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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Mol Biochem Parasitol. Author manuscript; available in PMC 2008 September 1.
Published in final edited form as:
PMCID: PMC1993804

BDA-410: A novel synthetic calpain inhibitor active against blood stage malaria


Falcipains, the papain-family cysteine proteases of the Plasmodium falciparum, are potential drug targets for malaria parasite. Pharmacological inhibition of falcipains can block the hydrolysis of hemoglobin, parasite development, and egress, suggesting that falcipains play a key role at the blood stage of parasite life cycle. In the present study, we evaluated the anti-malarial effects of BDA-410, a novel cysteine protease inhibitor as a potential antimalarial drug. Recombinant falcipain (MBP-FP-2B) and Plasmodium falciparum trophozoite extract containing native falcipains were used for enzyme inhibition studies in vitro. The effect of BDA-410 on the malaria parasite development in vitro as well as its anti-malarial activity in vivo was evaluated using the Plasmodium chabaudi infection rodent model. The 50% inhibitory concentrations of BDA-410 were determined to be 628 nM and 534 nM for recombinant falcipain-2B and parasite extract, respectively. BDA-410 inhibited the malaria parasite growth in vitro with an IC50 value of 173 nM causing irreversible damage to the intracellular parasite. In vivo, the BDA-410 delayed the progression of malaria infection significantly using a mouse model of malaria pathogenesis. The characterization of BDA-410 as a potent inhibitor of Plasmodium falciparum cysteine proteases, and the demonstration of its efficacy in blocking parasite growth both in vitro and in vivo assays identifies BDA-410 is an important lead compound for the development of novel anti-malarial drugs.

Keywords: Falcipain, Malaria, Protease, Cysteine protease inhibitor, Plasmodium falciparum

1. Introduction

Malaria, particularly the disease caused by Plasmodium falciparum, is one of the most important parasitic infections of humans and is responsible for over a million deaths each year. The control of malaria is being challenged by the increasing resistance to available drugs. Therefore, new drugs to block essential metabolic pathways of the parasite are urgently needed. Mounting evidence now suggests that proteolytic enzymes play key roles in the life cycle of the malaria parasite in various species [1, 2]. During the erythrocytic stage of malaria infection, degradation of host hemoglobin is believed to be an important requirement for the survival of the intracellular parasite [1, 2]. Although hemoglobin breakdown by parasite proteases is considered a critical step, the fact that some parasite strains digest variable amounts of hemoglobin and the bulk of amino acids generated from such breakdown are excreted from the infected erythrocyte raises some doubts about the physiological dependence of the intracellular parasite on hemoglobin-degradation for its biosynthetic needs [2, 3]. Nonetheless, two major classes of parasite proteases have been implicated in the degradation of hemoglobin. The aspartic proteases called plasmepsins, encoded by four genes in the Plasmodium falciparum, degrade hemoglobin at an acidic pH [4]. There are as many as six additional genes that also encode aspartic proteases in the parasite genome, although the precise function of their gene products is not yet known [5]. Recently, Goldberg and colleagues concluded that the parasite plasmepsins are not promising targets of anti-malarial drugs presumably due to their functional overlap with parasite cysteine proteases [2].

The malaria parasite cysteine proteases, termed falcipains, have also been extensively investigated as key mediators of hemoglobin degradation during the blood stage infection of malaria parasite [1]. The early interest in parasite cysteine proteases originated from their potential involvement in the invasion and rupture of infected erythrocytes [614]. Despite several attempts, a functional role of parasite proteases in the release of malaria parasite from infected erythrocytes remains controversial. For example, one study suggested that the parasite proteases are involved in the primary rupture of the parasitophorous vacuole membrane followed by a secondary rupture of the erythrocyte membrane [1517]. In contrast, other evidence suggests that the parasite egress is coordinated by the primary rupture of the erythrocyte plasma membrane followed by the secondary breakdown of the parasitophorous vacuole membrane [16, 17]. Irrespective of the precise mechanism of parasite release, it appears that parasite proteases are likely required for the degradation of host hemoglobin, as well as, play a functional role in the breakdown of the parasitophorous vacuole and erythrocyte membranes [1, 5].

Like plasmepsins, there are four falcipain genes in the Plasmodium falciparum genome; designated as FP1, FP2A, FP2B, and FP3. The genes for FP2A (also called FP2), FP2B (also called FP2′), and FP3 are located on parasite chromosome 11 whereas the FP1 gene is located on chromosome 14 [1]. The FP2A and FP2B enzymes are more than 97% identical, and show 66% amino acid identity with FP3 enzyme but, are distantly related to the FP1 enzyme with 36% sequence identity [1, 18]. A previous study used the double stranded RNA mediated knockdown of FP1 and FP2 (FP2A) suggesting a functional role of these proteases in the breakdown of hemoglobin and food vacuole abnormalities [19]. Recent applications of targeted gene disruption techniques indicated that the FP2 (FP2A) knockout parasites exhibit a defect in early trophozoite development. However, in the mature stage, the knockout parasite line was indistinguishable from the parent wild type parasite line [20]. It is noteworthy that the FP2A knockout parasites in this study showed some leakiness with a small amount of mRNA was still detectable in the knockout parasites [20]. The expression of plasmepsins and other falcipains was nearly normal in the FP2A knockout parasites [20]. Importantly, the FP2A knockout parasites were 50-fold more sensitive to pepstatin, an aspartic protease inhibitor [20]. The development of FP1 knockout parasite lines was independently reported by two groups demonstrating that this cysteine protease is not required for parasite invasion and growth in erythrocytes [21, 22]. These results were in contrast to an earlier report showing that the FP1 enzyme plays an essential role in the merozoite invasion of erythrocytes [23]. Interestingly, one FP1 knockout study demonstrated that the FP1 enzyme reduces oocyst production and therefore might play a functional role during parasite development in the mosquito gut [22]. More recently, Rosenthal and colleagues reported individual knockouts of FP1, FP2A, and FP2B [24]. Again, the parasite growth was nearly normal in the three individual knockouts with the exception of FP2A knockout parasites showing the food vacuole abnormalities as reported earlier [20, 24]. In the same study, attempts to knockout the FP3 gene were unsuccessful suggesting that this cysteine protease may play a critical and non-redundant role in the parasite life cycle [24]. It is likely that a lack of phenotype in the FP2B (FP′) knockout strain reflects a functional compensation by FP2A, which is 97% identical to FP2B. A double knockout of FP2A and FP2B has not been reported as yet.

The falcipains have emerged as viable drug candidates against the blood stage malaria infection, particularly after the recent findings that plasmepsins are not promising drug targets against malaria [1, 2]. Previously, a number of studies have explored the possibility of using cysteine protease inhibitors as potential anti-malarial drugs both in vitro and in vivo [23, 2527]. These studies included inhibitors that are both synthetic chemicals and peptidomimetic compounds [23, 2528]. Previously, we have shown that a 10 amino acid peptide derived from erythrocyte ankyrin containing the falcipain-cleavage site abolished all known functions of FP2A in vitro [28]. This study provided a framework indicating that inhibition of falcipains is an attractive anti-malarial approach irrespective of whether such inhibition blocks hemoglobin degradation by falcipains or prevents parasite release from infected erythrocytes. From a practical standpoint, the development of synthetic chemical compounds is clearly more desirable than the peptide-based inhibitors against falcipains. For example, peptidyl fluoromethyl ketone [11, 29, 30], vinyl sulfone [26, 31], and aldehyde [25] based inhibitors have been developed against falcipains showing potent inhibitory effects on the survival of the malaria parasite at nanomolar concentrations. We have recently reported a series of novel peptidomimetic cysteine protease inhibitors as potential anti-malarial agents [27]. Ideally, the promising anti-malarial drugs would be soluble, stable, and membrane permeable, with high selectivity towards cysteine proteases.

In this study, we have evaluated a novel cysteine protease inhibitor termed BDA-410 as a potential anti-malarial drug. BDA-410 was originally synthesized as an orally active compound to act as a potent and selective inhibitor of calpain activity [32]. It is currently evaluated as a therapeutic modality to protect against neuronal cell death in models of Alzheimer’s disease [32]. The BDA-410 (molecular weight 484.6 daltons) is a relatively selective inhibitor of calpain-1 (Ki value of 130 nM) rather than calpain-2 (Ki value of 630 nM). The IC50 values for inhibition of papain (400 nM), cathepsin B (16 μM), cathepsin D (91.2 μM), cathepsin G (100 μM), thrombin (100 μM), and proteasome 20S (100 μM) suggest the potential utility of BDA-410 as an inhibitor of cysteine proteases. Moreover, the mutagenicity and toxicology studies of BDA-410 are also consistent with its use in therapeutic applications.

2. Materials and methods

2.1. Protease inhibitor BDA-410

BDA-410 was kindly provided by Drs. Narihiko Yoshi and Hiroshi Kinoshita (Mitsubishi Pharma Corporation, Yokohama, Japan). The chemical name of this compound is (2S)-N-{(1S)-1-[(S)-Hydroxy(3-oxo-2-phenyl-1-cyclopropen-1-yl)methyl]-2-methylpropyl}-2-benzenesulfony-lamino-4-methylpentanamide (C26H32N2O5S, M.W. 484.61) (Fig. 1). The compound was shown to be >99% pure by high-pressure liquid chromatography. It is a potent and selective inhibitor of calpain protease activity.

Fig. 1
Chemical structure of cysteine protease inhibitor BDA-410.

2.2. Culture of malaria parasites and preparation of parasite extract

The 3D7 strain of Plasmodium falciparum was maintained in continuous culture in human erythrocytes using the established protocol of Trager and Jensen[33]. Parasite cultures maintained in 75-cm2 flasks at 37°C under 5% CO2, 5% O2, and 90% N2, contained a 5% suspension of fresh type O+ human erythrocytes in RPMI1640 medium supplemented with 25 mM HEPES, 30 mg/L hypoxanthine, 0.2% NaHCO3, and 0.5% Albumax II (Invitrogen). Ring-stage parasites were synchronized by using 5% Sorbitol treatment. Trophozoite and schizont-infected RBCs were enriched to greater than 95% parasitemia by centrifugation through 65% (v/v) Percoll (Sigma, St Louis, MO) gradients. RBCs infected with trophozoite and schizont were then incubated with 0.01% (w/v) Saponin in phosphate-buffered saline (PBS, pH 7.4) at 37°C for 10 minutes to lyse the erythrocyte membrane and were washed three times with the ice-cold PBS. Parasites were then lysed with 5 mM phosphate buffer, pH 8.0, and centrifuged at 100 000g for 30 min. The resultant soluble cytosolic extract is referred to as the “parasite extract” and was used in the enzyme inhibition assays.

2.3. Expression and purification of MBP-FP-2B

MBP-FP-2B fusion protein was expressed in Escherichia coli BL21 and affinity-purified using amylose affinity chromatography (New England Biolabs) as described before[18]. The protease activity of recombinant protein was measured by fluorometric quantification of the cleavage of fluorescent substrate, Benzyloxycarbonyl- Phe-Arg-7- amino-4 methyl coumarin (Z-Phe-Arg-AMC) [31].

2.4. Recombinant and native enzyme inhibition assays

Equal amounts (~0.1 uM) of MBP-FP-2B fusion protein or Plasmodium falciparum trophozoite extract containing falcipain activity were incubated with various concentrations of BDA-410 (added from the 100x stock in DMSO) in 0.1 M sodium acetate and 10 mM DTT, at a pH 5.5, for 30 minutes at room temperature. Fluorogenic substrate, Z-Phe-Arg-AMC, was then added to a final concentration 5 uM. A 96-well microplate reaction volume was used for spectrofluorometric determination of fluorescence intensity. The release of AMC was continuously monitored (excitation 355 nm; emission 460 nm) for over 30 minutes at room temperature in a Wallac 1420 multilabel counter. Enzyme activity was expressed as fluorescence over time. Multiple inhibitor concentrations were used in duplicate or triplicate in multiple experiments. The rates of substrate hydrolysis were determined and compared to those of negative controls containing equal concentrations of DMSO and with positive controls containing 10 uM of MDL28170. The 50% inhibitory concentrations (IC50 values) against falcipain-2B were determined from plots of activity over substrate concentration using the SigmaPlot software (Systat Software), with data being fitted by nonlinear regression.

2.5. In vitro malaria parasite inhibition assay

To assess the effect of BDA-410 inhibitor on the malaria parasite development in vitro, sorbitol synchronized 3D7 strain Plasmodium falciparum parasites were cultured in a 96-well plate with different concentrations of BDA-410 (added from the 100x stock in DMSO) for 48 hours beginning at the ring stage. The culture medium was then changed after 24 hours by maintaining an appropriate concentration of inhibitor. Giemsa stained smears of parasites were made after 48 hours, when negative control cultures contained nearly all ring-stage parasites. The number of new ring forms per 1000 erythrocytes was counted, and these counts were compared with those from controls containing an equal concentration of DMSO (1%). The 50% inhibitory concentrations (IC50 values) for parasite growth inhibition were calculated with the SigmaPlot software.

2.6. In vivo antimalarial activity assay

The Plasmodium chabaudi infection rodent model was used to investigate the blood schizonticidal activity of BDA-410. Normal C57BL/6 mice (4–6 weeks of age, 20–22 g of weight) were infected by intraperitoneal (i.p.) injection of 106 parasite-infected erythrocytes from a donor mouse with ~20% parasitemia. After infection was established for 3 hours, 0.2 ml of BDA-410 in PBS was administered to each mouse at a dose of 25 mg/kg of body weight through i.p. once on the day of infection (day 0), and then three times per day for subsequent 4 days (day 1–4) as a standard 4-day suppressive test. The course of malaria infection in BDA-410 treated and solvent control mice was monitored by daily counting of parasites in the Giemsa-stained smears that were obtained from the tail vein blood, and parasitemia was determined by counting 1,000 erythrocytes.

3. Results and Discussion

In the presented study, we evaluated the inhibitory activity of BDA-410 against falcipains present in the parasite extract. Cytosolic extracts were made from mature (trophozoite and schizont) stages of Plasmodium falciparum as described earlier [18]. The falcipain activity in the parasite extract, contributed by major cysteine proteases, was measured by the cleavage of a synthetic fluorescent substrate. The BDA-410 inhibited total falcipain activity with an IC50 value of 534 nM (Fig. 2A). We expressed the recombinant falcipain 2B (also called FP2′) fused to maltose binding fusion (MBP) in bacteria and examined its inhibition by BDA-410. The BDA-410 inhibited purified FP2B with an IC50 value of 628 nM (Fig. 2B) suggesting that the bulk of the falcipain activity in the crude extract may be attributed to FP2B-like proteases in the parasite extract (Fig. 2A). We also examined the effect of BDA-410 on the growth of Plasmodium falciparum in vitro. Synchronized parasites at the ring stage were incubated with BDA-410 and its inhibitory activity was compared with MDL 28170, an established potent irreversible inhibitor of malaria parasite growth in human erythrocytes [17]. MDL 28170 is a membrane-permeable peptidomimetic compound initially known to inhibit calpain protease activities [34]. Like MDL 28170, BDA-410 inhibited the malaria parasite growth in human erythrocytes in vitro (Fig. 3). Pycnotic parasites with altered morphology were observed after 24 hour, 30 hour, and 48 hour of parasite growth in the presence of BDA-410 (Fig. 3). Quantitatively, the BDA-410 compound inhibited malaria parasite growth in vitro with an IC50 value of 173 nM (Fig. 4A). It is noteworthy here that the treatment of malaria parasite with known cysteine protease inhibitors such as leupeptin and E64, which are relatively non-permeable to membrane compartments, causes food vacuole abnormalities but leaves the parasites viable for further growth and re-infection upon removal of these inhibitors [17]. These food vacuole abnormalities presumably result from the accumulation of undigested hemoglobin in the inhibitor-treated parasite food vacuoles that appear to be filled with erythrocyte cytoplasm. Our results suggest that the inhibition of malaria parasite growth by BDA-410 is similar to MDL 28170, but unlike leupeptin, causing irreversible damage to the intracellular parasite in vitro.

Fig. 2
(A, B) BDA-410 inhibits falcipain activity of Plasmodium falciparum in vitro. The IC50 value for falcipain is based on three independent determinations comparing the activity of BDA-410 and solvent control (DMSO) against Z-Leu-Arg-AMC as substrate. Inhibition ...
Fig. 3
Effect of BDA-410 on Plasmodium falciparum (3D7 strain) growth in vitro. Thin blood smears of highly synchronized ring-stage Plasmodium falciparum were made at 24 hour, 30 hour, and 48 hour after continuous incubation of parasites in BDA-410 (25 μM), ...
Fig. 4
(A, B). Inhibition of malaria parasite growth by BDA-410. (A) Inhibition of cultured Plasmodium falciparum in vitro. The IC50 value of BDA-410 for cultured parasites is based on three independent measurements comparing parasitemia in the presence of BDA-410 ...

To examine the effect of BDA-410 on the survival of malaria parasite in vivo, we used a mouse model infected with Plasmodium chabaudi. A standard 4-day suppressive regime, as described in the Methods section, was employed to test the effect of BDA-410 on parasite infection in vivo. BDA-410 rapidly cleared parasitemia in vivo resulting in ~72% inhibition with no obvious detrimental effects due to inhibitor treatment in infected mice (Fig. 4B). Importantly, all control infected mice with no BDA-410 treatment died around day 14 whereas all BDA-410 treated mice survived until around day 23 (Table I). Two of the eight BDA-410 treated mice survived until day 60 when the experiment was terminated. It is noteworthy that the BDA-410 injections in treated mice were stopped after day 4, and therefore it is likely that continued BDA-410 treatment might further prolong the survival rate of infected mice. Together, these results indicate that BDA-410 can function as a potent inhibitor of malaria parasite growth in erythrocytes both in vitro and in vivo, and identifies BDA-410 as a potential lead compound for further development as an anti-malarial drug targeting the parasite cysteine proteases. According to the information provided by Mitsubishi Pharma for the pharmacokinetics properties of BDA-410, the major metabolic sites for drug absorption are intestinal tract, liver, and blood. Using a rat model, the extent of drug absorption was ~50% with the bioavailability of 1.9–4.5% by carboxymethylcellulose suspension and 9% by tricaprylin suspension. In a dog model, the extent of absorption was ~70% with the bioavailability of 2.6% by solid dispersion with HPC (hydroxypropylcellulose) in tablet form. It is noteworthy here that although the parasite falcipains appear to be the primary targets of BDA-410, there remains a possibility that inhibition of other targets may also contribute to the overall inhibitory effects of BDA-410 on parasite growth and invasion in vivo.

Table I
BDA-410 suppresses Plasmodium chabaudi infection in mice in vivo

Although the precise biological function of each falcipain, and the potential overlap with other falcipains and plasmepsins, is poorly understood, intense interest in the biology of malaria proteases is largely fueled by their potential as novel drug targets. Recently, the crystal structure of FP2A complex with an irreversible inhibitor, iodoacetamide, was reported at 3.1A [35]. Interestingly, the crystal structure revealed the presence of four monomers of FP2A in the crystal, consistent with our recent demonstration that the recombinant FP2B, which is a paralog of FP2A, exists as a multimeric enzyme in solution [18, 35]. Clearly, additional structural studies on falcipains complexed with novel inhibitors will be needed to facilitate ongoing efforts to develop compounds that can selectively target falcipains and show relatively less inhibitory effects on host calpains and other cysteine proteases. This is an important issue since the calpain activity is most abundant in the mammalian erythrocytes [36]. The presence of calpain activity in erythrocytes raises two important concerns for future drug development. The first issue is whether the intracellular parasite can modulate the activity of erythrocyte calpain, which in turn contributes to its survival in vivo, and the second concern is that if the host calpain is essential for the survival of the erythrocyte, then its non-specific inhibition by falcipain inhibitors may limit their development as potential anti-malarial drugs. To address these issues, we generated a calpain-1 knockout mouse model and demonstrated that calpain-1 is not essential for the invasion and growth of the malaria parasite in vitro and in vivo [13, 36]. Importantly, the calpain-1 knockout mice are viable with no major developmental and hematological defects thus raising the hope that the anti-falcipain inhibitors could be developed as potential therapeutics against the intraerythrocytic stage of malaria infection. The calpain-2 cysteine protease, which is not present in the erythrocytes, appears to be more critical for the survival of the host as revealed by the lethal phenotype of its knockout in mice [37].

The completion of the Plasmodium falciparum genome sequencing project and the advent of gene knockout techniques has permitted selective silencing of the parasite genes thus raising the prospect of developing new anti-malarial drugs in the near future. Both falcipains and plasmepsins are likely targets for the anti-malarial drug development because of their potential involvement in the degradation of hemoglobin and parasite release from infected erythrocytes. Notwithstanding a lack of the precise mechanism of action of parasite proteases, it may be necessary to evaluate the efficacy of selective inhibitors of these proteases in the primate models of malaria infection in vivo. A combination therapy targeting the cysteine and aspartic proteases of Plasmodium falciparum may lead to the development of new therapeutics against drug-resistant strains of malaria infections in humans.


We are grateful to Drs. Narihiko Yoshii and Hiroshi Kinoshita of Mitsubishi Pharma Corporation, Yokohama, Japan, for their generous gift of BDA-410 compound for these studies. This work was supported by the NIH grant HL 51445 (AC).


phosphate-buffered saline
maltose binding protein
Dimethyl Sulfoxide
Benzyloxycarbonyl-Phe-Arg-7-amino-4 methyl coumarin
50% inhibitory concentration


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