A click chemistry-based proteomic approach reveals that 1,2,4-trioxolane and artemisinin antimalarials share a common protein alkylation profile

Article abstract of DOI:10.1002/anie.201512062

In spite of the recent increase in endoperoxide antimalarials under development, it remains unclear if all these chemotypes share a common mechanism of action. This is important since it will influence cross-resistance risks between the different classes. Here we investigate this proposition using novel clickable 1,2,4-trioxolane activity based protein-profiling probes (ABPPs). ABPPs with potent antimalarial activity were able to alkylate protein target(s) within the asexual erythrocytic stage of Plasmodium falciparum (3D7). Importantly, comparison of the alkylation fingerprint with that generated from an artemisinin ABPP equivalent confirms a highly conserved alkylation profile, with both endoperoxide classes targeting proteins in the glycolytic, hemoglobin degradation, antioxidant defence, protein synthesis and protein stress pathways, essential biological processes for plasmodial survival. The alkylation signatures of the two chemotypes show significant overlap (ca. 90 %) both qualitatively and semi-quantitatively, suggesting a common mechanism of action that raises concerns about potential cross-resistance liabilities. Clickable 1,2,4-trioxolane activity-based protein-profiling probes (ABPPs) were designed to retain antimalarial activity and alkylate the molecular targets of the blood stage of Plasmodium falciparum in situ. Comparison of the 1,2,4-trioxolane protein alkylation signature with the corresponding artemisinin ABPPs indicates that both drug classes target key proteins in the glycolytic, hemoglobin degradation, antioxidant defence and protein synthesis pathways.

Full text of DOI:10.1002/anie.201512062

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International Edition: DOI: 10.1002/anie.201512062
German Edition: DOI: 10.1002/ange.201512062
Antimalarials
A Click Chemistry-Based Proteomic Approach Reveals that
1,2,4-Trioxolane and Artemisinin Antimalarials Share a Common
Protein Alkylation Profile
Hanafy M. Ismail, Victoria E. Barton, Matthew Panchana, Sitthivut Charoensutthivarakul,
Giancarlo A. Biagini, Stephen A. Ward, and Paul M. OꢀNeill*
Abstract: In spite of the recent increase in endoperoxide
antimalarials under development, it remains unclear if all these
chemotypes share a common mechanism of action. This is
important since it will influence cross-resistance risks between
the different classes. Here we investigate this proposition using
novel clickable 1,2,4-trioxolane activity based protein-profiling
probes (ABPPs). ABPPs with potent antimalarial activity
were able to alkylate protein target(s) within the asexual
erythrocytic stage of Plasmodium falciparum (3D7). Impor-
tantly, comparison of the alkylation fingerprint with that
generated from an artemisinin ABPP equivalent confirms
a highly conserved alkylation profile, with both endoperoxide
classes targeting proteins in the glycolytic, hemoglobin degra-
dation, antioxidant defence, protein synthesis and protein stress
Figure 1. Artemisinin 1a, semi-synthetics 1b and 1c with structures of
frontline synthetic peroxide based antimalarials OZ277 (2b) and
pathways, essential biological processes for plasmodial sur-
vival. The alkylation signatures of the two chemotypes show
significant overlap (ca. 90%) both qualitatively and semi-
quantitatively, suggesting a common mechanism of action that
raises concerns about potential cross-resistance liabilities.
OZ439 (2c).
D
espite concerns about the recent emergence of drug-
resistance,[1] the artemisinins (1a–c, Figure 1) remain front-
line agents for the treatment of malaria.[2] Understanding the
mechanism of action of such an important class has been the
subject of intense research over the last two decades.[2] The
proposed mechanism of bioactivation of the class involves the
cleavage of the endoperoxide bridge by a source of Fe2+ or
heme. This cleavage results in the formation of oxy-radicals
that rearrange into primary or secondary carbon centered
radicals (or electrophilic carbocations through single-electron
transfer oxidation) (Scheme 1).[3]
Scheme 1. Iron-mediated fragmentation of endoperoxides to reactive
intermediates capable of reacting with parasite proteins. (Only the
secondary carbon-centered radical derived from artemisinin is
depicted.)
These reactive intermediates are proposed to alkylate
proteins and form adducts with essential parasite macro-
molecules that result in the rapid death of the parasite.
However, the detail of these important alkylation reactions
are sparse and the underlying hypothesis remains controver-
sial.[2]
The debate has broadened with the development of highly
active fully synthetic endoperoxides based on the pharmaco-
phore of artemisinin namely the trioxolanes (2a)[4] and the
tetraoxanes.[5] From the perspective of the underlying chem-
ical mechanism of activation of peroxides and from a cross-
resistance risk it is important to establish if these different
endoperoxide chemotypes share a common mechanism of
action or not.
[*] V. E. Barton, S. Charoensutthivarakul, P. M. O’Neill
Department of Chemistry, University of Liverpool
Liverpool, L69 7ZD (UK)
E-mail: pmoneill@liv.ac.uk
H. M. Ismail, M. Panchana, G. A. Biagini, S. A. Ward
Research Centre for Drugs and Diagnostics, Liverpool School of
Tropical Medicine
A study of 1,2,4-trioxolanes using monoclonal antibodies
has demonstrated parasite protein alkylation with both
OZ277 (2b) and OZ439 (2c).[6] However, the methods
Pembroke Place, Liverpool, L3 5QA (UK)
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201512062.
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employed in this work were unable to definitively identify the
targeted proteins.
mediated activation with no role for the copper in peroxide
activation during sample work-up as discussed previously.[9]
This Cu-free click reaction possesses comparable kinetics to
the Cu-catalyzed reaction and proceeds within minutes in live
cells with no apparent cytotoxicity issues.[8b] The complemen-
tary reporter tags used in our study can either be sourced
commercially or synthesized by literature procedures (Fig-
ure S1 in the Supporting Information).
In a recent study, Wang et al.[7] used a non-optimised
ART-alkyne activity-based protein-profiling probe that via
click-chemistry reactions was associated with some 124 P.
falciparum proteins.[7] In this study, a further 125 proteins are
reported as being identified in single replicate experiments
only, raising concerns of the specificity of the approach.[7]
Concurrently, using both ART-alkyne and azide optimized
probes we were able to identify 59 P. falciparum proteins with
high confidence that were specifically alkylated by ART
pointing towards a pleiotropic mechanism of drug action.[9] In
our study we deployed probes with reduced linker length and
lipophilicity compared to the probe used by Wang et al., and
we used both copper-dependent and copper-free reactions
over a shortened incubation time (optimized to 1 h as
originally described,[8] cf. Wang et al., reaction time of 3 h)
together with control non-peroxide probe partner equiva-
lents. These methodological differences help to improve the
specificity and pharmacological relevance of the alkylated
proteins identified. Significantly in our study only 6 proteins
were identified in less than two replicate experiments (cf. 125
in Ref. [7]) with an azide probe, demonstrating the improved
specificity of our approach using optimized active and control
probe based methodology.
Scheme 2a provides an overview for the synthesis of
alkyne probe P1 (6a) and azide probe P2 (7a) along with
control probes CP1 (6b) and CP2 (13b). The first step in the
Here, using our refined approach, we describe the rational
design of potent activity-based protein-profiling probes
(ABPPs) based on a 1,2,4-trioxolane antimalarial core in
order to characterize their malaria parasite protein alkylation
fingerprint. Significantly, we show that these synthetic 1,2,4-
trioxolanes and a semi-synthetic ART share an overlapping
parasite protein-alkylation signature suggestive of a common
mechanism of action for the endoperoxide class of antima-
larial.
The probes were designed with the alkyne/azide click
handle sited within the adamantane ring system since this is
the site (Scheme 1) of reactive C-radical/carbocation gener-
ation post activation by FeII. We also deployed a bio-
orthogonal copper-free “click” methodology via the use of
an azide analogue along with its negative control Figure 2.
The azide probes were included in our analysis to demon-
strate that the protein alkylation was solely due to iron
Scheme 2. Synthesis of probes and control molecules used in chemical
proteomic interrogation of drug activation.
synthesis of the trioxolane probes involved the Koch–Haaf
carbonylation of hydroxyl adamantanone to give the methyl
ester 3. Co-ozonolysis of oxime 4 with 3 provided trioxolane
5; hydrolysis of the methylester function of 5 followed by
EDC-mediated coupling of propargyl amine provided probe
P1 (6a) in good overall yield. Coupling of 3-azido-1-propyl-
amine to 6 provided the azide probe P2 (7a) as shown in
Scheme 1. The control probe CP1 (6b) was made by a similar
approach using diol 9 in a cyclisation reaction with 3 to
produce the corresponding carba ester analogue 10. Hydrol-
ysis of 10 and coupling of the resultant acid with propargyl
amine afforded 6b in good yield (70%). Azide 7b was made
in a similar manner by hydrolysis and coupling as shown in
Scheme 2b.
The active probes containing azide/alkyne functionality
retained potent antimalarial activity as determined by their
IC50 in vitro against P. falciparum 3D7 parasites (Figure S2
and Table S1). The non-peroxidic negative control probes
CP1 (6b) and CP2 (7b) had no appreciable activity (IC50
> 10 mm) in these assays,[10] confirming the essentiality of the
endoperoxide-bridge for drug activity and further validating
our probe plus control pair strategy for biologically relevant
target protein identification.
As a next step, in vitro cultures of P. falciparum 3D7 were
incubated with 1 mm of the alkyne trioxolane based probe P1
(6a) or its corresponding control CP1 (6b), for 6 hours, a time
already shown to be pharmacologically relevant, causing
Figure 2. Rational design of endoperoxide activity based probes P1
(6a) and P2 (7a) with structures of control non-peroxidic derivatives
CP1 (6b) and CP2 (7b).
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irreversible parasite toxicity.[7,9,10] Following incubation with
of an alkyne with an azide group in P2 (7a) had no
P1 (6a) alkylated proteins were extracted from erythrocyte
free parasites and tagged with Alexa Fluor 488 azide via
a click reaction. This was processed for 1D-Gel analysis as
described in the supporting information section. After
fluorescence imaging of the 1D-Gel, the strongest labeling
was observed in the region of 12–75 KDa (Figure S2c).
Importantly, labeling was not observed for the corresponding
“negative control” probe CP1 (6b) samples. Additional
experiments were carried out to investigate the lowest
concentration of reporter azide required to obtain maximum
labeling with minimum background on SDS gels (data not
shown). As depicted in Figure S2c, the Alexa Fluor 488 azide
at a concentration of 20 mm was able to distinguish the P1 (6a)
labeling profile from its corresponding control CP1 (6b).
To rule out the possibility that CuI may have led to
parasite independent (artifact) protein alkylation and to
further validate the results a more stringent bio-orthogonal
copper-free “click” methodology was employed using cyclo-
octyne reporter tags as depicted in Figure 3.[8b] Replacement
detrimental effect on the antimalarial potency of the triox-
olane azide probe as indicated in Figure S2, Figure 3 and
Table S1. However, the labeling profile intensity with P2 (7a)
was much higher compared to P1 (6a) (Figure 3c,d) suggest-
ing greater efficiency of the copper-free click reaction.[8b]
Reduction of P2 (7a) concentrations to 100 nm (LC90) had
no impact on the labeling pattern observed (Figure 3 f).
After validating the importance of the endoperoxide
bridge for protein alkylation using a 1D gel, we excluded the
gel electrophoresis step and advanced the method to direct
analysis of the alkylated protein matrix captured using an
“on-bead” trypsin digestion protocol as shown in Figure 3b
and Figure S3. Overall, multiple proteins critical to parasite
life were identified as trioxolane targets with the P2 (7a)
probe (Figure S3). No labeling was evident with CP2 (7b) the
negative control analogue or with the DMSO control (Fig-
ure S3). Having completed the analysis of the 1,2,4-trioxolane
proteome we carried out a head-to-head comparison of an
analogous ART activity based profiling probe (Figure 4).[9]
Figure 3. Labeling of parasite proteins (P. falciparum, 3D7 strain) using 7a. a) Chemical structure of ozonide azide probe (P2 (7a)) and deoxyether
analogue (CP2 (7b)) and their antimalarial activity against P. falciparum 3D7. b) General workflows of copper-free click methodology for in situ
parasite protein identification using azide trioxolanes probes as detailed mentioned in methodology section. c) Fluorescence image of 1D gel for
proteins labeled in situ with alkyne probes (P1 (6a) and CP1 (6b)) vs. azide probes (P2 (7a) and CP2 (7b)), note that no labeling occurs with
negative control alkyne (CP1 (6b)) and azide control (CP2 (7b)). d) Arbitrary fluorescence intensity measurements of the major protein bands
labeled and identified with 20 mm Alexa flour 488 azide for parasite proteins tagged with 1 mm of alkyne probe (P1 (6a)) vs. proteins tagged with
1 mm of azide probe (P2 (7a)) identified with 20 mm Click-IT Alexa Fluor 488 DIBO Alkyne. Fluorescence arbitrary units reveal higher sensitivity in
case of bio-orthogonal copper free click reaction, that is, P2 (7a) treatment. e) Gel image of P2 (7a) treatment vs. control, pre and post
coomassie stain with equal protein loading. f) Fluorescence image representing probe titration from 1 to 0.1 mm P2 (7a) probe; proteins identified
via copper free click reaction with Click-IT Alexa Fluor 488 DIBO Alkyne. No changes were observed in labeling profiles of the trioxolane-tagged
proteins with concentrations relevant to pharmacological concentration of the drug (100 nm). g) Titration of DIBO dye at various concentrations
up to 20 mm for parasite treated in situ with 1 mm P2 (7a). h) Time dependent increase of fluorescence signal for proteins tagged with 1 mm P2
(7a) and 20 mm Click-IT Alexa Fluor 488 DIBO Alkyne indicating that the maximum band intensities could be achieved after 1-hour of click
reaction incubation.
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Figure 4. Mass spectrometry experiments with azide trioxolane azide probe (P2 (7a)) vs. artemsinin azide probe (P3 (11a)). a) Venn diagram
demonstrating overlap between proteins identified with the endoperoxide probes, P2 (7a) and P3 (11a) respectively. (b) Percentage of the
glutathionylated proteins, which contains the GSH binding motif that was identified with endoperoxides probes P2 (7a) and P3 (11a) in light of
Kehr et al.[11] (c) Head to head comparison between proteins identified with P2 (7a) vs. P3 (11a). Proteins sorted according to their molecular
weight from high to low. Errors bars represented the standard deviation for protein quantity in each treatment calculated by dividing the
exponentially modified protein abundance index (emPAI)[16] for each protein by the total emPAI values (each treatment contain two replicate, for
accuracy each replicate is the average of four injections into the Orbitrap LC-MS/MS instrument).
Remarkably, as depicted in Figure 4, both the trioxolanes
probe P2 (7a) and ART probe P3 (11a) share strongly
overlapping protein-labeling profiles both qualitatively and
semi-quantitatively. From a total of 62 proteins confidently
identified with the two probes 53 of the proteins were tagged
with both P2 (7a) and P3 (11a) (Figure 4a), with no labeling
observed for control probes CP2 (7b) and CP3 (11b)
(Figure S3). From a mechanistic perspective it is important
to note that ca. 70% of the tagged proteins can be readily
glutathionylated (Figure 4b), a post translational modifica-
tion that can effect redox regulation and signal transduc-
tion.[11] For instance, EXP1, the membrane glutathione S-
transferase identified with both P2 (7a) and P3 (11a) was
reported to efficiently degrade cytotoxic hematin in malaria
parasite.[12] Interestingly, artesunate, a water-soluble deriva-
tive of ART, has been reported to inhibit the GST activity of
EXP1 in a hematin dependent manner at ca. 2 nm (IC50).[12]
PfEXP1 facilitates the conjugation of GSH with artesunate in
vitro.[12] We proposed that carbon centered radicals generated
from the reductive scission of the endoperoxide bridge from
either P2 (7a) or P3 (11a) alkylate proteins by C-radical
attack at the disulfide bridges of the glutathionylated proteins
identified in this study;[11,13] further work is on-going to
confirm this mode of reactivity. The major proteins in the
mass range 30 to 75 KDa identified for P2 (7a) are, PfLDH,
PfOAT and PfHGPRT, similar to that seen earlier with the
equivalent ART probes,[7,9] Collectively the data suggest that
1,2,4-trixolanes efficiently target plasmodial energy supply,
the antioxidant defence system and DNA synthesis. In
addition, ART has been shown to modulate a variety of
signaling pathways in cancer cells.[14] In the present study
many cytoskeletal proteins including a-tubulin, b-tubulin, and
actin 1 were labeled with trioxolanes and ART probes
suggesting a potential link between these endoperoxide
drugs and parasite cell structure, protein trafficking systems
and signal transduction.[9] Moreover, the global analysis of
protein alkylation generated through P2 (7a), for both
classes, that is, ART and trioxolanes, is consistent with the
“cluster bomb” hypothesis,[7,9,15] whereby Fe2+/heme-acti-
vated drug alkylates multiple redox-susceptible protein
targets functioning in multiple cellular pathways (Figure S4)
including the food vacuole, a site considered important for
iron dependent activation, and also in the cytosol (Figure S5).
To conclude, a chemical proteomic approach has for the
first time enabled formal identification of the key proteins
that are alkylated by the 1,2,4-trixolane class of antimalarial.
Significantly, the proteomic profile of 1,2,4-trioxolanes is
similar to the artemisinins suggesting that 1,2,4-trioxolanes
are multi-targeting like artemisinin and it remains to be seen
if a similar stress response and accumulation of ubiquinated
proteins occurs for this class of antimalarial in PfKelch13
resistant parasites.[17]
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Barton, S. Muangnoicharoen, P. G. Bray, J. Davies, B. K. Park, S.
Clearly, our data raises concerns of the potential cross-
resistance[18] between these two different antimalarial chemo-
types. Our optimised endoperoxide-ABPPs strategy has
generated a specific and robust set of tools to study potential
protein targets of the endoperoxide class of antimalarials.[19]
We are currently further refining this approach to accom-
modate a broader range of peroxide-based antimalarial
chemotypes. Work is also underway to establish the life-
cycle-dependent “endoperoxome” patterns in asexual and
sexual stages of P. falciparum parasite isolates with well-
characterized artemisinin drug resistance phenotypes to assist
in our understanding of this worrying clinical phenomenon.
Wittlin, R. Brun, M. Preschel, K. Zhang, S. A. Ward, Angew.
Chem. Int. Ed. 2010, 49, 5693 – 5697; Angew. Chem. 2010, 122,
5829 – 5833.
[6] J. Jourdan, H. Matile, E. Reift, O. Biehlmaier, Y. Dong, X. Wang,
P. Mäser, J. L. Vennerstrom, S. Wittlin, ACS Infect. Dis. 2016, 2,
54.
[7] J. Wang, C. J. Zhang, W. N. Chia, C. C. Loh, Z. Li, Y. M. Lee, Y.
He, L. X. Yuan, T. K. Lim, M. Liu, C. X. Liew, Y. Q. Lee, J.
Zhang, N. Lu, C. T. Lim, Z. C. Hua, B. Liu, H. M. Shen, K. S.
Tan, Q. Lin, Nat. Commun. 2015, 6, 10111.
[8] a) A. E. Speers, B. F. Cravatt, Chem. Biol. 2004, 11, 535 – 546;
b) J. M. Baskin, J. A. Prescher, S. T. Laughlin, N. J. Agard, P. V.
Chang, I. A. Miller, A. Lo, J. A. Codelli, C. R. Bertozzi, Proc.
Natl. Acad. Sci. USA 2007, 104, 16793 – 16797.
[9] H. M. Ismail, V. Barton, M. Phanchana, S. Charoensutthivar-
akul, M. H. Wong, J. Hemingway, G. A. Biagini, P. M. OꢀNeill,
S. A. Ward, Proc. Natl. Acad. Sci. USA 2016, 113, 2080.
[10] a) P. A. Stocks, P. G. Bray, V. E. Barton, M. Al-Helal, M. Jones,
N. C. Araujo, P. Gibbons, S. A. Ward, R. H. Hughes, G. A.
Biagini, J. Davies, R. Amewu, A. E. Mercer, G. Ellis, P. M.
OꢀNeill, Angew. Chem. Int. Ed. 2007, 46, 6278 – 6283; Angew.
Chem. 2007, 119, 6394 – 6399; b) W. Asawamahasakda, I. Ittarat,
Y. M. Pu, H. Ziffer, S. R. Meshnick, Antimicrob. Agents Chemo-
ther. 1994, 38, 1854 – 1858.
[11] S. Kehr, E. Jortzik, C. Delahunty, J. R. Yates 3rd, S. Rahlfs, K.
Becker, Antioxid. Redox Signaling 2011, 15, 2855 – 2865.
[12] A. M. Lisewski, J. P. Quiros, C. L. Ng, A. K. Adikesavan, K.
Miura, N. Putluri, R. T. Eastman, D. Scanfeld, S. J. Regenbogen,
L. Altenhofen, M. Llinas, A. Sreekumar, C. Long, D. A. Fidock,
O. Lichtarge, Cell 2014, 158, 916 – 928.
Acknowledgements
This work was funded by the Wellcome Trust through its
Strategic Support Fund award, Medical Research Council
Confidence in Concept funding, and through Mahidol–Liver-
pool PhD Scholarships to M.P. and S.C.. The work was also
supported from grants from the BBSRC (UK) (P.O.N., V.B. ,
S.A.W.; BB/C006321/1, BBS/B/05508, BBS/Q/Q/2004/06032,
and BBS/S/P/2003/10353) and in part by the EU (Antimal.
FP6 Malaria Drugs Initiative)
Keywords: antimalarial · artemisinin · chemical biology · probes
How to cite: Angew. Chem. Int. Ed. 2016, 55, 6401–6405
Angew. Chem. 2016, 128, 6511–6515
[13] a) C. H. Sohn, J. Gao, D. A. Thomas, T.-Y. Kim, W. A. God-
dard III, J. L. Beauchamp, Chem. Sci. 2015, 6, 4550 – 4560; b) K.
Becker, L. Tilley, J. L. Vennerstrom, D. Roberts, S. Rogerson, H.
Ginsburg, Int. J. Parasitol. 2004, 34, 163 – 189.
[14] C. Huang, Q. Ba, Q. Yue, J. Li, J. Li, R. Chu, H. Wang, Mol.
Biosyst. 2013, 9, 3091 – 3100.
[1] J. Straimer, N. F. Gnadig, B. Witkowski, C. Amaratunga, V.
Duru, A. P. Ramadani, M. Dacheux, N. Khim, L. Zhang, S. Lam,
P. D. Gregory, F. D. Urnov, O. Mercereau-Puijalon, F. Benoit-
Vical, R. M. Fairhurst, D. Menard, D. A. Fidock, Science 2015,
347, 428 – 431.
[15] R. G. Ridley, Nature 2003, 424, 887 – 889.
[16] Y. Ishihama, Y. Oda, T. Tabata, T. Sato, T. Nagasu, J. Rappsilber,
M. Mann, Mol. Cell. Proteomics 2005, 4, 1265 – 1272.
[17] C. Dogovski, S. C. Xie, G. Burgio, J. Bridgford, S. Mok, J. M.
McCaw, K. Chotivanich, S. Kenny, N. Gnadig, J. Straimer, Z.
Bozdech, D. A. Fidock, J. A. Simpson, A. M. Dondorp, S. Foote,
N. Klonis, L. Tilley, PLoS Biol. 2015, 13, e1002132.
[2] a) P. M. OꢀNeill, V. E. Barton, S. A. Ward, Molecules 2010, 15,
1705 – 1721; b) For a proposal that artemisinins may act by
impairing parasite redox homeostasis as a consequence of an
interaction between the drug and flavin adenine dinucleotide
(FADH) and/or other parasite flavoenzymes in the parasite see
R. K. Haynes, W. C. Chan, H. N. Wong, K. Y. Li, W. K. Wu,
K. M. Fan, H. H. Y. Sung, I. D. Williams, D. Prosperi, S. Melato,
P. Coghi, D. Monti, ChemMedChem 2010, 5, 1282 – 1289.
[3] G. H. Posner, D. Wang, J. N. Cumming, C. H. Oh, A. N. French,
A. L. Bodley, T. A. Shapiro, J. Med. Chem. 1995, 38, 2273 – 2275.
[4] a) J. L. Vennerstrom, S. Arbe-Barnes, R. Brun, S. A. Charman,
F. C. Chiu, J. Chollet, Y. Dong, A. Dorn, D. Hunziker, H. Matile,
K. McIntosh, M. Padmanilayam, J. Santo Tomas, C. Scheurer, B.
Scorneaux, Y. Tang, H. Urwyler, S. Wittlin, W. N. Charman,
Nature 2004, 430, 900 – 904; b) S. A. Charman, S. Arbe-Barnes,
I. C. Bathurst, R. Brun, M. Campbell, W. N. Charman, F. C.
Chiu, J. Chollet, J. C. Craft, D. J. Creek, Y. Dong, H. Matile, M.
Maurer, J. Morizzi, T. Nguyen, P. Papastogiannidis, C. Scheurer,
D. M. Shackleford, K. Sriraghavan, L. Stingelin, Y. Tang, H.
Urwyler, X. Wang, K. L. White, S. Wittlin, L. Zhou, J. L.
Vennerstrom, Proc. Natl. Acad. Sci. USA 2011, 108, 4400 – 4405.
[5] P. M. OꢀNeill, R. K. Amewu, G. L. Nixon, F. Bousejra ElGarah,
M. Mungthin, J. Chadwick, A. E. Shone, L. Vivas, H. Lander, V.
[18] Recent studies on other potential targets of artemisinin include
PfPI3K as
a resistance-associated early ring stage target,
although it may bind to artemisinin non-covalently. A. Mbengue,
S. Bhattacharjee, T. Pandharkar, H. Liu, G. Estiu, R. V. Stahelin,
S. S. Rizk, D. L. Njimoh, Y. Ryan, K. Chotivanich, C. Nguon, M.
Ghorbal, J. J. Lopez-Rubio, M. Pfrender, S. Emrich, N. Mohan-
das, A. M. Dondorp, O. Wiest, K. Haldar, Nature 2015, 520, 683 –
687.
[19] This work was first presented at the Royal Society of Chemistry
Biological & Medicinal Chemistry Sector Meeting on new
perspectives in DMPK held at The Chemistry Centre, Burling-
ton House, in London, February 2014 see http://blogs.rsc.org/
books/2014/03/06/best-poster-rsc-bmcs-meetin/.
Received: December 31, 2015
Revised: February 19, 2016
Published online: April 18, 2016
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