Activity-based protein profiling reveals that secondary-carbon-centered radicals of synthetic 1,2,4-trioxolanes are predominately responsible for modification of protein targets in malaria parasites

Article abstract of DOI:10.1039/c9cc03719e

Endoperoxide-containing antimalarials, such as artemisinin and the synthetic trioxolane OZ439, are prodrugs activated by heme to generate primary and secondary carbon-centered radicals. We employed activity-based protein profiling (ABPP) to show that the

Full text of DOI:10.1039/c9cc03719e

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Activity-based protein profiling reveals that
secondary-carbon-centered radicals of synthetic
1,2,4-trioxolanes are predominately responsible
for modification of protein targets in malaria
parasites†
Cite this:DOI: 10.1039/c9cc03719e
Received 14th May 2019,
Accepted 9th July 2019
DOI: 10.1039/c9cc03719e
Chunyan Wei,‡a Cheng-Xiao Zhao,‡bc Sheng Liu,‡b Jin-Hui Zhao,‡b Zi Ye,b
b
Heng Wang,*ab Shi-Shan Yu*b and Chong-Jing Zhang
*
rsc.li/chemcomm
Endoperoxide-containing antimalarials, such as artemisinin and the disadvantages of artemisinin are its rapid clearance in vivo,
synthetic trioxolane OZ439, are prodrugs activated by heme to limited supply and impractical de novo synthesis. These chal-
generate primary and secondary carbon-centered radicals. We lenges stimulated the development of synthetically-accessible and
employed activity-based protein profiling (ABPP) to show that the longer-acting trioxolanes, such as OZ277 and OZ439.7 Mechanism
secondary-carbon-centered radical of 1,2,4-trioxolanes is primarily of action (MOA) studies revealed that the unique endoperoxide
responsible for protein labeling in malaria parasites.
group in artemisinin and synthetic trioxolanes was first activated
by heme to generate carbon-centered radicals, which subse-
Malaria caused by the protozoal parasite Plasmodium falciparum quently alkylate multiple proteins in malaria parasites.8 During
and related species is a mosquito-borne and life-threating the activation of the endoperoxide by heme, two different radicals
infectious disease, which leads to nearly a half million deaths are generated, namely, primary and secondary carbon-centered
globally each year.1 The first small molecule drug used to treat radicals, each with their own unique properties (Fig. 1A and B).9
malaria was quinine, extracted from the cinchona tree.2 Due to The primary carbon-centered radical is sterically smaller while the
limited access to this natural source during the Second World secondary-carbon-centered radical is more stable.10 However, the
War, chloroquine, a synthetically-derived quinoline was identi- relative contributions of each radical for antimalarial activity and
fied as an anti-malaria drug.3 Chloroquine and its derivatives specific protein targets have not been elucidated. Herein we
were extensively used from the 1930s until the 1950s.4 Unfortu- determine the protein targets of each radical and further refine
nately, widespread resistance to chloroquine eventually developed,
prompting a search for next generation anti-malaria drugs.
Chinese scientists led by Youyou Tu discovered the fascinating
natural product artemisinin (Qinghaosu) and showed that it
possesses unparalleled efficacy for treatment of malaria including
complicated malaria caused by chloroquine-resistant plasmodial
strains.5 Artemisinin established the pivotal role of the endoper-
oxide as a unique and effective antimalarial pharmacophore.6 The
a Department of Microbiology and Parasitology, Institute of Basic Medical Sciences,
Chinese Academy of Medical Sciences & School of Basic Medicine, Peking Union
Medical College, 5# Dong Dan San Tiao, Beijing, 100005, China.
E-mail: wanghpumc@163.com
b State Key Laboratory of Bioactive Substances and Functions of Natural Medicines
and Beijing Key Laboratory of Active Substances Discovery and Drugability
Evaluation, Institute of Materia Medica, Peking Union Medical College and
Chinese Academy of Medical Sciences, Beijing, 100050, China.
E-mail: yushishan@imm.ac.cn, zhangchongjing@imm.ac.cn
c School of Pharmaceutical Science, Shanxi Medical University, Taiyuan,
Fig. 1 Potential reaction pathways of artemisinin (A) and a synthetic
trioxolane (B) activated by heme. (a) Rearrangement; (b) 1,5-H shift;
(c) cleavage. (C) The structures of probes used in this study, ZJH-1 and
ZJH-2. RiPA, radical-induced protein alkylation.
Shanxi, 030001, China
† Electronic supplementary information (ESI) available: Intermediates and meth-
ods, and supplementary and extended figures. See DOI: 10.1039/c9cc03719e
‡ These authors contributed equally to this work.
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our mechanistic understanding of endoperoxide-containing anti-
malaria molecules.
To answer the above-mentioned mechanistic questions, we
used activity-based protein profiling (ABPP) to capture the
alkylated proteins of each radical. This was accomplished by
designing two chemical probes containing an alkyne group.
Alkynes are typically used in ABPP because they are sterically
small and can be selectively conjugated to a fluorophore or an
affinity tag employing bioorthogonal click-chemistry.11 Artemisinin
is not an ideal substrate for these studies since both the radicals
remain in the same scaffold (Fig. 1A); thus it is impossible to
discern the specific proteins labeled by each radical regardless
of where the alkyne group is introduced. On the other hand, the
synthetic trioxolane provides an ideal scaffold for our proposed
investigation. After heme-mediated activation followed by rear-
rangement and hydrolysis, synthetic trioxolane is transformed
into either an adamantane ketone and a primary-carbon-centered
radical, or a cyclohexane ketone and a secondary-carbon-centered
radical (Fig. 1B). The protein targets of the two radicals can be
easily traced by selecting appropriate attachment sites for the
Fig. 2 The viability of malaria parasites treated with 1,2,4-trioxlane-based
probes. The viability of malaria parasite was measured in the presence of
different concentrations of compound 3 (A), ZJH-1 (B) and ZJH-2 (C).
(D) The calculated IC50 of the three compounds (mean Æ standard deviation).
alkyne group. Following this design principle, incorporation of and ZJH-2 were confirmed by NMR spectroscopy and high-
the alkyne into the cyclohexane part (ZJH-1, Fig. 1C) will capture resolution mass spectrometry (ESI,† Fig. S1–S14).
the protein targets of the primary-carbon-centered radical while
We measured the antimalarial activity of probes ZJH-1 and
attachment of the alkyne group into the adamantane part (ZJH-2, ZJH-2 against P. falciparum 3D7 in vitro. Synchronization of
Fig. 1C) will capture the protein targets of the secondary-carbon- parasite cultures with two rounds of 5% (w/v) D-sorbitol was
centered radical.
performed as previously described.12 Compound 3 was included
Following this rationale, we synthesized probes ZJH-1 and as a positive control. The parasite sensitivity of compound 3,
ZJH-2 (Scheme 1). Oxime 1 and 2 were synthesized via the ZJH-1 and ZJH-2 was measured using SYBR Green I as an
reaction between the corresponding ketone and methoxyamine. indicator.13 The excitation and emission wavelengths of SYBR
The Griesebaum co-ozonolysis of 4-(methoxycarbonyl)cyclohex- Green I were set as 485 and 528 nm, respectively. All three
anone with oxime 2 afforded trioxolane 3. Saponification of 3 compounds could effectively inhibit the parasite growth and the
followed by EDC-mediated coupling with propargyl amine pro- calculated IC50 values were 12.67 nM, 7.32 nM and 17.36 nM for
vided the probe ZJH-1. Probe ZJH-2 was synthesized in a similar ZJH-1, ZJH-2 and compound 3, respectively (Fig. 2). These results
manner as described.8c The chemical structures of probes ZJH-1 indicate that incorporation of an alkyne in probes ZJH-1 and
ZJH-2 did not perturb the antimalarial activity confirming that
these probes phenocopy the parent trioxolane scaffold and are
thus suitable for ABPP.
To assess the ability of probes ZJH-1 and ZJH-2 to label
proteins in malaria parasites, different concentrations of probes
ZJH-1 and ZJH-2 ranging from 2 to 20 mM were incubated with
the mixed stage of P. falciparum parasite cultures for 4 hours at
37 1C. Red blood cells were removed by lysis with 0.1% saponin
and parasites were pelleted down by centrifugation. The labeled
proteome in the parasites was obtained by sonication and the
probe-modified proteins were labeled with a fluorescent dye
(rhodamine-azide conjugate) using standard copper-catalyzed
click chemistry conditions. The treated proteome was resolved
by denaturing gel electrophoresis and visualized by in-gel fluores-
cence scanning (Fig. 3A and ESI,† Fig. S15). We observed that ZJH-2
efficiently labeled proteins at the lowest probe concentration
examined (2 mM), whereas ZJH-1 displayed virtually no labeling
at this concentration and could only label a few proteins at
the highest concentration evaluated (20 mM). This result indicates
that the secondary-carbon-centered radical predominates protein
labeling of synthetic 1,2,4-trioxolanes in malaria parasites. The
Scheme 1 Synthetic routes of probes ZJH-1 and ZJH-2.
poor protein labeling of the primary-carbon-centered radical
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Fig. 4 Reaction between ZJH-2 and GSH in the presence of heme.
(A) Reaction scheme of ZJH-2 and GSH in the presence of heme and
sodium ascorbate; (B) mass spectrum of the main product of ZJH-2-P-S.
Fig. 3 (A) In situ labelling with different concentrations of probes ZJH-1
or ZJH-2 in malaria parasites. (B) Volcano plots of proteins pulled-down by
the probe ZJH-1 (20 mM) and ZJH-2 (2 mM). The P-value was set at 0.05,
and the cut-off value of fold change was 2. (C) Venny analysis of the
labeled proteins for ZJH-1 and ZJH-2.
To further support the result of the chemical proteomics
study, we carried out an in vitro experiment to check the
products of radical-induced alkylation. Briefly, probes ZJH-1
could be ascribed to two causes. First, because of the steric and ZJH-2 were respectively incubated with hemin and glu-
hindrance, the coordination of heme to the endoperoxide likely tathione (GSH) in the presence of sodium ascorbate. We
occurs at the least hindered oxygen atom resulting in generation checked the reaction by LC-MS. The expected reaction products
of the secondary-carbon-centered one. Second, the primary- between the probes and GSH are shown in Fig. 4A and ESI,†
carbon-centered radical is less stable than the secondary carbon- Fig. S17A. In the LC-MS profiles, we found that the main
centered one.
products generated by secondary-carbon-centered radicals
To further support the result of gel-based imaging, we next (ZJH-1-P-S and ZJH-2-P-S) have a single ion peak in the mass
employed gel-free quantitative proteomics to pull down and spectra with the expected m/z ratio (Fig. 4B, ESI,† Fig. S16C, D
identify the corresponding protein targets of ZJH-1 and ZJH-2 in and S18). However, it is difficult to find the ion peak for the
parasites. Briefly, the alkyne labeled parasite proteome was product generated by the primary-carbon-centered radicals
reacted with biotin-azide, followed by enrichment with strepta- (ZJH-1-P-P and ZJH-2-P-P) (ESI,† Fig. S16D and S17E). This
vidin beads. Enriched beads were subjected to on-bead diges- result also indicated that the secondary-carbon-centered radicals
tion with trypsin to release peptide fragments of bound are produced much more than the primary-carbon-centered ones
proteins. Collected peptides were separated and detected by after the trioxolane group was activated by heme.
LC-MS/MS and the results were processed with the label-free
In conclusion, we designed two molecular probes to inves-
proteomics method to identify the protein targets of ZJH-1 and tigate the protein labeling capacity of primary and secondary
ZJH-2. During data processing, we used a strict cut-off fold carbon-centered radicals generated by synthetic trioxolanes.
change of 2 and a p-value of 0.05 as the qualification criterion. Both gel-based and gel-free proteomics methods demonstrated
Accordingly the probe ZJH-1 targeted 22 proteins at the concen- that the secondary-carbon-centered radical was responsible for
tration of 20 mM, while ZJH-2 targeted 197 proteins at the nearly all the protein labeling in situ. These results provide a
concentration of 2 mM (Fig. 3B, ESI,† sheet 6 and sheet 7 in more nuanced insight into the unique mode of action of the
proteomics data.xlsx). There were 14 proteins labeled by both endoperoxide-containing antimalarials. This study may also aid
`
ZJH-1 and ZJH-2 (Fig. 3C and ESI,† Table S1). These results the further development of potent anti-malaria drugs.
confirm that the secondary-carbon-centered radical is much
We greatly thank Prof. Courtney Aldrich of the University of
more effective in protein-labeling than the primary-carbon- Minnesota for careful editing of our manuscript. We also thank
centered one. The recent clinical study of OZ439 showed that the start-up funding from the State Key Laboratory of Bioactive
the drug’s peak plasma concentration is around 3 mM.14 Taken Substances and Functions of Natural Medicines, Institute of
together, our finding suggests that the secondary-carbon- Materia Medica, Peking Union Medical College, and CAMS
centered radical from trioxolane accounts for nearly all the Innovation Fund for Medical Sciences (CIFMS) (2017-I2M-4-
alkylated proteins in vivo.
005, 2016-I2M-1-019).
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Conflicts of interest
The authors declare that there is no conflict of interest in the
publication of this article.
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