onto a column of Amberlite
IRCꢀ86 ionꢀexchange resin (150 mg resin prewashed with 40% acetonitrile in water), and the
column was washed with a solution of 40% acetonitrile in water (350 ꢁL × 3). The combined
eluates were evaporated to dryness in a speedvac. The residues were resuspended in 240 ꢁL of a
solution of 20% acetonitrile in water, and their radioactive purity confirmed by thin layer
chromatography (TLC) analysis. Up to six such reaction mixtures were combined for preparative
scale synthesis. If needed, samples were purified using silica solid phase extraction columns (1
g/6 mL Strata Siꢀ1 SPE columns from Phenomenex) preꢀequilibrated with a solution of 10%
methanol in dichloromethane (DCM), and eluting with the same solution. Fractions containing
radioactivity were pooled, evaporated and treated as above. Purity was estimated to be 85–95%,
but yields were generally found not to exceed 30%. Final stocks of 0.1 ꢁCi/mL were prepared in
a solution of 10% acetonitrile in water and stored at ꢀ20 °C until needed.
Synthesis of PanAms. DL-4
'
-Deoxy-N-pentyl pantothenamide (dN5-Pan). dN5ꢀPan was
27
synthesized as previously described.
DL-4'-Deoxy-N-phenethyl pantothenamide (dNꢀPE-PanAm). dNꢀPEꢀPanAm was
synthesized by modification of the method used to prepare dN5ꢀPan. To a solution of 3,3ꢀ
dimethylꢀ2ꢀoxoꢀNꢀ(3ꢀoxoꢀ3ꢀ(phenethylamino)propyl)pentanamide (150 mg, 0.493 mmol) in
anhydrous methanol (MeOH) (10 mL) at 0 °C under an inert atmosphere was added sodium
borohydride (28.0 mg, 0.739 mmol) in small portions. The reaction mixture was stirred for 1 hour
at 0 °C, and left to stir overnight at room temperature (RT). The reaction was quenched at 0 °C by
the addition of sat. aqueous NH Cl (10 mL) and the MeOH was removed in vacuo. The aqueous
4
solution was extracted with ethyl acetate (EtOAc) (3 × 15 mL) and the combined organic extracts
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were dried (Na SO ), filtered and concentrated in vacuo to yield the target compound (143 mg,
2
4
9
4 %) as a white solid. R
s, ꢀ(CH ), 2.36 (2H, t, J = 6.0 Hz, ꢀCH
ꢀ), 3.64 (1H, d, J = 5.3 Hz, ꢀCHꢀ), 5.63 (1H, br s, ꢀNHꢀ), 6.80 (1H, br s, ꢀNHꢀ), 7.18ꢀ7.25
3H, m, arom) and 7.30ꢀ7.36 (2H, m, arom). OH proton not observed. δ (150 MHz; CDCl
5°C) 28.6, 37.6, 37.8, 38.2, 38.2, 43.3, 82.1, 129.2, 131.2, 131.3, 141.2, 173.9 and 175.5.
f
= 0.35 (10% MeOH in DCM). δ
H
(600 MHz; CDCl
3
; 25°C) 0.98 (9H,
3
)
3
2
ꢀ), 2.80 (2H, t, J = 6.9 Hz, ꢀCH
2
ꢀ), 3.50ꢀ3.60 (4H, m, ꢀ
2 2
(CH )
(
C
3
;
2
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0
1
2
3
4
5
6
7
8
9
0
+
+
(
HRMS) [M+H] 307.2023 (Calculated [C17
27 2 3
H N O ] = 307.2022).
3,3-Dimethyl-2-oxo-N-(3-oxo-3-(phenethylamino)propyl)pentanamide. To a solution of 3ꢀ
aminoꢀNꢀphenethylpropanamide (480 mg, 2.50 mmol) in DCM (20 mL) at 0 °C was added N,Nꢀ
diisopropylethylamine (449 µL, 2.58 mmol) dropꢀwise over 5 minutes under an inert atmosphere.
27
1ꢀHydroxybenzotriazole hydrate (59.3 mg, 0.439 mmol), 3,3ꢀdimethylꢀ2ꢀoxoꢀbutyric acid (358
mg, 2.75 mmol) and Nꢀ(3ꢀdimethylaminopropyl)ꢀN'ꢀethylcarbodiimide hydrochloride (480 mg,
2
.50 mmol) were then added consecutively and the reaction mixture was stirred overnight at RT.
The reaction was quenched by the addition of 3 M aqueous HCl (25 mL) and the organic layer
was washed with 3 M aqueous HCl (1 × 25 mL) and sat. aqueous NaHCO (1 × 25 mL). The
organic layer was dried (Na SO ), filtered and concentrated in vacuo before purification by flash
3
2
4
column chromatography (FCC) (2:1 EtOAc: Hexanes) afforded the amide (150 mg, 20%) as a
white powder. R = 0.35 (FCC conditions). δ (300 MHz; CDCl ; 25°C) 1.32 (9H, s, ꢀ(CH ),
2.35 (2H, t, J = 6.0 Hz, ꢀCH ꢀ), 2.79 (2H, t, J = 7.0 Hz, ꢀCH ꢀ), 3.49ꢀ3.57 (4H, m, ꢀ(CH ) ꢀ), 5.63
f
H
3
3 3
)
2
2
2 2
(
1H, br s, ꢀNHꢀ), 7.15ꢀ7.26 (3H, m, arom), 7.28ꢀ7.33 (2H, m, arom) and 7.49 (1H, br s, ꢀNHꢀ). δ
100 MHz; CDCl
71.0 and 203.3. (HRMS) [M+H] 305.1874 (Calculated [C H N O ] = 305.1865).
C
(
3
; 25°C) 26.5, 35.3, 35.5, 35.8, 40.9, 43.1, 126.8, 128.8, 128.9, 138.9, 160.6,
+
+
1
17 25
2
3
3-Amino-N-phenethylpropanamide.
To
a
solution
of
benzyl
3ꢀoxoꢀ3ꢀ
(phenethylamino)propylcarbamate (950 mg, 2.91 mmol) in MeOH (50 mL) at RT was added
0% palladium on carbon (Pd/C) (124 mg, 1.16 mmol). The reaction atmosphere was filled with
hydrogen (H ) gas and the reaction mixture was stirred overnight at RT. The reaction mixture was
filtered and concentrated in vacuo to give the amine (550 mg, 98%) as a whiteꢀyellow solid. R
product on baseline (10% MeOH in DCM). δ (600 MHz; CDCl ; 25°C) 1.55 (2H, br s, ꢀNH
ꢀ), 2.80 (2H, t, J = 7.0 Hz, ꢀCH ꢀ), 2.94 (2H, t, J = 6.0 Hz, ꢀCH
.49 (2H, q, J = 7.0 Hz, ꢀCH ꢀ), 6.98 (1H, br s, ꢀNHꢀ), 7.19ꢀ7.24 (3H, m, arom) and 7.27ꢀ7.33
1
2
f
=
),
H
3
2
2.27 (2H, t, J = 6.0 Hz, ꢀCH
2
2
2
ꢀ),
3
2
(2H, m, arom). δ
C
(150 MHz; CDCl
3
; 25°C) 38.3, 40.7, 41.0, 43.0, 129.0, 131.1, 131.4, 141.0 and
+
+
175.0. (HRMS) [M+H] 193.1343 (Calculated [C11
H
17
N
2
O] = 193.1341).
Benzyl 3-oxo-3-(phenethylamino)propylcarbamate. Phenethylamine (621 µL, 4.93 mmol)
and diethyl cyanophosphonate (748 µL, 4.93 mmol) were added to a solution of Cbzꢀβꢀalanine
(1.00 g, 4.48 mmol) in anhydrous dimethylformamide (7 mL) at RT under an inert atmosphere.
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The reaction mixture was cooled to 0 °C before triethylamine (1.31 mL, 9.41 mmol) was added.
The reaction mixture was stirred for 2 hours at 0 °C and left to stir overnight at RT. EtOAc (50
mL) was added and the organic layer was washed with 5% aqueous citric acid (3 × 10 mL), 1 M
aqueous NaHCO
Na SO ), filtered and concentrated in vacuo to afford the carbamate (1.45 g, 99%) as a white
solid. R = 0.30 (3:1 EtOAc: Hexanes). δ (300 MHz; CDCl ; 25°C) 2.34 (2H, t, J = 5.9 Hz, ꢀ
ꢀ), 2.78 (2H, t, J = 6.9 Hz, ꢀCH ꢀ), 3.43 (2H, q, J = 6.2 Hz, ꢀCH ꢀ), 3.48 (2H, q, J = 7.0 Hz, ꢀ
ꢀ), 5.09 (2H, s, ꢀCH ꢀ), 5.38 (1H, br s, ꢀNHꢀ), 5.52 (1H, br s, ꢀNHꢀ) and 7.16ꢀ7.36 (10H, m,
(75 MHz; CDCl ; 25°C) 35.6, 36.0, 37.2, 40.6, 66.6, 126.6, 128.0, 128.5, 128.6, 136.6,
38.8, 156.6 and 171.3. (HRMS) [M+H] 327.1705 (Calculated [C19
4'-Phospho-N-phenethyl pantothenamide (4'-phospho-NꢀPE-PanAm). To a solution of Nꢀ
phenethyl pantothenamide 4'ꢀO,Oꢀdibenzylphosphate (120 mg, 0.206 mmol) in MeOH (9 mL)
and H O (1 mL) at RT was added 10% Pd/C (31.3 mg, 0.294 mmol). The reaction atmosphere
was filled with H gas and the reaction mixture was stirred overnight at RT. The reaction mixture
was filtered and concentrated in vacuo to give the target compound (82.3 mg, 99%) as a clear oil.
= 0.06 (10% MeOH in DCM). δ (300 MHz; D O; 25°C) 0.76 (3H, s, ꢀCH ), 0.83 (3H, s, ꢀ
CH ), 2.26 (2H, t, J = 6.7 Hz, ꢀCH ꢀ), 2.66 (2H, t, J = 6.7 Hz, ꢀCH
3
(2 × 10 mL) and sat. aqueous NaCl (1 × 10 mL). The organic layer was dried
(
2
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
f
H
3
CH
2
2
2
2
CH
2
arom). δ
C
3
+
+
1
23 2 3
H N O ] = 327.1709).
2
2
R
f
H
2
3
3
2
2
ꢀ), 3.25ꢀ3.37 (4H, m, ꢀ(CH
2
)
2
ꢀ
)
, 3.48 (1H, dd, J = 5.0, 9.7 Hz, ꢀCH ꢀ), 3.68 (1H, dd, J = 4.7, 10.0 Hz, ꢀCH ꢀ), 3.87 (1H, s, ꢀCHꢀ
2
2
), 7.14ꢀ7.18 (2H, m, arom) and 7.22ꢀ7.30 (3H, m, arom). OH protons not observed. δ
C
(75 MHz;
D
2
O; 25°C) 19.2, 21.2, 35.1, 36.1, 38.9, 39.0, 41.2, 72.1, 75.0, 127.2, 129.3, 129.5, 139.8, 174.3
+
and 175.4. δP (161.9 MHz; D O; 25°C) 3.43. (HRMS) [M+H] 403.1625 (Calculated
2
+
[
C
17
H
28
N
2
O
7
P] = 403.1634).
N-Phenethyl pantothenamide 4'-O,O-dibenzylphosphate. Dibenzyl chlorophosphate was
prepared in situ by reacting Nꢀchlorosuccinimide (373 mg, 2.79 mmol) and dibenzylphosphite
(732 mg, 2.79 mmol) in anhydrous toluene (4 mL) under an inert atmosphere for 2 hours at RT.
3
3
The reaction mixture was filtered to remove the succinimide. To a solution of NꢀPEꢀPanAm
(300 mg, 0.931 mmol) in anhydrous pyridine (5 mL) at ꢀ40 °C under an inert atmosphere was
added dibenzyl chlorophosphate dropꢀwise with stirring. The reaction mixture was stirred for an
additional 2 hours at ꢀ40 °C and the mixture was placed in the ꢀ20 °C freezer overnight. The
2
reaction mixture was allowed to warm to RT and was subsequently quenched with H O (3 mL)
and concentrated in vacuo. The resulting crude residue was reꢀdissolved in EtOAc (40 mL) and
the organic layer was washed with 1 M aqueous H SO (2 × 10 mL), 1 M aqueous NaHCO (2 ×
2
4
3
10 mL) and sat. aqueous Na
2
SO
4
(1 × 10 mL). The organic layer was dried (Na
2
SO
4
), filtered and
concentrated in vacuo before purification by FCC (5% MeOH in DCM) afforded the compound
(
150 mg, 28%) as a yellow oil. R = 0.11 (FCC conditions). δ (600 MHz; CDCl ; 25°C) 0.80
f
H
3
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2
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8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
3H, s, ꢀCH ), 1.04 (3H, s, ꢀCH ), 2.34 (2H, t, J = 4.3 Hz, ꢀCH ꢀ), 2.76 (2H, t, J = 7.2 Hz, ꢀCH ꢀ),
3
3
2
2
3
.44ꢀ3.52 (4H, m, ꢀ(CH
2
)
2
ꢀ), 3.55 (1H, dd, J = 7.6, 10.3 Hz, ꢀCH
ꢀ), 5.03 (2H, d, J = 8.8 Hz, ꢀCH
1H, br t, J = 5.6 Hz, ꢀNHꢀ), 7.02 (1H, br s, ꢀNHꢀ), 7.16ꢀ7.23 (5H, m, arom) and 7.28ꢀ7.38 (10H,
m, arom). OH proton not observed. δ (150 MHz; CDCl ; 25°C) 19.4, 21.9, 36.0, 36.3, 36.7,
0.1, 41.4, 70.4, 70.5, 74.2, 74.3, 120.9, 124.3, 127.2, 128.7, 129.3, 129.4, 136.0, 139.5, 171.8
2
ꢀ), 3.88 (1H, s, ꢀCHꢀ), 3.98 (1H,
dd, J = 6.9, 9.5 Hz, ꢀCH
2
2 2
ꢀ), 5.05 (2H, d, J = 8.2 Hz, ꢀCH ꢀ), 6.16
(
C
3
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
and 173.2. δ
P 3
(161.9 MHz; CDCl ; 25°C) 0.79. (HRMS) [M+H] 583.2575 (Calculated
+
40 2 7
[C31H N O P] = 583.2573).
P. falciparum culture and growth assay. All malaria parasite experiments were performed using
the 3D7 strain of P. falciparum and were maintained in synchronous continuous cultures as
4ꢀ5
described previously. In general, growth assays were performed in media containing no
2
,4ꢀ5
pantetheinase activity, prepared as previously described.
However, the antiplasmodial activity
of 4'ꢀphosphoꢀNꢀPEꢀPanAm was also determined in media with pantetheinase activity present as
5
previously described. Doseꢀresponse curves were determined with twoꢀfold serial dilutions of 4'ꢀ
deoxyꢀPanAms (from 0.391 µM to 200 µM) used in triplicate in a final volume of 200 µL.
Experiments for the PanAms, N5ꢀPan (from 0.391 µM to 200 µM) and NꢀPEꢀPanAm (from 1.95
nM to 2 µM) were also included as controls for comparative reasons. The antiplasmodial activity
of 4'ꢀphosphoꢀNꢀPEꢀPanAm was also evaluated by testing twoꢀfold serial dilutions (from 0.156 to
2
0 µM) in triplicate in a final volume of 200 µL.
P. falciparum parasite isolation. Mature trophozoiteꢀstage parasites were isolated from the host
1
6
erythrocytes as described previously. Briefly, infected erythrocytes were treated with saponin
0.05% (w/v)) and centrifuged immediately for 8 minutes at 2,000 × g at RT. The supernatant
was discarded and the resulting pellet washed by resuspension in malaria saline (25 mM HEPES
pH 7.1), 125 mM NaCl, 5 mM KCl, 20 mM glucose and 1 mM MgCl ) followed by
(
(
2
centrifugation at 14,000 × g for 30 seconds. The supernatant was discarded and the pellet washed
an additional 3ꢀ4 times until the supernatant became clear. The isolated parasites were
resuspended in malaria saline and either used immediately or incubated at 37 °C until used.
Accumulation of radiolabeled compounds in isolated parasites. Accumulation of radiolabeled
16
compounds was measured essentially as described previously. Briefly, isolated parasites were
suspended in saline (25 mM HEPES (pH 7.1), 125 mM NaCl, 5 mM KCl, 20 mM glucose and 1
mM MgCl ) at 37 °C; for experiments with transporter blockers, either 400 ꢁM phloretin or 10
2
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ꢁ
M CCCP was included. Each reaction mixture contained a final concentration of 0.05 ꢀCi/mL
14
14
radiolabeled compound (equivalent to 1.0 ꢀM [ C]Pan or 0.9 ꢀM [ C]PanAms) and a final
7
8
parasite concentration ranging between 5 × 10 to 1 × 10 parasites/mL. Reactions were initiated
by the addition of the radiolabeled compound. At preꢀdetermined time points 200 ꢀL aliquots of
the reactions were removed in duplicate and layered onto 200 ꢀL of oil (5:4 mixture of dibutyl
phthalateꢀdioctyl pthalate). Accumulation of the radiolabeled compounds was then immediately
terminated by centrifugation at 15,800 × g for 2 minutes which facilitated sedimentation of the
cells below the oil and thereby separated them from the aqueous solution containing the
radiolabeled compound. The aqueous supernatant was aspirated and the tube rinsed 4 times with
water. The oil was removed by aspiration and the cell pellet lysed with 0.1% (v/v) Triton Xꢀ100
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(500 ꢀL) and deproteinized by the addition of 5% (w/v) trichloroacetic acid (500 ꢀL). After
centrifugation at 10,000 × g for 10 minutes the supernatant was transferred to scintillation vials in
preparation for scintillation counting.
1
4
For [ C]Pan accumulation experiments the amount of radioactivity trapped in the
extracellular portion of the cell pellet was estimated in a separate batch of cells treated with 400
14
µM phloretin in order to inhibit the Pan transporter. The phloretinꢀtreated cells were mixed with
14
[
C]Pan as described above. Aliquots were sampled immediately for centrifugation through the
oil mix and subsequent processing. The counts per minute (CPM) in the pellets derived from
phloretinꢀtreated cells were subtracted from those obtained in untreated samples providing a
14
measurement of the intracellular [ C]Pan, from which distribution ratios could be determined.
14
For [ C]PanAm accumulation experiments the total initial amount of extracellular radioactivity
was estimated by measuring the radioactivity in the supernatant of a “t=0” sample, i.e. a sample
prepared by centrifugation through the oil mix immediately after mixing. An intracellular water
1
6
volume of 28 fL for isolate P. falciparum parasites was used to calculate distribution ratios.
14
To determine the accumulation of [ C]Pan in the presence of nonꢀradiolabeled PanAms,
14
or [ C]PanAms in the presence of nonꢀradiolabeled Pan, experiments were performed as above,
except that the solution of radiolabeled compound used to initiate the reaction also included
increasing concentrations of the appropriate nonꢀradiolabeled compound. Each reaction mixture
14
contained a final concentration of either 0.1 ꢀCi/mL [ C]Pan (equivalent to 2.0 ꢀM) or 0.08
1
4
ꢀ
Ci/mL of the appropriate [ C]PanAm (equivalent to 1.45 ꢀM), a final parasite concentration
7
7
ranging between 3.2 × 10 to 7.2 × 10 parasites/mL, and varying concentrations of unlabeled
14
substrate. Reactions were stopped after either 20 or 60 minutes (for accumulation of [ C]Pan and
1
4
[
C]PanAms respectively), and the samples were processed exactly as described above. Doseꢀ
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
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5
5
6
response curves were determined relative to the total amount of labeled compound taken up by
parasites incubated in the absence of unlabeled compound.
P. falciparum lysate preparation. Parasite lysates were prepared from washed, saponinꢀisolated
14
intact P. falciparum trophozoites as described before. The parasites were lysed by suspension in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
cold 10 mM Tris (pH 7.4) and trituration (10×) through a 25ꢀgauge needle. Particulate matter was
removed from lysates in one of two ways: (i) lysates were centrifuged three times at 2,000 × g for
30 minutes at 4 °C; or (ii) lysates were centrifuged once at 17,000 × g for 30 minutes at 4 °C.
Lysates were stored at ꢀ20 or ꢀ80 °C until required.
14
14
Time-course of phosphorylation of [ C]Pan and [ C]PanAms by PfPanK. Parasite lysates were
used to assess the ability of PfPanK to phosphorylate radiolabeled Pan or PanAms over time. The
phosphorylation experiments were performed using a modified version of the Somogyi reagentꢀ
19
based method. Mixtures containing kinase buffer with a final composition of 50 mM Tris (pH
7.4), 5 mM ATP and 5 mM MgCl
2
and the radiolabeled compounds (0.1 ꢀCi/mL, equivalent to
2.0 ꢀM) to be tested were incubated for 10 minutes at 37 °C. The phosphorylation reaction was
7
initiated by the addition of parasite lysate equivalent to 2.0–2.5 × 10 cells/mL. The reaction
mixtures were incubated at 37 °C for 120 minutes, and at specific time points 50 µL of the
reaction mixtures was transferred to wells of Whatman 96ꢀwell UNIFILTER 350 plates
(
containing 0.45 µm PP filter bottom and short drip directors) in duplicate. Each well was preꢀ
loaded with 50 µL 150 mM Ba(OH) and the reaction mixture was rapidly mixed with this
solution by pipetting to terminate the phosphorylation reaction by protein precipitation. To each
well 150 mM ZnSO (50 µL) was added to allow precipitation of phosphorylated compounds.
2
4
Nonꢀphosphorylated compounds were removed by filtration under vacuum, leaving
phosphorylated compounds trapped on the filters of each well. The filters were washed with
water (2 × 200 µL), and subsequently 95% (v/v) ethanol (200 µL) and kept under vacuum for 30
minutes before being placed in an incubator to dry overnight at 37 °C. MicroScintꢀO (30 ꢀL) was
added to each well and immediately sealed (top and bottom) with TopSealꢀA Pressꢀon Adhesive
Sealing Film. The radioactivity in each well was measured immediately by scintillation counting
in a TopCount Microplate Scintillation and Luminescence Counter (Packard).
To determine total radioactivity so as to allow the conversion of radioactivity in CPM
values into molar concentrations, two 50 ꢁL aliquots of the reaction mixtures were mixed with
150 ꢁL MicroScintꢀO in the wells of a 96ꢀwell OptiPlate. The plate was thereafter sealed with
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TopSealꢀA Pressꢀon Adhesive Sealing Film and the radioactivity of each well measured by
scintillation counting as described above.
14
Doseꢀresponse curves for the inhibition of PfPanKꢀcatalyzed [ C]Pan phosphorylation by
nonꢀradiolabeled compounds were generated using the same procedure as for the timeꢀcourse
experiments. Phosphorylation was allowed to take place for 30–60 minutes (when a linear
reaction rate applies under control conditions) before 50 ꢁL aliquots were transferred to 96ꢀwell
Whatman UNIFILTER 350 plates in duplicate. The plates were processed as described above to
determine scintillation count. Control reactions containing 10 mM Tris (pH 7.4) served as blank
reactions containing no parasite lysate, whereas reactions containing no PanAm served as
positive controls that represented 100% phosphorylation.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Determining kinetic parameters for PfPanK phosphorylation. Kinetic parameters were
determined by performing phosphorylation reactions with each radiolabeled compound at
concentrations that varied between 0.005 and 5.0 ꢁM, depending on the substrate. Each
compound was incubated at a specific concentration in kinase buffer and after addition of lysate
allowed to be phosphorylated by PfPanK for 30 minutes at 37 °C in a similar fashion as described
above. Control reactions containing 10 mM Tris (pH 7.4) were used as blank reactions containing
no parasite lysate. Kinetic parameters (Vmax and either K
M
and K0.5, as appropriate) were
determined by fitting either the MichaelisꢀMenten or Hill equations to the curves generated from
the initial rated data obtained in this manner.
Modeling of PanAms into PfPanK. The structure of PfPanK was predicted by homology
modeling, using the human PanK3 (PDB code: 3SMS) bound with N7ꢀPan and ADP as the
template structure. The biological unit 3SMS was prepared on the PDB website (www.rscb.org)
in order to model the homoꢀdimer. The sequence of PfPanK and human PanK were aligned
49
against the PFAM seed alignment for type II PanKs (PF03630) using TꢀCOFFEE, followed by
manual adjustments to minimize gaps and insertions. Homology models of PfPank including
50
ligands N7ꢀPan and ADP were built using MODELLER 9.13. Considerable portions of PfPanK
do not align with cognate sequences. In order to keep the total atomic system a manageable size,
the Nꢀterminus (first 18 residues) and the loop from His37 to Asp61 were left unmodeled or
deleted prior to subsequent modeling. The Schrödinger software suite (2015ꢀ1) was used for
subsequent docking and molecular dynamics. N7ꢀPan was modified in situ to generate the NꢀPEꢀ
PanAm series. NꢀPEꢀPanAm (as well as the αꢀ and homo counterparts) were subsequently reꢀ
51
docked into each active site using the GLIDE module. Highꢀscoring poses of each compound
were chosen for both sites to prepare the system for molecular dynamics with DESMOND using
De Villiers et al.
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