A prodomain peptide of Plasmodium falciparum cysteine protease (falcipain-2) inhibits malaria parasite development.
ABSTRACT Falcipain-2 (FP-2), a papain family cysteine protease of Plasmodium falciparum, is a promising target for antimalarial chemotherapy. Designing inhibitors that are highly selective for falcipain-2 has been difficult because of broad specificity of different cysteine proteinases. Because propeptide regions of cysteine proteases have been shown to inhibit their cognate enzymes specifically and selectively, in the present study, we evaluated the inhibitory potential of few falcipain-2 proregion peptides. A 15 residue peptide (PP1) inhibited falcipain-2 enzyme activity in vitro. Studies on the uptake of PP1 into the parasitized erythrocytes showed access of peptide into the infected RBCs. PP1 fused with Antennapedia homeoprotein internalization domain blocked hemoglobin hydrolysis, merozoite release and markedly inhibited Plasmodium falciparum growth and maturation. Together, our results identify a peptide derived from the proregion of falcipain-2 that blocks late-stage malaria parasite development in RBCs, suggesting the development of peptide and peptidometric drugs against the human malaria parasite.
- SourceAvailable from: cshlp.org[show abstract] [hide abstract]
ABSTRACT: The search for novel antimalarial drug targets is urgent due to the growing resistance of Plasmodium falciparum parasites to available drugs. Proteases are attractive antimalarial targets because of their indispensable roles in parasite infection and development, especially in the processes of host erythrocyte rupture/invasion and hemoglobin degradation. However, to date, only a small number of proteases have been identified and characterized in Plasmodium species. Using an extensive sequence similarity search, we have identified 92 putative proteases in the P. falciparum genome. A set of putative proteases including calpain, metacaspase, and signal peptidase I have been implicated to be central mediators for essential parasitic activity and distantly related to the vertebrate host. Moreover, of the 92, at least 88 have been demonstrated to code for gene products at the transcriptional levels, based upon the microarray and RT-PCR results, and the publicly available microarray and proteomics data. The present study represents an initial effort to identify a set of expressed, active, and essential proteases as targets for inhibitor-based drug design.Genome Research 05/2003; 13(4):601-16. · 14.40 Impact Factor
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ABSTRACT: New drugs to treat malaria are urgently needed. Cysteine proteases of malaria parasites offer potential new chemotherapeutic targets. Cysteine protease inhibitors block parasite hemoglobin hydrolysis and development, indicating that cysteine proteases play a key role in hemoglobin degradation, a necessary function of erythrocytic trophozoites. These inhibitors also block the rupture of erythrocytes by mature parasites, suggesting an additional role for cysteine proteases in the hydrolysis of erythrocyte cytoskeletal proteins. Recent studies have shown that the repertoire of cysteine proteases of malaria parasites is larger than was previously realized. Plasmodium falciparum, the most virulent human malaria parasite, expresses three papain-family cysteine proteases, known as falcipains. All three proteases are expressed by trophozoites and hydrolyze hemoglobin at acidic pH, suggesting roles in this process. Falcipain-2 also hydrolyzes ankyrin at neutral pH, suggesting additional activity against erythrocyte cytoskeletal targets. Multiple orthologs of the falcipains have been identified in other plasmodial species. Analysis of orthologs from animal model rodent parasites identified similar features, but some noteworthy biochemical differences between the cysteine proteases. These differences must be taken into account in interpreting in vivo experiments. A number of small molecule cysteine protease inhibitors blocked parasite hemoglobin hydrolysis and development, and inhibitory effects against parasites generally correlated with inhibition of falcipain-2. Some compounds also cured mice infected with otherwise lethal malaria infections. Current research priorities are to better characterize the biological roles and biochemical features of the falcipains. In addition, efforts to identify optimal falcipain inhibitors as antimalarials are underway.Current Pharmaceutical Design 02/2002; 8(18):1659-72. · 3.31 Impact Factor
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ABSTRACT: Papain-family cysteine proteases of the malaria parasite Plasmodium falciparum, known as falcipains, are hemoglobinases and potential drug targets. Available data suggest that papain-family proteases require prodomains for correct folding into functional conformations. However, in prior studies of falcipain-2, an Escherichia coli-expressed construct containing only a small portion of the prodomain refolded efficiently, suggesting that this enzyme differs in this regard from other papain-family enzymes. To better characterize the determinants of folding for falcipain-2, we expressed multiple pro- and mature constructs of the enzyme in E. coli and assessed their abilities to refold. Mature falcipain-2 refolded into active protease with very similar properties to those of proteins resulting from the refolding of proenzyme constructs. Deletion of a 17-amino acid amino-terminal segment of the mature protease yielded a construct incapable of correct folding, but inclusion of this segment in trans allowed folding to active falcipain-2. The prodomain was a potent, competitive, and reversible inhibitor of mature falcipain-2 (K(i) 10(-10) m). Our results identify a chaperone-like function of an amino-terminal segment of mature falcipain-2 and suggest that protease inhibition, but not the mediation of folding, is a principal function of the falcipain-2 prodomain.Journal of Biological Chemistry 05/2002; 277(17):14910-5. · 4.65 Impact Factor
A Prodomain Peptide of Plasmodium falciparum Cysteine Protease (Falcipain-2) Inhibits
Malaria Parasite Development
Reshma Korde,†Ashima Bhardwaj,†Rita Singh,‡Anand Srivastava,†Virander S. Chauhan,†Raj K. Bhatnagar,†and
International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India, Department of Zoology,
Delhi UniVersity, Delhi-110007
ReceiVed June 22, 2007
Falcipain-2 (FP-2), a papain family cysteine protease of Plasmodium falciparum, is a promising target for
antimalarial chemotherapy. Designing inhibitors that are highly selective for falcipain-2 has been difficult
because of broad specificity of different cysteine proteinases. Because propeptide regions of cysteine proteases
have been shown to inhibit their cognate enzymes specifically and selectively, in the present study, we
evaluated the inhibitory potential of few falcipain-2 proregion peptides. A 15 residue peptide (PP1) inhibited
falcipain-2 enzyme activity in vitro. Studies on the uptake of PP1 into the parasitized erythrocytes showed
access of peptide into the infected RBCs. PP1 fused with Antennapedia homeoprotein internalization domain
blocked hemoglobin hydrolysis, merozoite release and markedly inhibited Plasmodium falciparum growth
and maturation. Together, our results identify a peptide derived from the proregion of falcipain-2 that blocks
late-stage malaria parasite development in RBCs, suggesting the development of peptide and peptidometric
drugs against the human malaria parasite.
Malaria proteases are attractive antimalarial targets due to
their critical roles in parasite infection and development. In silico
approaches on parasite genome have identified 92 putative
proteases that are grouped into five major classes (aspartic,
cysteine, metallo, serine, and threonine) in the Plasmodium
falciparum genome.1Among these proteases, cysteine and
aspartic proteases of P. falciparum known as falcipains and
plasmepsins respectively, play an important role in parasite food
assimilation by their ability to degrade hemoglobin. Because
hemoglobin degradation is an essential step in the parasite life
cycle, blocking of these proteases offers an attractive target to
arrest development of malaria parasite.2–4
Studies with protease inhibitors have suggested that malaria
cysteine proteases are important for the hydrolysis of hemo-
globin, erythrocyte rupture, and erythrocyte invasion.5,6In P.
falciparum, four falcipains (falcipain-1, falcipain-2 and -2′, and
falcipain-3) have been described at intraerythrocytic stage of
parasite life cycle and investigations are on to elucidate their
physiological role(s).7–10These enzymes belong to the papain
superfamily with a number of unique features. Genes encoding
falcipain homologues have also been identified in other plas-
One of the difficulties in developing chemotherapeutic
inhibitors against cysteine proteases is the ubiquitous presence
of these enzymes in viruses, prokaryotes, and higher organisms
and the identity of their catalytic mechanism.12These enzymes
also have a broad substrate specificity, which makes it difficult
to develop inhibitors that specifically inhibit individual protein-
ases.13As a consequence of these limitations, propeptide regions
of different cysteine proteases are being employed to specifically
inhibit individual protease.13–17Prosegment of proteases, espe-
cially cysteine proteases, have been shown to play a role in
protein folding, intracellular trafficking, secretion of mature
protease, and in the control of proteolytic activity by masking
the active site.16,18–20Like many proteases, parasite cysteine
proteinases are also synthesized as inactive zymogens that are
converted to the mature form upon proteolytic cleavage and
the release of the proregion polypeptide.21,22Recent studies on
the mapping of the proregion peptides have revealed that
subpeptides derived from the proregions inhibit their cognate
enzymes in a selective manner.13For example, propeptide
derived from cathepsin L of Fasciola hepatica is a selective
inhibitor of the parasite cathepsin L and does not inhibit human
cathepsin L.23Similarly, peptides containing pentapeptide
sequence (LCGTV) from the proregion of trypanosomal cysteine
proteinase, congopain, were found to be competitive inhibitors
of the corresponding trypanosomal cysteine proteinases but did
not inhibit rat cathepsins B and L.24,25
Of the four identified cysteine proteases of P. falciparum,
falcipain-2 is the most intensely studied enzyme, and recent
functional and structural data suggest that it is an attractive target
for therapeutic intervention.26,27In comparison to other papain
family proteases, falcipain-2 possesses an unusually long
prosequence that is 2-3 times larger than in related enzyme
prosequences.21Unlike other related proteases, falcipain-2 does
not require its prodomain for acquiring a catalytically competent
conformation.21,28Nevertheless, recombinant proregion of fal-
cipain-2 has been shown to inhibit falcipain-2 activity ef-
fectively.29Considering the uniqueness and sequence divergence
of falcipain-2 proregion, we explored the possibility of using
small prodomain peptides of falcipain-2 for evaluating their
antimalarial activity. A series of peptides from the prosegment
of falcipain-2 were designed, and their effects on falcipain-2
catalytic activity and parasite development in vitro were studied.
This study establishes the inhibitory potential of a peptide within
* To whom correspondence should be addressed. Phone: 91 11 26741358.
Fax: 91 11 26742316. E-mail: firstname.lastname@example.org. Mailing address:
International Centre for Genetic Engineering and Biotechnology, Aruna Asaf
Ali Marg, New Delhi 110067, India.
†International Center for Genetic Engineering and Biotechnology.
‡Department of Zoology, Delhi University.
J. Med. Chem. 2008, 51, 3116–3123
10.1021/jm070735f CCC: $40.75
2008 American Chemical Society
Published on Web 05/08/2008
the prosegment of falcipain-2 that has the potential of being
used as a therapeutic molecule.
Inhibition of Falcipain-2 Activity by Synthetic Proregion
Derived Peptides. To delineate inhibitory segments in the
falcipain-2 proregion sequence, we designed five peptides (PP1,
PP2, PP3, PP4, and PP5)acorresponding to the -89aa to -44aa
of the falcipain-2 proregion (Figure 1). This segment was chosen
to design small peptides because it includes a conserved motif
ER(F/W)N(I/V)N that is considered to be a functional unit
involved in the inhibition of cathepsin L-like cysteine pro-
teases.30Table 1 depicts the sequence of each peptide and their
molecular masses. To analyze the effect of prodomain peptides
on falcipain-2 activity, active recombinant falcipain-2 was
refolded and purified to near homogeneity by affinity chroma-
tography and ion-exchange chromatography using a protocol
described previously by Shenai et al., 200021(Figure 2A,B).
The refolded protein was enzymatically active as it cleaved
Z-FR-AMC, a substrate of falcipain-2, and enzymatic activity
of the protein was significantly inhibited by a broad spectrum
inhibitor of cysteine protease, E-64 (Figure 2C). The effect of
different proregion peptides on enzyme activity was analyzed
by preincubating the active falcipain-2 with each of these
peptides before addition of the substrate. The proteolytic activity
of falcipain-2 was significantly inhibited by peptide PP1, while
peptides PP2 and PP3 inhibited the enzyme activity marginally
(Figure 2C). Consequences of interaction of peptides with
purified enzyme were monitored kinetically and biophysically.
Vmaxof the substrate Z-Phe-Arg-AMC was estimated to be 120
pmol/min. Inhibition of enzyme activity by PP1 peptide was
linear with time and concentration (Figure 2D). Inhibition
constant (Ki) was calculated by double reciprocal plot and was
3.8 µM (Table 2). A peptide, PP5, corresponding to falcipain-
2′, that contains two substitutions in the primary sequence (A-
V, M-T), also inhibited the falcipain-2 activity with a similar
Ki value. To know whether PP1 peptide specifically inhibits
the cognate enzyme or it also affects the activities of other
falcipains, PP1 peptide was incubated with active recombinant
falcipain 1. As shown in Figure 2C (inset), PP1 peptide did not
inhibit the falcipain-1 activity. An unrelated Ova peptide (OVA)
also failed to inhibit the falcipain-2 activity (Figure 2C).
Together, these results suggested that the peptide PP1, corre-
sponding to the subpeptide of the proregion of falcipain-2,
inhibited the enzyme activity specifically. Circular dichroism
of peptide PP1 and purified enzyme produced a significant
diminution in the elepticity of falcipain-2 protein, while an
unrelated peptide, Ova peptide, did not produce any such change,
suggesting an interaction between PP1 peptide and the falci-
pain-2 protein (Figure 3).
Uptake of Falcipain-2 Proregion Peptide by P. falciparum
Infected RBC’s and Inhibition of Falcipain-2 Activity. Peptides
have been shown to get access into the parasitized erythrocytes
selectively.31To investigate the translocation of PP1 peptide
into the infected RBCs, PP1 peptide was labeled with FITC
and FITC-PP1 peptide was incubated with RBC infected with
P. falciparum 24 h postinvasion. The parasites treated with the
FITC labeled peptides were washed and visualized by confocal
microscopy at three different time intervals. Parasites inside
RBC at trophozoite and schizont stages were observed to
accumulate fluorescent PP1 peptide, whereas uninfected RBCs
did not permit the accumulation of PP1 peptide (Figure 4).
Because fluorescent PP1 peptide was able to translocate into
the P. falciparum infected erythrocytes, we examined the ability
of FITC-PP1 peptide to block the falcipain-2 mediated hydroly-
sis of Z-FR-AMC substrate by confocal microscopy. The
hydrolysis of Z-FR-AMC was measured quantitatively by
capturing the blue fluorescence generated as a result of release
of free AMC by cysteine proteases. As shown in Figure 4A,
PP1 treated parasites showed significant reduction in blue
fluorescence in comparison to the untreated parasites. Quantita-
tive measurements by confocal microscopy showed up to ∼50%
block in substrate hydrolysis in PP1 peptide treated infected
erythrocytes over untreated infected cells (Figure 4B). These
results demonstrated that FITC-PP1 peptide enters into infected
aAbbreviations: PP1, propeptide 1; PP2, propeptide 2; PP3, propeptide
3; PP4, propeptide 4; PP5, propeptide 5; Ant PP1, propeptide 1 with
Antennapedia homeoprotein internalization domain; FITC-PP1, fluorescein
isothyocyanate lableled propeptide 1; OVA, ovalbumin peptide.
Figure 1. Schematic representation of falcipain-2 protein and its proregion peptides spanning the region -89 aa to -44 aa. Numbering is based
on the position of first amino acid (Q) of falcipain-2 mature region.
Table 1. Amino Acid Sequences of Synthetic Falcipain-2 Proregion and Control Peptides Used in the Present Studya
peptideamino acid sequencepositionmolecular mass (Da)
-89aa to -75aa
-74aa to -60aa
-59aa to -48aa
-63aa to -44aa
-89aa to -75aa
-89aa to -75aa
-89aa to -75aa
aThe position of each proregion peptide with respect to first amino acid (Q) of the falcipain-2 mature region is indicated. Molecular mass of each peptide
in Daltons is presented.
P. falciparum Cysteine Protease Inhibits Malaria ParasiteJournal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3117
erythrocyte and blocks the hydrolysis of Z-Phe-Arg-AMC, a
substrate of falcipain-2.
Effect of Falcipain-2 Proregion Peptide on P. falciparum
Development. We next investigated the effect of PP1 peptide
on parasite growth and development in vitro by hypoxanthine
uptake assay and by microscopic examination. Although PP1
peptide was able to access the intraerythrocytic parasites as
shown previously, to enhance its uptake further, a 16aa sequence
of Antennapedia (Ant) homeoprotein was coupled to the NH2-
terminus of PP1. The Antennapedia internalization sequence has
been known to facilitate the uptake of peptides into P.
falciparum and mammalian cells.32,33As shown in Figure 5,
both PP1 and Ant-PP1 peptides inhibited parasite growth in vitro
in a dose dependent manner. However, differences in the
quantum of inhibition by these peptides were noteworthy. At
100 µM concentration, PP1 inhibited parasite growth up to 50%,
whereas at the same concentration, Ant-PP1 inhibited the growth
up to 80%. Peptides PP3, PP4, Ant, and OVA did not show
any effect on parasite development. We also investigated the
effects of these peptides on the total parasitemia by counting
the Giemsa stained smears microscopically. Treatment of
parasites with PP1 and Ant-PP1 considerably reduced the total
parasitemia (∼50-70%) in comparison to the parasites treated
with control OVA peptide. To visualize the effect of PP1 and
Ant-PP1 peptides on parasite development, a transgenic parasite
line expressing green fluorescent protein (GFP) fused with a
region of knob associated histidine rich protein, KAHRP
(KAHRP(-His)-GFP) was treated with the PP1 peptide. At 100
µM PP1 concentration, significant cessation in growth as
indicated by loss of GFP fluorescence was observed in the
treated parasites (Figure 5). These results were similar to that
observed after the treatment of transgenic parasites with E-64
(Figure 5B,C). The cessation of growth observed by microscopic
examination correlated with the [3H] hypoxanthine uptake assay.
These results suggested that PP1 inhibits falcipain-2 and
consequently arrests the growth and development of the parasite.
Effect of Falcipain-2 Proregion Peptide on P. falciparum
Morphology. Cysteine protease inhibitors have been shown to
cause morphological abnormalities, especially in the food
vacuole of malaria parasites.9,34Treatment of parasites at late
ring or early trophozoite stage with PP1 resulted in a phenotype
similar to that seen in cysteine protease inhibitor-treated
parasites, whereby the food vacuole of early trophozoites was
swollen and darkly stained. The abnormal food vacuole also
showed tight clumps of malaria pigment that appeared to be
different from the normal hemozoin (Figure 6). In PP1 treated
parasites, we also observed several incompletely ruptured
parasites after 56 h postinvasion of the first cycle of treatment.
These schizonts contained spherical clusters of merozoites,
enclosed in a delicate membrane covering (Figure 6). Similar
clusters have been previously reported in parasite cultures
wherein the parasite cysteine proteases especially falcipain-2
were inhibited by either cysteine protease inhibitors or by
cognate siRNAs.6,35These results indicate that, in addition to
its role in hemoglobin degradation, falcipain-2 also plays a role
in merozoite release. Falcipain-2 has been previously shown to
cleave the two erythrocyte skeletal proteins, ankyrin and band
4.1.32It is likely that cleavage of cytoskeletal elements by
falcipain-2 destabilizes the erythrocyte membrane that in turn
leads to its rupture and release of merozoites.
Inhibition of Hemoglobin Degradation by Falcipain-2
Proregion Peptide. To know whether the above-mentioned food
vacuole abnormalities seen in PP1 treated parasites were due
to a block in globin hydrolysis, parasites were incubated with
PP1 peptide for 24 h (beginning at the ring stage) and their
proteins were analyzed by SDS-PAGE. Compared to the wild
type (WT) parasites, PP1-treated and E-64-treated parasites
showed accumulation of undigested hemoglobin. Taken together,
our data indicates that a proregion peptide (PP1) inhibits the
falcipain-2 activity, which in turn blocks the hydrolysis of
hemoglobin in P. falciparum trophozoite.
Parasite encoded cysteine proteinases from the genus Try-
panosoma and Plasmodium have been reported as potential
targets for chemotherapeutic inhibitors.11,36In P. falciparum,
the use of cysteine protease inhibitors such as peptidyl fluo-
romethyl ketone, vinyl sulfone, aldehyde, and nonpeptide
inhibitors has supported the development of cysteine protease
Figure 2. Expression and activity analysis of recombinant falcipain-
2. (A) Coomassie blue stained 12% SDS-polyacrylamide gel of Ni2+-
NTA purified falcipain-2. Lanes 1-8, elutes from Ni2+-NTA column.
Lane M, molecular mass markers (kDa). Arrow indicates the position
of the recombinant protein. (B) Coomassie stained 12% SDS-PAGE
of refolded falcipain-2 protein. (C) Inhibition of recombinant falcipain-2
activity by falcipain-2 prodomain peptide (PP1). Proteolytic activity
of falcipain-2 was measured at pH 5.5. Activity was analysed for the
release of fluorescence units measured over a period of 30 min. Error
bars represent SD of the results from three different sets of experiments.
AFU, arbitary fluorescence units. The inset shows the effect of PP1 on
falcipain-1 activity. (D) Dose-dependent activity profile of falcipain-2
incubated with varying concentrations of PP1 peptide over a period of
Table 2. Kinetic Constants
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11Korde et al.
inhibitors as antimalarials11,37,38Recent analysis has shown that
the antiparasitic effects of cysteine protease inhibitors correlate
with potent inhibition of falcipain-2 and falcipain-3 in P.
falciparum and their homologous enzymes in P. ViVax and P.
Vinckei.39,40Extensive studies are now underway to develop
specific inhibitors corresponding to these enzymes for antima-
larial therapy.3,11One of the limitations of developing specific
inhibitors to a particular class of proteases is the conservation
of active site residues in each class of proteins and broad
substrate specificity.25On the other hand, prodomain of pro-
teinases have been shown to be unique for each enzyme and
inhibition of proteases by their corresponding prosequence is
highly specific.13,24This specificity of prodomains inhibition
of cognate enzyme is being exploited to develop a new
generation of specific peptide inhibitors.
A large number of recent reports have shown that the parasitic
proteases can be inhibited by their cognate prodomains. Several
of these reports have employed E. coli expressed and purified
protein of vector or parasite origin.15,17,23,25,29,41In the present
study, we evaluated the potential of synthetic peptides corre-
sponding to falcipain-2 proregion (-89aa to -44aa) for the
antiparasitic activity. This region of prodomain was selected
based on previous observations, suggesting that an evolutionary
conserved R-helical motif in the proregion of cathepsin L-like
proteases is likely to be involved in the inhibition of enzyme
activity.42Of these peptides, only PP1 (-89aa to -75aa)
significantly inhibited the falcipain-2 activity in a dose dependent
manner. A corresponding peptide from the proregion of falci-
pain-2′ that has two amino acid substitutions (A-V and M-T)
also showed similar inhibitory activity. The peptide, PP1, was
highly selective as it failed to inhibit falcipain-1 activity.
Surprisingly, PP4 peptide, which included the conserved ER(F/
W)N(I/V)N motif, did not inhibit falcipain-2 activity in the
present study as previously reported by Karrer et al.30,42
Import of proteins and macromolecules into malaria infected
erythrocytes has been the subject of contention among different
groups.43Recently, the access of fluorescent peptides with
molecular mass ranging from 653 to 3146 Da into the parasitized
erythrocyte has been investigated in detail, and the results
Figure 3. Analysis of interaction of PP1 with falcipain-2 by circular dichorism (CD). Far-ultraviolet CD spectrum of falcipain-2 in presence of
different concentrations of PP1 peptide.
Figure 4. Inhibitory effect of PP1 peptide on substrate hydrolysis in
the intracellular malaria parasite. (A) Confocal fluorescence imaging
of P. falciparum trophozoite to show uptake and block in hydrolysis
by PP1 peptide in the parasitized RBC. Panel 1, uptake and hydrolysis
of fluoregenic substrate, Z-F-R-AMC. Note the presence of blue
fluorescence in parasitophorous membrane and food vacuole, indicating
the hydrolysis of substrate by native falcipain-2. Panel II, uptake of
FITC PP1 peptide and its inhibitory effect on subtrate hydrolysis. (B)
Quantitative analysis of fluorescence activity in the parasitzed RBCs
to show inhibition in hydrolysis of Z-F-R-AMC by PP1 peptide.
P. falciparum Cysteine Protease Inhibits Malaria ParasiteJournal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3119
revealed that peptides up to a molecular mass of 2365 Da are
transported into the parasite.31We studied the uptake of
substrate, Z-Phe-Arg-AMC and FITC labeled PP1 peptide by
P. falciparum parasite and subsequently analyzed the effect of
PP1 peptide on substrate hydolysis within the parasite using
confocal microscopy. Both the substrate and FITC PP1 peptide
were able to access the intracellular parasites. It appears that
the substrate and labeled peptide are transported through the
membrane channels from the outside directly to the parasites
as no such uptake was seen inside the uninfected erythrocytes.
The substrate hydrolysis was observed inside the parasite in
PV as well in the food vacuole in the wild type parasite, thereby
indicating the presence of falcipain-2 or other cysteine proteases
in these sites. The fluorescent peptide PP1 was also detected in
the intracellular compartments of P. falciparum trophozoites and
schizonts. Treatment of parasites with PP1 peptide resulted in
a block in Z-Phe-Arg-AMC hydrolysis inside the parasites.
These results suggest that the small peptides can enter into the
parasitized RBCs and can produce their biological affects
Recently, small peptides have been successfully used to
inhibit merozoite invasion and block the parasite growth and
development. Haldar and co-workers showed that peptides
designed to inhibit GRs protein function, considerably reduced
parasitemia in P. falciparum cultures in vitro and in P. berghei
infection in vivo.44,45Likewise, Hanspal and co-workers showed
that an ankyrin peptide that blocks the cleavage of ankyrin
mediated by falcipain-2 markedly inhibits the parasite growth
and maturation.32Because PP1 peptide inhibited the native
falcipain-2 activity in the intracellular parasites, we further
examined the effect of PP1 peptide on the growth and develop-
ment of P. falciparum. To do so, a modified PP1 peptide (Ant-
PP1) that contains an Antennapedia internalization sequence at
its NH2 terminus was synthesized. This design has been
previously used to deliver peptides into HeLa cells and into
infected RBCs.32Ant-PP1 inhibited the falcipain-2 catalytic
activity in a similar way as that of PP1 peptide. Both the peptides
showed almost same potency (Kifor PP1 and Ant-PP1 were
∼3 µM). Addition of each of these peptides to P. falciparum
culture resulted in marked inhibition of parasite growth and
development as determined by3[H] hypoxanthine assay, mi-
croscopic examination, and the counting of Giemsa stained
smears. Less than 20% new ring stage parasites were observed
in Ant-PP1 peptide treated culture in comparison to the control
culture containing the same final concentration of OVA peptide.
Interestingly, Ant-PP1 peptide was 1.5 fold more potent in
inhibiting parasite growth than the PP1 peptide which may be
a consequence of increased uptake of Ant-PP1 peptide within
the parasite as a result of presence of the Antennapedia
A number of previous studies have demonstrated that
treatment of malaria parasites with cysteine protease inhibitors
results in morphological abnormalties at trophozoite stage; the
food vacuole appears to be enlarged and filled with undigested
globin.34,46Recently, knock out or RNAi studies suggest that
these food vacuole abnormalities arise due to inhibition or
complete loss of falcipain-2 activity.9,35In addition to its role
in hemoglobin degradation, falcipain-2 has also been suggested
to be involved in erythrocyte rupture.32,35In the present study,
treatment of parasites with either PP1 or Ant-PP1 peptides
produced distinct food vacuole abnormalities similar to that
observed with other cysteine protease inhibitors. We also
observed few parasites stuck at the schizont stage 48 h after
either PP1 or Ant-PP1 treatment. A control peptide OVA did
not produce these abnormalities even at 4-fold higher concentra-
tion. Taken together, our study identifies a falcipain-2 proregion
peptide that inhibits parasite growth and development by
blocking the hemoglobin hydrolysis.
In summary, our results validate falcipain-2 as a potential
new chemotherapeutic target. In addition, this study also
establishes the antimalarial potential of a prosegment of
falcipain-2 that may contribute to the design of proteinase-
directed antiparasitic drug(s) of therapeutic interest.
Although proregions of parasitic proteases have been previ-
ously shown to inhibit their cognate enzyme activites,15,23,25,29
here we show for the first time the use of a synthetic prodomain
peptide as an antimalarial effector. Even though PP1 peptide
inhibited the P. falciparum growth and development with a Ki
value in the micromolar range, this study has potential for the
development of peptide and peptidometric drugs. It has been
earlier shown that interplay of multiple falcipains and plasme-
psins (aspartic proteases) is responsible for the hemoglobin
degradation inside the malaria parasite. It will be useful to design
Figure 5. Effect of PP1 peptide on the growth and development of P.
falciparum. (A) Inhibition of3[H] hypoxanthine uptake in cultured P.
falciparum parasites by falcipain-2 proregion peptide, PP1. Synchro-
nized cultures of parasite infected RBCs at ring stage were incubated
with different concentrations of PP1 peptide for 24 h in a 96-well
microtiter plate. Approximately 1 µCi of3[H] hypoxanthine was added
in each well, and cultures were maintained for an additional 24 h. The
cells were harvested, and3[H] hypoxanthine was quantified using a
scintillation counter. OVA peptide was used as a control. (B)
Fluorescence image analysis after 3 h of treatment and (C) after 10 h
of treatment, to show the effect of PP1 peptide on the development of
KAHRP (-His) transgenic parasite line. Note the loss of fluorescence
in E-64 and PP1 peptide treated parasites. In each panel, the first image
(left to right) represents fluorescence signal from GFP chimeric protein,
second is the bright field image, third is an overlay of these two images,
fourth is the DAPI stained image. Panel I, untreated parasites; panel
II, E-64 treated parasites; panel III, PP1 peptide treated parasites.
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 Korde et al.
synthetic prodomain peptides against multiple food vacuole
enzymes and then explore their potential to control malaria
Materials and Methods
Peptide Synthesis. The peptides, PP1, PP5, Ant-PP1, PP2, PP3,
and Ova peptide were synthesized by the solid-phase synthesis
method using the Fmoc procedure. PP4, Ant-PP1, and FITC PP1
peptides were obtained from Peptron, South Korea, and Genscript
Corporation, NJ. All the peptides obtained were purified by
semipreparative HPLC using a Bondapak C18 column. The
molecular mass and purity of the synthesized peptides was checked
by amino acid analysis and mass spectroscopy with MALDI-TOF.
The stock solutions of the peptides were prepared in DMSO/water
(1:1), and concentrations were measured spectrophotometrically.
Parasite Strains, Culture, and Peptide Treatment. Plasmo-
dium falciparum strains, 3D7 and KAHRP (-His)-GFP strains were
cultured with human erythrocytes (4% hematocrit) in RPMI media
(Invitrogen, USA) containing gentamycin sulfate, NaHCO3, and
L-glutamine in vitro using protocol described previously.47The
strain KAHRP (-His)-GFP is a transgenic cell line that is transfected
with a construct in which a region (of knob-associated histidine-
rich protein (KAHRP) gene encoding the first 60 amino acids was
joined upstream of GFP coding sequence.48Trangenic strain is
maintained in media containing 0.25 nM WR99210 drug. Both the
wild type and transgenic parasites were synchronized using sorbitol
treatment.49To assay the inhibitory effect of prodomain peptide(s),
synchronized parasites cultures were adjusted to 4% hematocrit with
1% infected erythrocytes, and 0.2 mL of these cultures at the early
ring stage were treated with prodomain peptides in a 24-well culture
plate in triplicate. Parasites were maintained further for 24 or 48 h.
For the microscopic analysis, smears were made from each well,
stained with Giemsa, and examined for abnormalities as well as
the number of new rings formed. The number of ring-stage parasites
per 2000 RBCs was determined for each well. Parasites with
enlarged food vacuoles and very little hemozoin accumulation were
scored as abnormal.3[H] hypoxanthine uptake assay was performed
as described previously by Malhotra et al.50
Preparation of Recombinant Falcipain-2 Protein. Recombi-
nant falcipain-2 protein was prepared by a protocol described by
Shenai et al. and Kumar et al.21,28
Enzyme Assay and Kinetic Analysis. Fluorimetric assay for
falcipain-2 activity was carried out as described previously by
Kumar et al..28Briefly, in 3 mL of assay buffer (100 mM sodium
acetate pH 5.5 10 mMDTT) containing 200 nM enzyme, fluoregenic
substrate Z-F-R-AMC was added at 7 µM concentration and the
release of 7-amino-4-methyl coumarin (AMC) was monitored
(excitation 355 nm; emission 460 nm) over 30 min at RT using a
LS50B Perkin-Elmer fluorimeter. To analyze the effect of peptide(s)
on enzyme activity, recombinant falcipain-2 was preincubated with
Figure 6. Prodomain peptide, PP1 blocks hemoglobin degradation in P. falciparum. (A) Effect of PP1 peptide on parasite morphology. Light
microscope image of parasitized RBCs after 36 h of treatment. Note the presence of food vacuole abnormalities in E-64 and PP1 treated parasites.
(B) Effect on globin hydrolysis by PP1 peptide treatment. Lane 1, molecular mass markers; lane 2, wild type parasite (control); lanes 3 and 4, E-64
(1 and 10 µM) treated parasites; lanes 5, 6, and 7, PP1 (50, 100, and 150 µM) treated parasites; lane 8, pepstatin (1 µM) treated parasites.
P. falciparum Cysteine Protease Inhibits Malaria ParasiteJournal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3121
each peptide for 10 min at room temperature. Remaining activity
was determined using the fluorogenic substrate.
Rate of hydrolysis at varied concentration of substrate Z-F-R-
AMC was determined at constant enzyme concentration (200 nM)
in 3 mL reaction volume. Kinetic constants km, Vmax, and Kivalues
were determined using PRISM software (Graph Pad, San Diego).
CD Spectroscopy. The CD spectra of the PP1 peptide, falci-
pain-2 protein, and falcipain-2 protein + PP1 peptide were measured
at room temperature (25 °C) between 190 and 300 nm using a Jasco
J-715 spectrapolarimeter (Jasco, Easton, MD). The path length of
the cell was 1 cm. Experiments were performed in 10 mM
phosphate buffer, pH 7.0. All the spectra are averages of multiple
Confocal Microscopy. To study the uptake of peptides into the
infected erythrocytes, 3D7 parasites at 4-5% parasitemia were
incubated with the FITC PP1 peptide at 24-28 h post invasion for
10 h. After the incubation, the cultures were washed with complete
medium and incubated with the substrate Z-F-R-AMC for 10 min.
The treated parasitized cells were immediately mounted on a glass
slide with a coverslip and visualized using a Nikon TE 2000-U
microscope. To capture FITC peptide, an excitation of 488 nm was
used. Emitted light was collected through a band-pass filter at
505-530 nm. The release of AMC was captured using the UV
band-pass filter. Transmitted light observations were performed
during the experiments in order to follow the integrity of the cells.
Fluorescence arbitary units were acquired from an average of
selected whole parasites.
Analysis of Globin Hydrolysis in P. falciparum Parasites. The
effects of PP1 peptide or E-64 on hemoglobin hydrolysis in malaria
parasites were determined as previously described.35Briefly P.
falciparum cultures were treated with these inhibitors for 24 h at
ring stage. After the treatment, the infected erythrocytes were
collected by centrifugation and lysed using 0.15% saponin in PBS
on ice for 10 min. The lysed samples were centrifuged and washed
three times with ice-cold PBS to remove erythrocyte cytoplasmic
contents. Parasite pellets were solubilized in Laemmli buffer and
separated on 15% SDS-PAGE. The gels were stained with
Coomassie Brilliant Blue R250. Approximately 1 × 106parasites
were loaded in each well.
Acknowledgment. We thank Krishan Kumar and Aseem
Mishra for assistance in protein purification and CD spectros-
copy respectively. We are thankful to Charu Tanwar and Shahid
Jameel for enabling us to use the Confocal Microscope Facility
at ICGEB, New Delhi, funded through an International Senior
Research Fellowship of the Wellcome Trust (UK). We thank
Dr. Pushkar Sharma, NII, for his help and Dr. Roy, NII, for
help and discussions on CD spectroscopy. We thank the Rotary
Blood Bank for providing O positive human red cells. We also
thank Department of Biotechnology, India, for the financial
support for this project.
Supporting Information Available: Table of purity of all target
compounds; reverse phase HPLC profile of PP1; mass spectrometry
data of PP1; reverse phase HPLC profile of Ant PP1; mass
spectrometry data of Ant PP1. This information is available free
of charge via the Internet at http://pubs.acs.org.
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