Opinion

Artemisinins and the biological basis

for the PfATP6/SERCA hypothesis
Sanjeev Krishna, Serena Pulcini, Farrah Fatih and Henry Staines
Centre for Infection, Cellular and Molecular Medicine, St. Georges University of London, Cranmer Terrace, London, UK, SW17 0RE

With the advent of artemisinin resistance, it is timely to

revisit the biological basis for the controversial suggestion that this class of antimalarial exerts its activity by
inhibiting a calcium ATPase (PfATP6) that is most similar
to sarcoplasmic endoplasmic reticulum calcium ATPases
(SERCAs). Herein, evidence is discussed that relates to
this hypothesis as alternative suggestions for how artemisinins might act have been reviewed elsewhere.
Artemisinins: mode of action
Several years ago the hypothesis that artemisinins might
act as antimalarial agents by inhibiting a single target, the
Plasmodium falciparum sarcoplasmic, endoplasmic,
calcium ATPase PfATP6 [1], led to the prediction that
amino acid polymorphisms in this sequence might be
associated with resistance to these agents [1]. These predictions were based on biochemical assays after heterologous expression and molecular modelling techniques
[1]. The value of this PfATP6 hypothesis for action of
artemisinins is now examined in light of recent evidence
for clinically relevant artemisinin resistance [2,3] as well
as in the wider context of action against SERCA pumps as
targets for artemisinins in non-plasmodial cells. The
PfATP6/SERCA hypothesis has generated many testable
corollaries and continues to be useful in design of experiments in the field of epidemiology, biology and drug development as well as areas such as oncology [4].
Alternative hypotheses for antiparasitic properties of artemisinins include parasite heme, electron transport chain
in mitochondria or other proteins as targets, but these
cannot coherently accommodate other observations. These
include ring-stage killing of parasites, killing of non-heme
generating parasites, perturbations in calcium metabolism
that are seen with artemisinins and genetic association
studies between polymorphisms in PfATP6 and artemisinin susceptibilities.
Genetic predictions
Following from expression studies of PfATP6 in Xenopus
laevis oocytes showing that artemisinins inhibited
PfATP6, it becomes important to discover how artemisinins might interact with this target. Available information
from thapsigargin, which is a potent and highly SERCAspecific inhibitor, has been obtained in mammalian
SERCA with which it has been co-crystallised. This information was used as a basis for molecular modeling of
PfATP6 [1]. Amino acid mutations in the predicted thapCorresponding author: Krishna, S. (s.krishna@sgul.ac.uk).

sigargin binding pocket that could modulate sensitivity to

artemisinins when PfATP6 was tested in vitro were used as
examples of the more general principle that artemisinin
sensitivity in this target might change with certain amino
acid substitutions.
Some months later, evidence from field isolates related
increased IC50 values to artemisinins to mutations in
PfATP6 [5]. Since those observations, there have been
extensive studies in many geographic areas that have
identified polymorphisms in PfATP6 (Table 1)
(Figure 1). For a subset of variants in field studies, IC50
values have been determined against different artemisinin
derivatives (Table 1). More information that summarizes
frequencies of sequence polymorphisms from parasites in
different geographical locations is now available and illustrates structuring of PfATP6 gene diversity and provides
useful sequence alignments [6].
Some PfATP6 sequence polymorphisms are associated
reproducibly with increases in IC50 values to artemisinins.
For example, geographically dispersed isolates in French
Guiana carrying the S769N (Figure 1) mutation have
increased IC50 values to artemether when experiments
have been carried out on isolates collected over three years
(2002, 2003 and 2005) [57]. The same mutation has not
been associated with increased IC50 to dihydroartemisinin
(DHA) when assayed in a single African isolate, suggesting
that either the mutation only alters sensitivity to artemether, or that the genetic background of the isolate is also
important in modulating artemisinin sensitivity [8]. A
different mutation (H243Y, Figure 1) might modulate
sensitivity to DHA in African isolates (Table 1) [8]. Interestingly, those substitutions that are associated with an
increase in IC50 to an artemisinin (a total of five summarised in Table 1, although a recent (re-) analysis identifies
13 haplotypes of PfATP6 that are associated with
elevations in IC50 values to either artemether or artesunate) are located in regions that border functionally conserved domains in PfATP6, such as the nucleotide binding
domain (Figure 1). There is no a priori reason to suppose
that mutations should inevitably be associated with the
predicted thapsigargin pocket because the interactions
between artemisinins and SERCAs still need more
detailed investigations.
The highly variable primary sequence of PfATP6 also
includes at least 25 other amino acid substitutions. Their
distribution is also apparently non-random (Figure 1) with
many substitutions in regions that are not conserved
between SERCAs and that are inserts in PfATP6 (see
[9] for discussion on inserts and [6]). It might be that some

1471-4922/$ see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2010.06.014 Trends in Parasitology, November 2010, Vol. 26, No. 11

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Table 1. Polymorphism in the PfATP6 gene of Plasmodium falciparum

Region(s) (%
of parasites with
mutations)

Thailand
Africa,Angola
Engineered line
(increased normalised IC50%)
Senegal (18.6%), Angola,
Cameroon, Ghana, Cote
DIvoire, Nigeria, Kenya,
Congo Dem. Rep.,
Madagascar, Niger, China,
Zanzibar (31%), Tanzania
(39%), Gambia, Liberia,
Malawi, Sudan, Uganda
Senegal
Cameroon
Cameroon
French Guiana
Senegal, Niger (4.7%)
French Guiana
French Guiana
French Guiana

French Guiana
French Guiana
French Guiana

French Guiana
French Guiana
French Guiana
French Guiana

Senegal
Senegal

Non-synonymous
nucleotide
substitution
Wild type

Amino acid
substitution

T266C
C727T

I89T
H243Y
L263E

G1291A

E431K

G1291A/C1868A
G1294A
G1291A/ G1294A
A1721C/G2306A
C1868A
G2306A
G2306A/T2694A
T1204G /A1612G/
A1721C/G2306A/
T2694A
T1204G/ A1612G/1
T2694A
T2694A
T1329C/G2306A/
T2694A
T1614C/G2306A/
T2694A
A1706G/ T2694A
T1204G/ A1706G/
G2306A/ T2694A
G1291A/G2306A/
T2694A
T1204G/T1614C/
A1721C/G2306A/
T2694A
T1204G/C1868A
G2306A

E431K/A623E
E432K
E431K/ E432K
Q574P/S769N
A623E
S769N
S769N/ I898
L402V/ S538R/
Q574P/S769N
I898
L402V/ S538R/
I898
I898
D443/ S769N/
I898
S538/ S769N/
I898
N569S/ I898
L402V/N569S/
S769N/ I898
E431K/S769N/
I898
L402V/ S538/
Q574P/S769N/
I898
L402V/ A623E
S769N

Increase of SNPs frequency after ACT implementation

Year of widespread ACT use
Region(s)
Niger
Niger, Senegal, Angola
Zanzibar, Tanzania

2005
2005
2005

Artemisinin
IC50 median
[range] (nM)

Artemether
IC50 median
[range] (nM)
5.6[1.355.8]

nd a
nd
30 (7G8)
20 (D10) p = 0.02
nd

nd
nd
nd
nd

Artesunate
IC50 median
[range] (nM)
0.25 [0.1718.4]
5.46 [0.6861.1]
3.38 [0.8129.9]
nd
0.03 (7G8)
0.01 (D10)
20.8

nd
nd
nd
nd
nd
nd
nd
nd

nd
nd
nd
116.8
>b
58.8 [38.2100]
2050
2050

44.7
nd
nd
nd
nd
nd
nd
nd

nd
0.50
3.67
nd
nd
nd
nd
nd

[5,6]
[42]
[42]
[5]
[5,41]
[5]
[6]
[6]

nd

2050

nd

nd

[6]

nd
nd

2050
2050

nd
nd

nd
nd[

[6]
[6]

nd

50100

nd

nd

[6]

nd
nd

50100
>100

nd
nd

nd
nd

[6]
[6]

nd

>100

nd

nd

[6]

nd

>100

nd

nd

[6]

nd
nd

nd
nd

2045
>30

nd
nd

[6]
[6]

Mutation detected
(% SNP positive)
A623E 0.0% vs 2.4%
L402V E431K A623E 771E
E431K N569K A630S

DHA IC50
median
[range] (nM)
0.68 [0.131.8]

Ref.

nd
4.2; 6.4
33 (7G8)
18 (D10) p = 0.02
1.38

[20]
[8,41]
[1,19]

Increase of frequency compared

with little or no ACT use
p= 0.053
nd
nd

SNPs where no functional consequences have been sought or found

R37K, W141V, V229I, G421E, N463S, N465S, S466N, T539I, K561N, N569I, N569K, G585D, T602I,
L610I, K611N, N612Y, A621D, A621S, A623T, A630S, G632E, T635A, T635K, E637G, G639D, S641G,
E643Q, N644I, K649E, N683K, K716R, I723V, H747R, H747Y, K771E, K776N, K783E, R809G,
G884 (R)c, I987T, V1168I (H) c
Synonymous SNPs found
D252, Y441, N460, N465, D537, S538, S557, T570, R756, L758, R801, I885, I898, I959, I987, K1030, C1031

[1,5,19,20]

[5,6,41,
42,43]

Ref.
[15]
[41]
[43]

Ref.
[6,8,15,16,41,43,44,46]

Ref.
[6,8,15,16,43,44,45,46]

nd= not determined.

> = a reported increase in IC50 value
c
reported nucleotide not found in 3D7 gene sequence
b

amino acid substitutions in inserts are less likely to be of

functional relevance to different aspects of PfATP6 activity
[9], including inhibitor susceptibility. An L263E mutation
that was engineered in laboratory experiments has not
been identified in field isolates, which is perhaps to be
518

expected as this mutation was derived on theoretical

grounds. Sustained drug pressure has selected for
mutations that modulate susceptibility of parasites to
the antifolates [1012], for example, as well as establishing
the contribution to drug resistance of key mutations in

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Figure 1. PfATP6 sequence showing three functional domains based on mammalian SERCA structures (see Table 1 for references). SNPs are indicated in boxes with yellow
backgrounds if they have functional consequences and white boxes if there are no functional consequences. Predicted transmembrane spanning regions are numbered.
Highly conserved region between SERCAs and PfATP6 are highlighted in light blue. Abbreviations: A domain, actuator domain; N domain, nucleotide binding domain; P,
phosphorylation domain.

pfcrt [13] and pfmdr1 copy number [14]. Similar selection

pressure exerted by artemisinins might result in significant changes in some PfATP6 mutation frequencies over
time, if these mutations can alter susceptibility to artemisinins of PfATP6.
Recent studies show that with sustained use of ACTs,
there are indeed changes in frequencies of some mutations
in PfATP6 (Table 1). In one study, there was an increase in
the frequency of A623E mutation in isolates from Niger
also after ACT use [15] (p = 0.053). Interestingly, this
substitution was previously reported in Senegal to be
associated with increased IC50 values to artesunate, adding weight to the suggestion that it can be of functional
significance in artemisinin resistance [5]. In Peru, a
reported increase in the frequency of a single amino acid

deletion mutation in PfATP6 [16] was originally thought to

be associated with ACT use, but this finding was geographically confounded and has not been confirmed on
reanalysis (Table 1) [17].
Combinations of substitutions in particular haplotypes
might also synergise in their effects on modulating parasite sensitivity to artemisinins, as suggested by results
from Senegal and French Guiana [5] and discussed in
greater detail [6].
Methodological limitations associated with the high
degree of polymorphism of PfATP6 sequences, the variability associated with IC50 assays for artemisinin derivatives, and the influence of different genetic backgrounds of
parasites in different countries might make it harder to
establish statistically significant associations between a
519

Opinion
sequence polymorphism and a resistance phenotype in
vitro. Nevertheless, studies assessing associations between polymorphisms in PfATP6 and sensitivity to artemisinins have already given useful insights, and can
contribute to understanding of resistance to artemisinins
as well as to their mechanism of action.
Clinical resistance to artemisinins and PfATP6
Resistance to antimalarials in P. falciparum has so far
arisen either through changes in susceptibility of the
target of an antimalarial by mutation or through decreased
delivery of drug to target by the action of drug resistance
pumps. These general mechanisms should also apply to the
artemisinin class of antimalarials, with some evidence that
changes in target susceptibility are associated with resistant phenotypes in vitro (summarised before). A quiescence
mechanism for an artemisinin tolerant phenotype has
recently been identified, where parasites do not display
changes in IC50 values assessed by conventional assays.
However, after in vitro selection with drug, parasites
recrudesce when drug is removed after remaining developmentally arrested at the ring stage [18].
It is also becoming clearer from in vivo studies that even
as there might be evidence for prolonged parasite clearance
times for patients receiving artesunate in Western
Cambodia, correlating this phenotype with elevated
IC50 values to artemisinins in vitro is challenging [3].
Indeed, the magnitude of increase in IC50 values to dihydroartemisinin (artesunates active metabolite) from
patients in Pailin, where some parasites are considered
to be artemisinin resistant, was relatively small (median
value of 2.3 nM IQR (1.1 to 3.2 nM) compared with a
median value of 1.5 nM (IQR 0.7 to 2.2 nM) for sensitive
parasites from Wang Pha (p = 0.04). There was no correlation in measures of parasite clearance times and IC50
estimations. The phenotype of response to artesunate was
at variance when compared with DHA with median IC50
(IQR) values of 1.9 (1.3 to 3.4) nM for Pailin parasites
compared with 3.2 (1.7 to 4.1) nM (p = 0.07) for Wang Pha
parasites. Not all parasites could be assayed reliably (only
18/40 in Pailin and 32/40 in Wang Pha) [3].
In contrast to these findings in a separate study from
this region, there was consistency between increased IC50
levels and prolonged parasite clearance times in vivo in 2
subjects in whom adequate drug levels were determined at
particular time points [2]. In those parasites, the fulllength sequence of PfATP6 was not determined.
Several conclusions can be drawn from these types of
studies. As with transgenic lines harbouring a mutation in
PfATP6 (L263E), there is considerable variability in IC50
values determined on the same parasite line [19]. This
variability is an intrinsic property of the parasite and not
a consequence of assay methodologies, which have been
standardised. Variability might also be larger in parasites
carrying mutations that are associated with elevated IC50
values to artemisinins, compared with those carrying wild
type sequences [19]. In analysis of susceptibility to artemisinins, normalization of IC50 values with the IC50 values of
control parasite lines (PfATP6 L263) revealed significantly
decreased susceptibility to artemisinin and dihydroartemisnin in 263E parasites, but not to artesunate [19].
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Second, in attempting to relate relatively small changes

in in vitro IC50 values to prolongations in parasite clearance times, it might be that small shifts in in vitro phenotype can influence clinical outcomes. If that is the case,
then mechanisms other than decreased sensitivity of target sequences to artemisinins become of clinical relevance,
such as the changes in IC50 associated with increased
pfmdr1 copy number [20] or quiescence mechanisms
[18]. In any case, many more parasite isolates with
relevant phenotypes will need to be assayed to assess if
particular mutations in PfATP6 (or any other sequence)
are associated with the artemisinin resistance phenotype.
It is therefore probably premature to conclude that PfATP6
does not contribute to emerging resistance to the artemisinin based malaria treatments in Cambodia [3]. Furthermore, sample size calculations indicate that with the
published values for dispersion of IC50 s in artemisinins,
each parasite might have to be assayed many times to
determine with confidence if there is a significant change in
IC50 compared with a control line [19].
Consequences of inhibiting PfATP6
Calcium signaling is implicated in the regulation of many
processes in the parasites life cycle, including secretion of
adhesins, motility, cellular invasion and egress, and intracellular development [2122]. Given these diverse roles,
mechanisms that maintain calcium homeostasis are central to parasite survival and are tightly controlled by
proteins mediating signal transduction or other activities
after interacting with free intracellular calcium. Approximately 30 calcium interacting proteins have been identified after sequence analysis in Plasmodia, and these
include three assigned as being active calcium transporters on the basis of signature sequences and functional
assays; PfATP6, PfATP4 (a Golgi-like calcium pump that
may also be present at the plasma membrane) and a
calcium/proton exchanger [23].
In erythrocytes, the parasite maintains a free cytosolic
calcium concentration of 100 nM, and has at least two
functionally distinct intracellular calcium stores: an ERlike store that releases calcium after inhibition by mammalian SERCA inhibitors and a separate acidic store that
releases calcium after application of modulators of proton
gradients [2425]. PfATP6 has been localised to the ER [26]
using fluorescent thapsigargin. Thus, inhibiting PfATP6
should reduce the capacity of the ER to take up free
calcium, leading to elevated cytosolic free calcium levels.
Whereas several parasite studies using fluorometric techniques showed little or no effect of 12 mM thapsigargin
[24,27,28], another study with higher concentrations (
5 mM), reported increased cytosolic free calcium levels.
This finding is consistent with a prediction based on the
PfATP6 hypothesis for artemisinin action as well as the
prediction that thapsigargin can have difficulty in accessing intracellular parasites [29].
A recent study [30] showed that artesunate (4 nM)
transiently increased cytosolic free calcium in single cell
studies of P. falciparum, using Fluo-4. Thapsigargin
(10 mM) also increased free calcium but not after the
addition of artesunate suggesting that artesunate depletes
ER calcium stores [30]. Consistent with these findings,

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micromolar concentrations of artemisinin reduced the

effect of thapsigargin on calcium mobilisation in isolated
P. chabaudi parasites imaged with Fluo-3.
Studies on another apicomplexan parasite, Toxoplasma
gondii, have identified a thapsigargin-sensitive SERCA
orthologue (TgSERCA) [23,31]. This parasite is also
susceptible to artemisinins, with the synthetic derivative
artemisone killing at nanomolar concentrations [3233].
Yeast cells with defective endogenous calcium ATPase
activity were rescued by expression of TgSERCA, which
rendered them sensitive to artemisinin inhibition [32].
Furthermore, artemisinin triggered a calcium-dependent
secretion of cellular invasion proteins and altered intracellular calcium oscillations [32,34]. These findings independently support the hypothesis that artemisinins act on
calcium homeostatic mechanisms and have implicated
TgSERCA as a target for artemisinins. Resistance to artemisinins in this system (or indeed others) might not be
related to changes in the sequence of the target, but to
other mechanisms [32,34] and does not, of course, preclude
the PfATP6/SERCA hypothesis.
Variability of inhibition of SERCAs by artemisinins
In the original description of inhibition of PfATP6 by
artemisinins, mammalian SERCA from rabbit skeletal
muscle preparations was not inhibited by relatively high
(50 mM) concentrations of artemisinin [26]. In a later study
these muscle preparations were resistant to inhibition by
artemether (up to 10 mM) but susceptible to (1 mM produced approximately 40% inhibition of activity) [35], once
again highlighting that important differences in function
are associated with relatively minor chemical differences
between artemisinin derivatives. Combining different
classes of antimalarial also modulated the ability of derivatives to inhibit SERCA [35].
Artemisinin dimers have more recently been shown to
inhibit mammalian SERCA at micromolar concentrations
[36] when tested on purified and reconstituted SERCA
preparations. Artemisinin itself could not inhibit this preparation [36], whereas SERCA from a human colon cancer
cell line, HT29 (among others), was inhibited by artemisinin (with an estimated IC50 of 1 mM) [37]. A dansylated
artemisinin derivative co-localises to ER structures in a
Hep3B cell line [38].
PfATP6 has been expressed in yeast in preparation for
structural studies. Reconstitution experiments of these
highly purified preparations have confirmed that PfATP6
is a SERCA-type calcium transporting ATPase [39]. However, it has somewhat lower activity than mammalian
SERCAs in these experimental conditions. Reconstituted
PfATP6 was virtually insusceptible to inhibition by thapsigarin, although it is predicted to be sensitive (Figure 2),
only showing 50% inhibition with 45 mM thapsigargin.
By contrast, mammalian SERCAs are generally inhibited
within the range of 0.3 to 30 nM thapsigargin and show
50% inhibition with 20 mM BHQ (5-i-tert-butyl)-1, 4-benzohydroquinone, which inhibits mammalian SERCA at
concentrations < 10 mM. Neither was it inhibited by
100-500 mM concentrations of artemisinins. Thapsigargin
kills parasites with an IC50 of 2.6 mM, highlighting the
discrepancy between properties of purified, reconstituted

Figure 2. (a) Sequence alignment of thapsigargin coordinating residues in

different species. In red (invariant) and pink (highly conserved) aminoacid
residues. Underlined (both plants) and in blue residues that vary between
thapsigargin sensitive and insensitive SERCAs. (b) Binding mode of the
artemisinin (grey) and thapsigargin (yellow) modeled in the predicted binding
site of PfATP6 [40]. (c) Figure based on models for predicting antimalarial activity
(Log RA) of artemisinin derivatives using calculated free energy of binding [40].

enzyme and more physiologically relevant PfATP6 assays.

This is further emphasized by the fact that thapsigargin
might access intracellular parasites with difficulty [29] so
that IC50 values should be higher than inhibitory constants for purified enzymes. Purified PfATP6 is not behaving in a qualitatively similar way to mammalian SERCAs
in these assays. It will be interesting to assay BHQ derivatives for their capacity to kill parasites.
Collectively these results confirm that the ability of
artemisinins to inhibit SERCAs of diverse sources is often
dependent on the methodologies used to study this inhibition. Purified enzymes might not reflect the parasiticidal
properties of inhibitors and cannot rule out a direct effect of
artemisinins on PfATP6 in parasites.
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Opinion
Conclusions
Several years ago, it was asserted by some that artemisinin
resistance was very unlikely to emerge soon. Unfortunately, as our studies on PfATP6 predicted, artemisinin
resistance has already appeared, and in some studies is
linked to changes in sequence for PfATP6. Further work is
needed to better understand the nature of interactions
between artemisinins and SERCA-type transporters.
There is added impetus to carry out these types of studies
as indications for using artemisinins in diverse areas of
medicine are increasing [4].
Acknowledgements
SK is funded by the European Commission projects ANTIMAL (Grant No.
018834) and MALSIG (Grant No. 223044) as well as the Wellcome Trust
(grant no. 074395). HMS is a Wellcome Trust Career Development Fellow
(grant no. 07441), sponsored by SK. SP is an INTERMAL Training Fellow
sponsored by the EU and FF is an MRC funded PhD student.

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