The antimalarial natural product symplostatin 4 is a nanomolar inhibitor of the food vacuole falcipains

Article abstract of DOI:10.1016/j.chembiol.2012.09.020

The marine natural product symplostatin 4 (Sym4) has been recognized as a potent antimalarial agent. However, its mode of action and, in particular, direct targets have to date remained elusive. We report a chemical synthesis of Sym4 and show that Sym4-treatment of P. falciparum-infected red blood cells (RBCs) results in the generation of a swollen food vacuole phenotype and a reduction of parasitemia at nanomolar concentrations. We furthermore demonstrate that Sym4 is a nanomolar inhibitor of the P. falciparum falcipains in infected RBCs, suggesting inhibition of the hemoglobin degradation pathway as Sym4's mode of action. Finally, we reveal a critical influence of the unusual methyl-methoxypyrrolinone (mmp) group of Sym4 for potent inhibition, indicating that Sym4 derivatives with such a mmp moiety might represent viable lead structures for the development of antimalarial falcipain inhibitors.

Full text of DOI:10.1016/j.chembiol.2012.09.020

Chemistry & Biology
Article
The Antimalarial Natural Product
Symplostatin 4 Is a Nanomolar Inhibitor
of the Food Vacuole Falcipains
Sara Christina Stolze,1,5 Edgar Deu, Farnusch Kaschani, Nan Li, Bogdan I. Florea, Kerstin H. Richau, Tom Colby,
2,5
1,5
4
4
3
3
3
4
2
1,
Renier A.L. van der Hoorn, Hermen S. Overkleeft, Matthew Bogyo, and Markus Kaiser *
Zentrum f u¨ r Medizinische Biotechnologie, Fakult a¨ t f u¨ r Biologie, Universit a¨ t Duisburg-Essen, Universit a¨ tsstr. 2, 45117 Essen, Germany
1
2Department of Pathology and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford,
CA 94305, USA
3Max Planck Institute for Plant Breeding Research, Carl-von-Linn e´ -Weg 10, 50829 Cologne, Germany
4Faculty of Science, Leiden Institute of Chemistry, Bio-organic Synthesis, Gorlaeus Laboratories, Einsteinweg 55, 2333 CC Leiden,
The Netherlands
5These authors contributed equally to this work
*Correspondence: markus.kaiser@uni-due.de
http://dx.doi.org/10.1016/j.chembiol.2012.09.020
SUMMARY
Due to an impressive structural diversity in combination with
often potent bioactivities, natural products have proven to
The marine natural product symplostatin 4 (Sym4) be valuable lead structures for drug discovery (Mayer et al.,
has been recognized as a potent antimalarial agent. 2010). Their further development into drugs is, however, often
hampered by a lack of knowledge of their mode of action.
Consequently, several strategies for the identification of the
However, its mode of action and, in particular, direct
targets have to date remained elusive. We report
a chemical synthesis of Sym4 and show that Sym4-
treatment of P. falciparum-infected red blood cells
direct targets of bioactive natural products have been developed
in the past few years (Lomenick et al., 2011; Rix and Superti-
Furga, 2009). Among them, proteome labeling strategies such
(
RBCs) results in the generation of a swollen food
as activity-based protein profiling (ABPP) have evolved into
reliable tools for the identification of protein targets of potentially
bioreactive natural small molecules (B o¨ ttcher et al., 2010;
Cravatt et al., 2008; Deu et al., 2012; Heal et al., 2011; van der
vacuole phenotype and a reduction of parasitemia
at nanomolar concentrations. We furthermore dem-
onstrate that Sym4 is a nanomolar inhibitor of
the P. falciparum falcipains in infected RBCs, sug- Hoorn et al., 2004).
gesting inhibition of the hemoglobin degradation
In 2009, the cyanobacterial secondary metabolites symplosta-
pathway as Sym4’s mode of action. Finally, we tin 4 (Sym4, Figure 1A) and gallinamide A were independently
isolated from the species Symploca sp. and Schizothrix sp.,
respectively (Linington et al., 2009; Taori et al., 2009).
reveal a critical influence of the unusual methyl-
methoxypyrrolinone (mmp) group of Sym4 for potent
inhibition, indicating that Sym4 derivatives with
such a mmp moiety might represent viable lead
structures for the development of antimalarial falci-
pain inhibitors.
Subsequent total syntheses of these two natural products and
structural characterizations revealed that both compounds are
in fact identical (Conroy et al., 2010, 2011). Subsequent biolog-
ical evaluations of Sym4 demonstrated their potent antimalarial
properties: In fact, gallinamide A (and, therefore, Sym4), as
well as three chemically synthesized diastereomers that differed
INTRODUCTION
only in the stereochemistry of their N-terminal isoleucine residue,
turned out to be potent nanomolar growth inhibitors of the
Malaria is a devastating disease that affects approximately malaria parasite P. falciparum (strain 3D7 and W2, IC50s of
15 million patients annually, among whom around 650,000 36–100 nM) (Conroy et al., 2010, 2011; Linington et al., 2009).
2
die (WHO, 2011). The spread of the disease can normally be Notably, no lysis of red blood cells (RBCs) was observed during
controlled by a combination of vector control and chemo- Sym4 treatment even at the highest tested concentrations
therapy. However, there is widespread resistance of the malaria (>25 mM) (Conroy et al., 2010), indicating that its antiparasitic
parasite to most front-line therapeutics. In recent years, an effect is not due to permeabilization of the RBC membrane.
emergence of resistance against artemisinin-based combination The molecular basis of this antimalarial activity, however, re-
therapy, which is the standard of care recommended by the mained elusive.
WHO for uncomplicated malaria, has furthermore been noted
Sym4 (Figure 1A) thereby displays several structural features
in Southeast Asia (Dondorp et al., 2009). Consequently, alterna- that are only rarely found in natural products. For example,
tive chemotherapeutic strategies for combating malaria are Sym4 features a (4S)-amino-(2E)-pentenoic acid that is linked
required at a constantly increasing rate (Guiguemde et al., with a methyl-methoxypyrrolinone (mmp) unit at its C-terminal
2
012; Wells et al., 2009).
end and an isocaproic acid moiety involved in an ester bond
1546 Chemistry & Biology 19, 1546–1555, December 21, 2012 ª2012 Elsevier Ltd All rights reserved

Chemistry & Biology
Falcipains as Direct Targets of Symplostatin 4
Figure 1. Structure and Synthesis of Sym4 and Sym4 Derivatives
(
A) Molecular structure of the parent natural product symplostatin 4 (Sym4).
B) Reagents and conditions: (a) 1. NH(OMe)Me*HCl, Et N, DCC, DCM; 2. LiAlH
O; 2. Meldrum’s acid, EDC, DMAP, DCM; 3. CH
CN, 99% (4), 85% (5); (e) 1. SOCl , MeOH/DCM; 2. BocNMeIleOH, DCC, DMAP, DCM,
5%; (f) TFA/DCM (1:1); 2. MeI, DIEA, DMF, 38%; (g) TFA/DCM (1:1); 2. Propargyl bromide, DIEA, CH CN, 63%; (h) 1. LiOH, THF/MeOH/H O; 2. 4 (after cleavage
); (i) 1. LiOH, THF/MeOH/H O; 2. 5 (after cleavage with TFA/
, TBTA, TCEP, H O, 23% (RhSym4), 67% (RhSym4
(
3
4
, Et
2
O, (EtO)
2
P(O) = CHCOOMe, DCM, 59%; (b) 1. LiOH, THF/MeOH/H
2
O; 2.
HAlaOMe*HCl, HOBt, HBTU, DIPEA, DCM, 46%; (c) 1. LiOH, THF/MeOH/H
THF, 90%; (d) 1. TFA/DCM (1:1); 2. BocLeuOH, HOBt, HBTU, DIPEA, CH
2
3
CN, reflux; 4. PPh , DEAD, MeOH,
3
3
2
5
3
2
mmp/Ala
mmp/Ala
with TFA/DCM [1:1]), HBTU, HOBt, DIPEA, CH
3
CN, 39% (Sym4
), 53% (hSym4
, CuSO
2
mmp/Ala
DCM [1:1]), HBTU, HOBt, DIPEA, CH
3
CN, 51% (Sym4), 64% (hSym4); (j) RhN
3
4
2
).
Chemistry & Biology 19, 1546–1555, December 21, 2012 ª2012 Elsevier Ltd All rights reserved 1547

Chemistry & Biology
Falcipains as Direct Targets of Symplostatin 4
with an N-terminally dimethylated isoleucine residue. The HBTU/HOBt activation and acetonitrile as the solvent (Jou
Michael system in the (4S)-amino-(2E)-pentenoic acid unit is, et al., 1997).
thus, potentially bioreactive (Drahl et al., 2005); in fact, covalently
The two different N-terminal depsipeptide fragments—i.e.,
binding cysteine protease inhibitors, proteasome as well as one building block with an N-dimethylated isoleucine residue
GAPDH inhibitors, with such Michael acceptor units have been (7) and the other one with an N-methyl-propargyl-modified
reported (Clerc et al., 2009a, 2009b; Groll et al., 2008; Kaschani isoleucine moiety (8)—were synthesized next. To this end,
et al., 2012; Powers et al., 2002). In Sym4, this chemical moiety is (S)-2-hydroxy-isocaproic acid was converted into a methyl
uniquely linked to a highly rigid mmp group, which could influ- ester and coupled with N-methylated Boc-isoleucine, thereby
ence the bioreactivity and/or target specificity of this natural yielding the desired depsipeptide 6. The Boc group was cleaved
product.
The favorable biological activities and intriguing structural group with either a methyl (7) or a propargyl group (8).
features of Sym4 raise the question of the underlying mode of For fragment assembly, the corresponding N- and C-terminal
and the resulting intermediate modified at its N-terminal amino
action of this antimalarial natural product. To this end, an eluci- building blocks were then coupled with each other.
dation of the direct molecular target(s) and of the structural Accordingly, the N-terminal dipeptide fragments 7 and 9 were
determinants for bioactivity is highly desirable. Therefore, in first C-terminally deprotected by saponification with lithium
the present study, we chemically synthesized Sym4 and a set hydroxide and then directly coupled to the Boc-deprotected
of analogs and characterized their antimalarial properties. In C-terminal tripeptide fragments 4 or 5, using HBTU/HOBt
addition, we identified falcipains as Sym4’s molecular targets activation. This approach delivered the desired natural product
and investigated the role of the mmp group for bioactivity.
Sym4 (51%) and the derivatives for structure-activity rela-
tionship studies (i.e., the alkyne-tagged derivative hSym4
mmp/Ala
RESULTS
[64%] as well as the corresponding Sym4
[39%]
[53%] derivatives without the mmp group).
mmp/Ala
and hSym4
Chemical Synthesis of Sym4 and its Derivatives
Finally, the propargyl-modified analogs hSym4 and
mmp/Ala
In order to obtain the required chemical probes for the target hSym4
were tagged with a rhodamine fluorophore,
+
identification studies as well as for the synthesis of Sym4 using a Cu -mediated Huisgens [3+2] cycloaddition and Rh-N
3
derivatives lacking the mmp group, we devised a convergent, (Kolb et al., 2001; Speers et al., 2003). This approach resulted
fragment-based approach that was used to synthesize Sym4 in the generation of two fluorescent probes RhSym4 and
mmp/Ala
as well as C- and N-terminally modified Sym4 derivatives (Fig- RhSym4
ure 1; Supplemental Experimental Procedures available online).
that were later used in the ABPP experiments.
To this end, we retrosynthetically divided Sym4 and the corre- Biological Assays Reveal the Critical Role of the mmp
sponding derivatives into two fragments, i.e., an N-terminal Group of Sym4 for Antimalarial Activity
depsipeptide moiety and a C-terminal tripeptide residue. Such With these compounds in hand, we first evaluated the effect of
mmp/Ala
an approach is beneficial because a ‘‘combinatorial’’ coupling Sym4 and Sym4
on malaria parasite replication during
of differently modified N- and C-terminal fragments allows an effi- the erythrocytic cycle. To this end, a culture of P. falciparum
cient, cost-effective, and rapid generation of various Sym4 D10 parasites at ring stage and 2% parasitemia was treated
derivatives.
With this plan in mind, we started our synthesis with the tions of Sym4 and Sym4
for 75 hr (i.e., one and a half life cycles) with different concentra-
mmp/Ala
(Figure 2A). After 75 hr, the
generation of two different C-terminal fragments that varied in parasites reached schizont stage with completed DNA re-
the presence or absence of the mmp group (Figure 1B). Accord- plication, thereby allowing an efficient differentiation between
ingly, Boc-Ala-OH was converted to an a,b-unsaturated methyl infected and noninfected RBCs via propidium iodide staining
ester 1 in 59% yield using a protocol introduced by Pollini and and subsequent parasitemia quantification via flow cytometry
coworkers (Benetti et al., 2002). In the next step, ester 1 was as described previously (Deu et al., 2010). Sym4 (EC50
=
mmp/Ala
deprotected using lithium hydroxide and coupled with alanine 0.7 ± 0.2 mM) was 40-fold more potent than Sym4
methyl ester hydrochloride to obtain dipeptide intermediate (EC50 = 27 ± 7 mM), indicating that the C-terminal mmp group
. Dipeptide 2 was deprotected C-terminally with lithium significantly contributes to the antiparasitic activity of this
hydroxide and the resulting intermediate was coupled with compound.
2
Meldrum’s acid, yielding a highly reactive enol intermediate
In order to better understand the mechanism of action of
that rearranged into a pyrrolidine-2,4-dione moiety upon reflux Sym4, we next investigated whether treatment with these
in acetonitrile. The use of Mitsunobu conditions (DEAD, meth- compounds induces a specific phenotype. To this end, ring
anol, and triphenylphosphine) then transformed this pyrroli- stage parasites were treated with different concentrations of
mmp/Ala
dine-2,4-dione into the desired mmp-modified dipeptide 3 in Sym4 and Sym4
. After 24 hr, their morphology was
0% yield (Patino et al., 1992). The final C-terminal tripeptide visualized by Giemsa-stained thin blood smears. In this assay,
9
building block 5 was then obtained in 85% yield by Boc depro- cells treated with 0.1 mM Sym4 already showed a distinct swollen
tection of 3, followed by a coupling step with Boc-Leu-OH and food vacuole phenotype (Figure 2B, upper panel). On the other
mmp/Ala
HBTU/HOBt activation and acetonitrile as the solvent (Jou et al., hand, a 100-fold higher concentration of Sym4
was
1
997). In contrast, the tripeptide building block 4 that lacks required to cause the same effect (Figure 2B, lower panel). In
a mmp group but features, instead, an alanine residue was many cases, such a phenotype is caused by accumulation of
obtained by acidic Boc deprotection of 2 and subsequent nondigested hemoglobin or oligopeptides in the food vacuole,
coupling of the free amine to Boc-Leu-OH, again with generally due to inhibition of proteases involved in this pathway
1548 Chemistry & Biology 19, 1546–1555, December 21, 2012 ª2012 Elsevier Ltd All rights reserved

Chemistry & Biology
Falcipains as Direct Targets of Symplostatin 4
mmp/Ala
mmp/Ala
Figure 3. Labeling Pattern of RhSym4 and RhSym4
Labeling of intact parasites with RhSym4 and RhSym4
. Purified
schizonts were pretreated with 10 mM of the nonfluorescent compounds
mmp/Ala
mmp/Ala
Sym4, hSym4, Sym4
, hSym4
, or DMSO. This was fol-
mmp/Ala
lowed by 1 hr incubation with 10 mM RhSym4 or RhSym4
. The
labeling reaction was stopped by boiling the samples in gel electrophoresis
loading buffer. Proteins were resolved by SDS-PAGE and fluorescent proteins
were visualized with a flatbed fluorescence scanner. The figure was assem-
bled using two regions of the same gel. Lanes irrelevant to the conclusion were
omitted (indicated by white box).
Figure 2. Comparison of the Biological Activities of Sym4 and
Sym4
mmp/Ala
(
A) Sym4 inhibits P. falciparum growth at ring stage with an EC50 = 0.7 ± 0.2 mM
See also Figures S3 and S4.
mmp/Ala
and thus at significantly lower concentrations than Sym4
mM). Error bars indicate SD from three independent experiments.
B) Sym4 induces a swollen food vacuole phenotype (indicated by arrows)
(EC50 = 27 ±
7
(
at concentrations as low as 0.1 mM, thereby indicating inhibition of the
10 mM. However, at concentration of 10 mM and above an accu-
mulation of mature schizonts was observed (Figure S1). This
effect was not permanent as these parasites were able to egress
and invade RBCs normally if cultured for another 24 hr in the
presence of Sym4 (Figure S1). Therefore, we assume that this
delay in rupture results from an inhibition of the hemoglobin
degradation by Sym4, which is known to slow down parasite
hemoglobin degradation pathway (Rosenthal, 2004, 2011). Intriguingly,
mmp/Ala
Sym4
, the Sym4 derivative with alanine instead of the methyl-
methoxypyrrolinone unit, requires concentrations of 10 mM to achieve the
same phenotype.
See also Figure S1.
(Rosenthal, 2004, 2011). In fact, two distinct food vacuole development.
defects have been reported in the literature: (1) A red swollen
food vacuole that is observed when proteases involved at the Labeling of Infected Blood Samples with RhSym4
initial stages of hemoglobin degradation are inhibited (falcipains Reveals One Major Target for Sym4
0
2
/2 /3). This results in an accumulation of undigested hemo- To clarify whether Sym4 covalently modifies its targets, we
globin that still contains the heme group (red color). (2) A clear compared the labeling patterns of P. falciparum-infected blood
mmp/Ala
swollen food vacuole was recently reported due to an inhibition samples treated with RhSym4 and RhSym4
. To this
of aminopeptidases (Harbut et al., 2011), which does not block end, intact schizonts were incubated with the rhodamine-
the initial breakdown of hemoglobin but results in the accumula- modified compounds in the presence or absence of their respec-
tion of undigested peptides (clear). In the case of Sym4 treat- tive nonfluorescent derivatives. After incubation, proteins were
ment, we observed a red food vacuole defect, which indicates isolated and resolved by denaturing PAGE, and the fluorescently
that Sym4 blocks the initial stages of hemoglobin degradation. labeled proteins were visualized using a flatbed fluorescence
We did not observe lysis of RBCs with either of the two scanner (Figure 3). In the case of RhSym4, two strongly labeled
compounds, which is in accordance with previously published protein bands in the 28 kDa region became visible. The labeling
data (Conroy et al., 2010). Finally, a dead phenotype was event was sensitive to preincubation with the natural product
observed at 100 mM (Figure 2B). This is most likely due to Sym4, as well as with the closely related analog hSym4,
a general toxic effect caused by the reactivity of the (4S)- indicating that the interaction is indeed dependent on the struc-
amino-(2E)-pentenoic acid unit found in this class of compounds. ture of the compounds and not an artifact caused by the rhoda-
To determine whether Sym4 had any effect on parasite egress mine group. On the other hand, the same experiment with
mmp/Ala
from infected RBCs or erythrocyte invasion, we then treated RhSym4
revealed only weak labeling in the 28 kDa
trophozoites for 24 hr with different concentrations of Sym4 region. Importantly, these results correlate well with the different
mmp/Ala
and analyzed the parasites morphology by Giemsa-stained potency of Sym4 and Sym4
thin blood smears (Figure S1). No effects were observed below (Figures 2A and 2B).
in the parasite assays
Chemistry & Biology 19, 1546–1555, December 21, 2012 ª2012 Elsevier Ltd All rights reserved 1549

Chemistry & Biology
Falcipains as Direct Targets of Symplostatin 4
Competitive Activity-Based Protein Profiling Identifies
Falcipains as Sym4 Targets
The formation of a food vacuole defect by Sym4, the known bio-
reactivity of vinyl amino acid derivatives toward cysteine prote-
ases, and the labeling of proteins at 28 kDa by RhSym4 strongly
suggested that one or more of the food vacuole’s falcipains (FP2,
0
FP2 , and FP3) (Rosenthal, 2004, 2011) might be targets of
Sym4. These proteases are plasmodial papain-like cysteine
proteases (PLCPs) that are known to migrate at 28 kDa in an
SDS-PAGE and are important for the proper development of
the parasite in the blood stages (Greenbaum et al., 2002b; Pan-
dey et al., 2006). FP1 is the only falcipain outside of the food
vacuole and is believed to play a role in RBC invasion (Green-
0
baum et al., 2002b). FP2, FP2 , and FP3 are involved in the
hemoglobin degradation pathway, which is the main source of
amino acids for the developing parasite (Sijwali and Rosenthal,
2
004). Importantly, inhibition of these three proteases causes
the same phenotype as the one observed for Sym4 treatment
i.e., a swollen food vacuole and block of parasite replication;
(
Figure 2) (Moura et al., 2009; Rosenthal et al., 1988). The pheno-
type observed with Sym4 is also reminiscent of the initial
phenotype reported for the P. falciparum FP2 knockout strain.
Parasites lacking FP2 undergo a transient food vacuole swelling
at trophozoite stage that resolves in the schizont stage due to
concomitant expression of FP3 (Sijwali and Rosenthal, 2004;
Sijwali et al., 2006).
0
Figure 4. Characterization of Sym4 Specificity Using Activity-Based
Protein Profiling with Cy5-DCG-04
In order to probe if Sym4 targets falcipains 2, 2 , and 3, we
performed competitive ABPP experiments with the activity-
based PLCP probe Cy5-DCG-04 (Greenbaum et al., 2000). In
competitive ABPP experiments, biological samples are first pre-
incubated with the small-molecule inhibitors of interest, followed
by visualization of residual enzyme activity by a fluorescent
probe (Clerc et al., 2011; Cravatt et al., 2008; Fonovi cꢀ and
(
A) P. falciparum D10 lysates (in 50 mM NaOAc, pH 5) were preincubated for
mmp/Ala
3
0 min with different concentrations of Sym4 or Sym4
, followed by
addition of 1 mM Cy5-DCG-04. After 1 hr, the reaction was stopped by adding
SDS-PAGE loading buffer and boiling for 5 min. Proteins were resolved by
SDS-PAGE and visualized by in-gel detection of fluorescence on a flatbed
0
fluorescence scanner. Sym4 inhibited FP2/2 /3 at low nanomolar concentra-
mmp/Ala
tions and FP1 at concentrations above 1.6 mM. In contrast, Sym4
was
Bogyo, 2008; Jeffery and Bogyo, 2003). In general, such an
approach allows the evaluation of inhibitory potency and speci-
ficity under physiologically relevant conditions.
0
a much weaker FP2/2 /3 inhibitor and did not inhibit FP1 at the concentrations
used. DPAP1, dipeptidyl aminopeptidase
Greenbaum et al., 2002b).
1 (Arastu-Kapur et al., 2008;
To this end, parasite lysates were incubated first with different
mmp/Ala
(B) Sym4 inhibits the falcipains in living parasite. A culture of P. falciparum at
trophozoite stage was treated for 1 hr with different concentrations of Sym4.
Parasite pellets were separated from RBCs by saponin lysis and residual PLCP
activities were measured by lysing the pellets in acetate buffer containing 1%
NP40 and 1 mM of Cy5-DCG-04. After 1 hr, the reaction was stopped by adding
SDS-PAGE loading buffer. Proteins were resolved by SDS-PAGE and visual-
ized by in-gel detection of fluorescence on a flatbed fluorescence scanner.
concentrations of Sym4 or Sym4
and then labeled with
Cy5-DCG-04. Subsequently, proteins were separated by elec-
trophoresis and visualized by in-gel fluorescence detection (Fig-
ure 4A). The advantage of using Cy5-DCG-04 is that its targets in
P. falciparum-infected RBCs have already been identified in
previous studies (Greenbaum et al., 2002a, 2002b; Pandey
et al., 2006). Therefore, a reduction in the intensity of one of
the known signals reveals this PLCP as a target of the tested
compounds. Satisfyingly, Sym4 was found to be an astonish-
0
Sym4 inhibited falcipains 1, 2/2 , and 3 at high, low, and medium nanomolar
0
FP1
FP2/2
FP3
concentrations (IC50
= 140 ± 23 nM, IC50
= 8.5 ± 1.3 nM, IC50
= 22 ±
8
nM), respectively. Sym4 did not inhibit DPAP1 at 1 mM.
See also Figures S2–S5.
0
ingly potent inhibitor of FP2, FP2 , and FP3, inhibiting these
proteases at low nanomolar concentrations (1.5 nM). FP1 was proteases during the erythrocytic cycle and are involved in
also inhibited, but at significantly higher concentrations hemoglobin degradation (Klemba et al., 2004; Deu et al., 2010)
mmp/Ala
(
>1.6 mM). In contrast, Sym4
proved to be much less and parasite egress (Arastu-Kapur et al., 2008), respectively.
0
potent, inhibiting Cy5-DCG-04 labeling of FP2, FP2 , and FP3 Competitive ABPP with either the probe Cy5-DCG04 or the
only at concentrations above 25 mM, while no inhibition DPAP activity-based probe FY01 showed that neither DPAP1
of FP1 labeling was observed. Interestingly, hSym4 and (Figure 4) nor DPAP3 (Figure S3) is inhibited by Sym4. Conse-
mmp/Ala
hSym4
showed an inhibition pattern similar to their quently, these experiments indicate that Sym4 does not act as
corresponding parent compounds (Figure S2), indicating that a general inhibitor of P. falciparum cysteine proteases but rather
the N-terminal part of the protein is not essential for target preferentially inhibits food vacuole falcipains.
inhibition.
After these encouraging results, we turned our attention to the
Another class of plasmodial PLCPs is the dipeptidyl amino- determination of the inhibition potency of Sym4 in intact para-
peptidases (DPAPs). DPAP1 and DPAP3 are essential cysteine sites. To this end, purified schizonts were treated with different
1550 Chemistry & Biology 19, 1546–1555, December 21, 2012 ª2012 Elsevier Ltd All rights reserved

Chemistry & Biology
Falcipains as Direct Targets of Symplostatin 4
concentrations of Sym4 for 1 hr, RBCs were lysed with saponin, its direct molecular targets and (2) characterize the underlying
and the residual activity of PLCPs in the parasite pellets was structure-activity relationships and correlate them to the antima-
again labeled with Cy5-DCG-04 (Figure 4B). This assay larial activities.
confirmed that Sym4 is a nanomolar inhibitor of falcipains in
intact parasites, although this time we also observed potent inhi- Synthesis of Sym4 and Derivatives
bition of FP1. Quantification of the gel labeling pattern thereby We first developed a convergent synthesis to Sym4 and deriva-
0
revealed a preferential inhibition of FP2/2 over FP3 or FP1 tives. In order to establish a synthetic route that allows rapid
FP3
0
FP2/2
FP1
(
IC50
= 8.5 ± 1.3 nM, IC50
= 22 ± 8 nM, IC50
= 140 ± access to different Sym4 derivatives, a modular synthesis was
devised. Thus, in contrast to previous synthetic approaches,
2
3 nM; Figure 4B).
To further characterize the inhibitory potency of Sym4, we which relied on a stepwise assembly of different amino acid
subsequently determined the kinetics of substrate turnover building blocks, we chose a fragment condensation strategy
inhibition, using recombinantly produced FP2 or FP3 at different to assemble the Sym4 framework. Consequently, two different
inhibitor concentrations (Figure S5). As expected, this experi- N- and C-terminal fragments were synthesized via solution
ment confirmed that Sym4 is an irreversible inhibitor of these phase peptide synthesis and merged to four different Sym4
two falcipains. Moreover, and in accordance with the competi- derivatives. This approach was used to synthesize not only
tive ABPP experiments, FP2 turned out to be more potent than the natural product Sym4, but also derivatives lacking the
ꢀ1
ꢀ1
FP3 (FP2: k
= kobs / [I] = 7,030 ± 250 M
i
= kobs / [I] = 58,600 ± 1,400 M
s
versus FP3: C-terminal mmp group. In addition, derivatives with a propargyl
group at the N terminus were generated, which enabled the
ꢀ1
ꢀ1
k
i
s ).
In order to evaluate the bioreactivity pattern of Sym4, we next attachment of a fluorescent reporter rhodamine via a copper-
investigated if Sym4 also inhibits mammalian PLCPs or the pro- catalyzed click reaction in an additional reaction step (RhSym4
mmp/Ala
teasome. To this end, we performed additional competitive and RhSym4
ABPP experiments with bone marrow-derived mouse macro-
Altogether, the developed synthetic route allows a rapid and
phages in vivo. This mammalian cell line was preincubated facile generation of Sym4 and derivatives, using ‘‘standard’’
).
mmp/Ala
with 0.5 mM and 5 mM of Sym4, hSym4, Sym4
or hSym4
,
peptide chemistry manipulations. However, because malaria
mmp/Ala
. Then, selected residual protease activities mostly affects people in third world countries that cannot afford
were determined by incubating protein extracts with a broad- expensive medication, the cost of goods of a synthesis is always
band PLCP ABP (Greenbaum et al., 2002a) (Figures S4A and an important issue. Although the prices for the required starting
S4B) or with a proteasome-specific ABP (Kolodziejek et al., materials (i.e., mainly amino acids and other peptide chemistry
2
011) (Figures S4C and S4D). In these assays, Sym4, hSym4, reagents) have decreased significantly in the last few years,
mmp/Ala
mmp/Ala
Sym4
, or hSym4
did not inhibit the protea- the overall costs of the chemical synthesis of Sym4 derivatives,
some (Figures S4C and S4D). In contrast, we observed a concen- even if established on an industrial scale, will most probably
tration-dependent inhibition of cathepsins in these experiments. exceed the generally accepted $2 limit. Thus, for a chemothera-
Both Sym4 and hSym4 were found to inhibit cathepsin L at peutic utilization of Sym4, further compound optimization, in
0
.5 mM (Figure S4A). At the 5 mM concentration, cathepsins B, regard not only to its bioactivities but also to the cost of
H, S, and Z were also inhibited. As observed with the falcipains, synthesis, is still required; along these lines, Sym4 derivatives
the Sym4 derivatives lacking the mmp group were less active. featuring a structurally less complex N-terminal fragment but
mmp/Ala
mmp/Ala
Sym4
or hSym4
did not inhibit any cathepsins maintaining the biologically relevant C-terminal moieties might
at 0.5 mM, but did inhibit cathepsin L at 5 mM (Figure S4B). We be more appropriate drug candidates.
then tested the labeling properties of the fluorescent probes
mmp/Ala
RhSym4 and RhSym4
also with bone marrow-derived Falcipain Inhibition Is Responsible for the Antimalarial
mouse macrophages (Figure S4E). Interestingly, the obtained Activity of Sym4
labeling profile indicates, as expected from the competitive The evaluation of the biological activity of chemically synthe-
ABPP experiments, that the predominant target of Sym4 in sized Sym4 confirmed that this natural product is a potent anti-
mammalian cells is cathepsin L.
malarial compound, as was previously reported (Conroy et al.,
010, 2011). In phenotypic assays, we subsequently showed
2
DISCUSSION
that Sym4 causes a distinct food vacuole defect at nanomolar
concentrations (Figure 2). These findings guided our following
Half of the human population is threatened by malaria, and the target identification studies using competitive ABPP, which
urge to develop novel antimalarial therapies is becoming more demonstrated all four P. falciparum falcipains as targets of
and more pressing (Guiguemde et al., 2012). Compounds with Sym4 in intact parasites. However, we observed different inhibi-
0
antimalarial activity are frequently reported in the literature tion potencies: Sym4 most potently inhibited FP2/2 , followed by
(Nogueira and Lopes, 2011), but further development of these FP3 and FP1. Among the different falcipains, FP3 is the only one
compounds into drugs is often hampered by their difficult chem- for which knockout attempts have been unsuccessful, which
ical synthesis (or limited availability via natural product isolation) suggests it is the most important falcipain during P. falciparum
0
and/or their unknown mode of action.
blood stages. FP1 and FP2 knockout strains have no apparent
The natural product symplostatin 4 (Sym4) has been identified phenotype, with FP1 not being essential in the erythrocytic stage
as another promising antimalarial compound (Conroy et al., of malaria (Sijwali et al., 2004), while the FP2 knockout showed
2010, 2011). To investigate the mode of action of this promising a marked food vacuole defect (Sijwali et al., 2004; Sijwali and
0
antimalarial agent, we synthesized several analogs to (1) identify Rosenthal, 2004). To the best of our knowledge, the FP2/2
Chemistry & Biology 19, 1546–1555, December 21, 2012 ª2012 Elsevier Ltd All rights reserved 1551

Chemistry & Biology
Falcipains as Direct Targets of Symplostatin 4
double knockout has not been attempted, so we cannot rule out tested concentration; Figure S4C). However, a similar assay
that inhibition of both FP2 homologs is lethal to parasites. With testing for activity against mammalian cathepsins using
this caveat in mind, our findings suggest that FP3 inhibition might BODIPY-DCG04 (Greenbaum et al., 2002a) showed that Sym4
be the crucial factor for the antimalarial activity of Sym4. also reacts with mammalian cathepsins. Our assays indicated
Although we observed the formation of a food vacuole defect that among the different cathepsins, Sym4 preferentially reacts
at 0.1 mM Sym4, parasite killing (i.e., the antimalarial activity) with cathepsin L. In an uninfected mammalian cell line, Sym4
required 7-fold higher concentrations (EC50 = 0.7 mM). The inhibits a large portion of cathepsin L at a concentration of
different concentrations required to induce parasite killing on 0.5 mM and thus at a concentration comparable to the observed
the one hand, and the formation of the food vacuole defect on antimalarial effect of Sym4. In contrast, the cathepsins B, H, S,
the other hand, might at least partly result from the falcipain inhi- and Z required ten times higher concentrations (i.e., 5 mM) to
bition profile of Sym4. Although at lower concentrations Sym4 reach the same inhibition level (Figure S4A). Although the
0
first inhibits FP2/2 and thus causes a food vacuole phenotype, observed ‘‘off-target’’ inhibition of cathepsin L is not desirable,
higher concentrations are required for supplementary FP3 inhibi- a recent report indicated that only a high fractional inhibition of
tion, thereby inducing parasite killing. In any case, it is important cathepsins (i.e., more than 95% inhibition) leads to a consider-
to note that Sym4 inhibits falcipains in intact parasites with IC50
s
able biological effect (M e´ thot et al., 2008). Nevertheless, these
in the low to medium nanomolar range, which is much lower than findings indicate that further studies are required to evaluate
the observed EC50 of 0.7 mM in the replication assay. Several the impact of off-target cathepsin inhibition for the further
reasons might explain the discrepancy between these two utility of Sym4 as a guiding structure for the development of
values. First, high fractional and sustained inhibition of falcipains antimalarials.
might be required to impair parasite replication. This has been
observed for DPAP1, where sustained inhibition of more than thesized rhodamine analog RhSym4 strongly labels the food
0% of DPAP1 activity for several hours is required to effectively vacuole falcipains in intact parasites (Figure 3). To the best of
Finally, as a spin-off of our studies, we observed that the syn-
9
block parasite replication (Deu et al., 2010). Therefore, the our knowledge, this is the first activity-based probe that discrim-
required concentrations of Sym4 for impairing P. falciparum inates between the food vacuole falcipains and FP1, and the first
0
replication via inhibition of falcipains needs to be much higher fluorescent activity-based probe that labels FP2, 2 , and 3 in
than the actual IC50 value. The fact that Sym4 does not irrevers- intact cells. Therefore, development of probes based on the
ibly block egress also indicates that Sym4 does not inhibit other Sym4 scaffold might prove to be very useful tools for imaging
cysteine proteases involved in schizont rupture, such as DPAP3 PLCP activity in cells.
(
Figure S3) or human calpain-1 (Chandramohanadas et al., 2011;
Millholland et al., 2011). Second, the intramolecular Sym4 The mmp Group Is Critical for Efficient Inhibition
concentration and thus the ‘‘reservoir’’ of ‘‘free’’ Sym4 that is of Falcipains
available to target falcipains might be lower than anticipated, In order to better understand the structure-activity relationship of
thereby resulting in less efficient falcipain inhibition. Several Sym4, we tested different N- and C-terminal derivatives for their
mechanisms might lead to such a reduced Sym4 concentration. antimalarial activity. We found that changes at the N-terminal
For example, although our studies indicate that falcipains are cell end were well tolerated and had only a slight effect on bioactivity.
permeable, the uptake of extracellular Sym4 into the cytosol For example, the hSym4 derivative showed a similar inhibition
might be hampered or slowed down. In addition, it is reasonable pattern as Sym4 (Figures S2 and S4B). These findings indicate
to assume that off-target binding to other proteins or cell compo- that the N-terminal part of Sym4 is amenable for at least slight
nents reduce the amount of ‘‘free’’ Sym4; although some of modifications and not essential for target recognition. These
these binding events might be reversible, the reaction of Sym4 findings are indeed corroborated by previous synthetic studies
with host cathepsins such as cathepsin L (Figure 4A), for of Sym4 derivatives (Conroy et al., 2010, 2011).
example, is most probably irreversible, thereby persistently
On the other hand, replacement of the C-terminal mmp group
mmp/Ala
reducing Sym4 levels. Finally, intracellular Sym4 might also be with an alanine (Sym4
) results in severe loss of antima-
metabolically or chemically instable and thus intracellular larial potency. This is very interesting because the mmp group is
Sym4 levels could be subject to a continuous decline. Interest- rarely found in natural products, and most falcipain inhibitors re-
ingly, in our replication assay parasites were treated at ring ported so far have peptide-like structures at the C-terminal end,
mmp/Ala
stage, while for example FP3 is known to be expressed at similar to the Sym4
derivative. In parasite assays,
proved to be a much weaker inhibitor of the falci-
mmp/Ala
schizont stage (i.e., more than 24 hr after treatment). At this Sym4
point, Sym4 concentration may already be significantly reduced, pains than Sym4 (Figure 4), which explains its reduced antima-
thereby explaining the observed bioactivity difference between larial activity (Figure 2). Furthermore, our results indicate that
falcipain inhibition in intact parasites and the EC50 on infected the mmp group has a decisive influence on not only the potency
red blood cells.
of falcipain inhibition, but also for other PLCPs (Figures S4A and
Despite these drawbacks, our experiments revealed Sym4 as S4B). Although we have not further investigated the molecular
one of the most potent falcipain inhibitors reported so far basis of the different activity patterns, we anticipate that different
(
Teixeira et al., 2011). This potency is accompanied by a distinct factors might contribute to the observed increase of activity. For
selectivity pattern. By performing comparative ABPP ex- example, the mmp group might occupy a distinct binding pocket
periments with a specific proteasome probe in living cells in the active site of falcipains. The resulting increase in binding
(
MVB003), we could verify that Sym4 does not inhibit the affinity and thus local concentration of Sym4 would conse-
mammalian proteasome at concentrations up to 5 mM (highest quently favor the subsequent irreversible reaction with the active
1552 Chemistry & Biology 19, 1546–1555, December 21, 2012 ª2012 Elsevier Ltd All rights reserved

Chemistry & Biology
Falcipains as Direct Targets of Symplostatin 4
site cysteine, resulting in an overall improved inhibition rate.
Alternatively, the mmp group may also increase the overall
harvested 36 hr postinvasions. Parasites were released and isolated from
RBCs after saponin lysis of the RBC membrane. Proteins were extracted
from parasites by treatment with PBS containing 1% NP40. The soluble
fraction was subsequently separated from the insoluble one by centrifugation
chemical reactivity of Sym4. In fact, we observed a stronger
mmp/Ala
background labeling for RhSym4 versus RhSym4
(Fig-
(
Arastu-Kapur et al., 2008).
ure 3), suggesting that higher chemical reactivity may contribute
to the improved inhibition rate. On the other hand, it has been
frequently noticed that attaching fluorescent groups like rhoda-
mine or BODIPY to electrophiles like Michael systems or epox-
ides increases the off-target labeling of these ABPs when
compared to strictly biotinylated ABPs. Taken together, the
mmp group enhances the overall falcipain inhibition rate but
may also increase off-target effects. Therefore, future chemical
derivatizations of the Sym4 structure aiming at the improvement
of its inhibitory potential will have to be complemented by selec-
tivity studies.
Phenotypic Characterization of Sym4-Treated Parasites
A synchronized culture of the parasite at ring stage (2% parasitemia) was
treated with different concentrations of Sym4, Sym4 analogs, or DMSO. After
2
4 hr, phenotypic effects were observed by Giemsa-stained thin blood
smears. The food vacuole defect is clearly visible as an enlargement of this
acidic organelle. This is due to the inhibition of proteases in the food vacuole
that leads to the accumulation of undigested hemoglobin.
P. falciparum Replication Assay
A total of 200 ml synchronous D10 parasites at ring stage (2% parasitemia and
0
.5% hematocrit) were treated with the corresponding compounds and
cultured in 96-well plates for 75 hr until the DMSO controls reached schizont
stage. Quantification was performed by flow cytometry as previously
described (Wang et al., 2011). All fluorescence activated cell sorting measure-
ments were taken on a BD FACScan flow cytometer (Becton Dickinson). EC50
values for parasite death were obtained by fitting the parasitemia values to
a dose-response curve.
Finally, it is important to note that falcipains have been recog-
nized as potential targets for the development of antimalarials
for quite some time, and numerous compound classes have
evolved that address these enzymes (Teixeira et al., 2011).
Among them, peptidic small molecules featuring an electrophilic
warhead such as epoxides or Michael acceptor systems have
proven to be potent inhibitors. Despite intense efforts, none of
these compounds could be developed into a drug and, there-
fore, interest in falcipain inhibitors has slightly decreased in the
last few years. Sym4 is also based on an a,b-unsaturated
Michael acceptor system that is, however, C-terminally linked
to a mmp group, resulting in a structurally novel scaffold that
has, so far, not been investigated. Indeed, our findings indicate
that this unusual combination results in much more potent
inhibitors, suggesting that further studies, for example with
N-terminally modified, less peptidic derivatives, might represent
a feasible approach to finally tackle these drug targets.
Labeling of Cysteine Protease Activity with ABPs and Competition
of Labeling by Protease Inhibitors
Cy5-DCG04, a cell-impermeable fluorescently tagged activity-based probe
was used to label the activity of DPAP1 and falcipains in lysates. To test the
potency of Sym4 and analogous compounds, parasite lysates (diluted 1:10
2
in acetate buffer [50 mM sodium acetate, 5 mM MgCl , and 5 mM DTT at
pH 5.5]) were pretreated with the corresponding compounds for 30 min
followed by labeling for 1 hr with 1 mM of Cy5-DCG04. The reaction was
stopped by boiling the samples in SDS-PAGE loading buffer. Proteins were
resolved by SDS-PAGE and the different cysteine proteases were detected
using a Typhoon 9410 flat-bed fluorescence scanner (Amersham Biosciences,
GE Healthcare).
Labeling of proteins from intact parasites with RhSym4 and
mmp/Ala
RhSym4
was performed by incubating purified schizonts with
different concentration of RhSym4 for 1 hr in PBS followed by boiling of the
samples in loading buffer and separation of the different proteins by SDS-
PAGE. Labeled bands were detected using the Typhoon 9410 scanner.
To measure falcipain inhibition in living parasites, a culture of P. falciparum
at 20% parasitemia and 2% hematocrit was treated with different concentra-
tions of Sym4. After 1 hr of treatment, a 1 ml aliquot of culture was lysed with
SIGNIFICANCE
In summary, we have reported a total synthesis of Sym4 and
the rational synthesis of a series of analogs. Sym4 causes
a food vacuole phenotype in Plasmodium-infected RBCs
and inhibits pathogen replication with an EC50 of 0.7 mM.
We subsequently identified the falcipains as direct molec-
ular targets of Sym4, thereby explaining its potent antima-
larial properties. Finally, we elucidated the C-terminal mmp
unit as a critical component for potent inhibition of falci-
pains. Therefore, our findings not only provide the mecha-
nistic basis for the observed potent antimalarial properties
of the marine natural product symplostatin 4 (Sym4), but
they may also serve as a valuable guide for the future rational
design of potent falcipain inhibitors. Although an assess-
ment of the drug-like properties of Sym4 has yet to be
done, we anticipate that the synthesis of Sym4-like deriva-
tives opens new possibilities to the design of tool com-
pounds to investigate and develop antimalarial chemothera-
peutics that combat this devastating disease.
0
.15% saponin in PBS buffer, the parasite pellets were harvested, and residual
cysteine protease activity was detected by labeling with 1 mM of Cy5-DCG04
for 1 hr in acetate buffer containing 1% NP40. After SDS-PAGE, the labeling of
0
FP1, FP2/2 , FP3, and DPAP1 was quantified using the ImageJ software
(Abr a´ moff et al., 2004) and fitted to a sigmoidal dose response curve to obtain
IC50 values.
i
K Determination Using a Biochemical Activity Assay for FP2 and FP3
Inhibition values of recombinantly produced FP2 and FP3 were measured at
ꢁ
2
5 C in assay buffer (200 mM sodium acetate, 10 mM DTT, 0.01% Triton-X,
3
.625% glycerol, pH 6) containing 25 mM of Z-LR-AMC. Substrate turnover
was measured for 2 hr in a 96-well plate at 460 nm (lexcitation = 350 nm and
an emission cutoff filter at 435 nm) in a Spectramax M5 plate-reader (Molecular
Devices).
i
Accurate k values (corresponding to kobs / [I]) for Sym4 (second order rate
constant of inhibition) were obtained by incubation of FP2 or FP3 with different
concentrations of Sym4 and by recording the decrease in activity over time.
The rates of substrate turnover relative to the DMSO controls (v/v
to a simple irreversible inhibitor model:
0
) were fitted
EXPERIMENTAL PROCEDURES
ki
/
E + I
E ꢀ I
(Equation 1)
Parasite Culture, Harvesting, and Lysate Preparation
D10 P. falciparum were cultured and kept synchronous as previously
described (Arastu-Kapur et al., 2008). Trophozoite stage parasites were
v
=
expðꢀ kobs$½Iꢂ$tÞ:
v
o
Chemistry & Biology 19, 1546–1555, December 21, 2012 ª2012 Elsevier Ltd All rights reserved 1553

Chemistry & Biology
Falcipains as Direct Targets of Symplostatin 4
Synthesis of Sym4 and Sym4 Derivatives
Deu, E., Verdoes, M., and Bogyo, M. (2012). New approaches for dissecting
protease functions to improve probe development and drug discovery. Nat.
Struct. Mol. Biol. 19, 9–16.
The synthesis and characterization of compounds 1 to 8 as well as Sym4 and
Sym4 derivatives are described in Supplemental Experimental Procedures.
Dondorp, A.M., Nosten, F., Yi, P., Das, D., Phyo, A.P., Tarning, J., Lwin, K.M.,
Ariey, F., Hanpithakpong, W., Lee, S.J., et al. (2009). Artemisinin resistance in
Plasmodium falciparum malaria. N. Engl. J. Med. 361, 455–467.
SUPPLEMENTAL INFORMATION
Supplemental Information includes five figures and Supplemental Experi-
mental Procedures and can be found with this article online at http://dx.doi.
org/10.1016/j.chembiol.2012.09.020.
Drahl, C., Cravatt, B.F., and Sorensen, E.J. (2005). Protein-reactive natural
products. Angew. Chem. Int. Ed. Engl. 44, 5788–5809.
Fonovi cꢀ , M., and Bogyo, M. (2008). Activity-based probes as a tool for func-
tional proteomic analysis of proteases. Expert Rev. Proteomics 5, 721–730.
ACKNOWLEDGMENTS
Greenbaum, D., Medzihradszky, K.F., Burlingame, A., and Bogyo, M. (2000).
Epoxide electrophiles as activity-dependent cysteine protease profiling and
discovery tools. Chem. Biol. 7, 569–581.
The authors would like to thank Dr. Rosenthal for providing recombinant FP2
and FP3. This research was funded by the Max Planck Society and the
Deutsche Forschungsgemeinschaft HO 3983/4-1 (to R.H.), by an ERC starting
grant (No. 258413, to M.K.), and NIH Grants R01EB005011 and R01AI078947
Greenbaum, D., Baruch, A., Hayrapetian, L., Darula, Z., Burlingame, A.,
Medzihradszky, K.F., and Bogyo, M. (2002a). Chemical approaches for func-
tionally probing the proteome. Mol. Cell. Proteomics 1, 60–68.
(to M.B.).
Greenbaum, D.C., Baruch, A., Grainger, M., Bozdech, Z., Medzihradszky, K.F.,
Engel, J., DeRisi, J., Holder, A.A., and Bogyo, M. (2002b). A role for the
protease falcipain 1 in host cell invasion by the human malaria parasite.
Science 298, 2002–2006.
Received: April 23, 2012
Revised: September 5, 2012
Accepted: September 27, 2012
Published: December 20, 2012
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