Inhibition of malaria parasite Plasmodium falciparum development by crotamine, a cell penetrating peptide from the snake venom

Accepted Manuscript Title: Inhibition of malaria parasite Plasmodium falciparum development by crotamine, a cell penetrating peptide from the snake venom Author: S. El Chamy Maluf C. Dal Mas E.B. Oliveira P.M.S. Melo A.K. Carmona M.L. Gazarini M.A.F. Hayashi PII: DOI: Reference:

S0196-9781(16)30013-4 http://dx.doi.org/doi:10.1016/j.peptides.2016.01.013 PEP 69595

To appear in:

Peptides

Received date: Revised date: Accepted date:

4-12-2015 15-1-2016 19-1-2016

Please cite this article as: El Chamy Maluf S, Dal Mas C, Oliveira EB, Melo PMS, Carmona AK, Gazarini ML, Hayashi M.A.F.Inhibition of malaria parasite Plasmodium falciparum development by crotamine, a cell penetrating peptide from the snake venom.Peptides http://dx.doi.org/10.1016/j.peptides.2016.01.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Inhibition of malaria parasite Plasmodium falciparum development by crotamine, a cell penetrating peptide from the snake venom El Chamy Maluf S.a, Dal Mas C.b, Oliveira E.B.c, Melo P.M.S.a, Carmona A.K.a, Gazarini M.L.d,*, Hayashi M.A.F.b,*

a

Departamento de Biofísica, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil; bDepartamento de Farmacologia, Universidade Federal de São Paulo (UNIFESP), São Paulo, Brazil; cDepartamento de Bioquímica e Imunologia, Universidade de São Paulo (USP-RP), Ribeirão Preto, Brazil; dDepartamento de Biociências, Universidade Federal de São Paulo (UNIFESP), Santos, SP, Brazil.

*Corresponding authors:

Mirian A. F. Hayashi, Ph.D. Departamento de Farmacologia, Universidade Federal de São Paulo (UNIFESP), Rua 3 de maio 100, Ed. INFAR, 3rd floor, CEP 04044-020, Tel +55-11-5576 4447/FAX +5511-5576 4499, São Paulo, Brazil; e-mail: [email protected] or [email protected] and Marcos L. Gazarini, Ph.D. Departamento de Biociências, Universidade Federal de São Paulo (UNIFESP), Rua Silva Jardim 136, CEP 11015-020, Santos, SP, Brazil. e-mail: [email protected] ou [email protected]

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Graphical Abstract

Schematic figure showing the preferential affinity of crotamine for P. falciparum infected erythrocytes, which may have increased negative charge exposure compared to the liquid net neutral surface of uninfected erythrocytes due to the extensive host cell remodeling mediated by parasites. The localization of crotamine in parasitophorous vacuole (PV), as well as in acidic digestive vacuole (DV) and nucleus are also indicated.

Highlights     

Crotamine is a cationic natural peptide with anti-plasmodial activity; Crotamine has anti-plasmodial activity against Plasmodium falciparum; Crotamine inhibits the development of the P. falciparum in a dose-dependent manner; Crotamine was observed in the parasite nucleus and parasitophorous vacuole; Crotamine may disrupt the parasite acidic compartments H+ homeostasis;

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Abstract We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC50 value of 1.87 µM], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H+ homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles.

Abbreviations: RBC, red blood cell; iRBC, infected RBC; AO, acridine orange; Cy3, Cyanine 3 dye;

Keywords: Plasmodium, parasites, crotamine, antimalarial, acidic compartments, peptide trafficking.

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1. Introduction

Malaria is a critical human infection and it is responsible for the death of nearly a million people every year [1]. The search for new antimalarial compounds is crucial, since drug resistance is spreading quickly in the existing parasite population [2-5]. In contrast to the increased number of methods currently available to identify new compounds with antimalarial activity as described in recent reports, actually very few innovative contributions to drug discovery can be found in the field [6]. The Plasmodium life cycle involves two well-known hosts, the arthropod mosquito vector and the vertebrate host, for the sexual and asexual stages, respectively. During the asexual stage, which is the main target for the antimalarial studies, crucial biochemical and physiological changes, necessary for the P. falciparum development, are observed in erythrocytes [7-9]. The increased permeability of the erythrocyte membrane to different metabolites during the asexual stage favors the entrance of several inhibitors with potential as antimalarial drugs [10-15]. The acidic compartments, in addition to the endoplasmic reticulum and mitochondria, present in Plasmodium cells, possess an important role in the intracellular ionic homeostasis (i.e. Ca2+ and H+) [9,16-17]. They are also the key elements to provide, during the Plasmodium development, the necessary environment for the functions of vital enzymes [18]. For instance, the hemoglobin degradation, which is dependent of the action of different proteases as falcipains and plasmepsinas, occurs in acidic compartments, and therefore, the ion homeostasis is undoubtedly crucial [8, 19, 20]. The acidic compartments are also described as the local of accumulation of antimalarials, as chloroquine and derivatives, which kill the parasite by altering the hemoglobin metabolism and the formation of hemozoin crystals [21]. However, the 4

increasing widespread resistance to chloroquine claims the search for alternative compounds able to inhibit the parasite development [22]. Crotamine

is

a

polypeptide

of

42

amino

acid

residues

(sequence

YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG, MW = 4889.81 Da and pI of about 9.5) isolated from the venom of the South American rattlesnake Crotalus durissus terrificus [23]. The high content of basic amino acid residues and the presence of three disulfide bonds confer to this toxin a stable 3D structure with high exposure of positive charges and an amphipathic characteristic [24,25]. These structural features stimulated us to investigate the cell penetrating properties of crotamine, which showed a unique high specificity and affinity for actively proliferating cells compared to quiescent non-proliferating or non-tumoral mammalian cells [26- 29]. In addition, crotamine is also capable of carrying genes and other molecules into cells [30-32]. Taking this into account, the commercial use of crotamine as a biomarker of proliferating cells and/or its employment as a carrier of bioactive molecules into cells was already protected by our group (PCT/BR06/000052; US-2008-0181849-A1; 1866332 EPO Bulletin No. 37/14 in Sept 10th, 2014). Biochemical, molecular and cellular studies showed that the cell penetrating ability of crotamine is dependent on its positive net charge and its affinity for negatively charged surfaces [30,31]. Moreover, crotamine also shows cytotoxic effect that involves the disruption of lysosomes and the consequent release of the vesicles contents, as the free calcium and cathepsins, which may trigger cell apoptosis and leading us to suggest the acid compartments of cells as the primary intracellular target of crotamine [31,33]. Crotamine also presents specificity towards the cell membranes of microorganisms, which is also dependent on their membrane surface negative charges [27,34],

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reinforcing again the importance of the negative net charges on surface for the biological activities and functions of crotamine [34]. Therefore, considering that Plasmodium acidic compartments are the local of accumulation of antimalarials and that their ionic homeostasis are crucial for the parasite development, in the present study, the potential antimalarial effect of crotamine was explored in a Plasmodium falciparum model. Both selective internalization into infected red blood cells (iRBCs) and intracellular localization of crotamine were visualized by confocal microscopy. In addition, crotamine affected parasite development in a dose-dependent manner, more likely due to the disruption of the P. falciparum parasites H+ homeostasis, as evaluated by flow cytometry and by the observed changes in lysosomotropic acridine orange (AO) fluorescence in infected erythrocytes, respectively. Therefore, we believe that the results presented here provide interesting insight for a novel potential structural model for the development of new antimalarial peptidomimetics based on disruption of the H+ homeostasis of P. falciparum parasites.

2. Materials and Methods 2.1 Ethics statement This study was approved by the Ethics Committee of the Universidade Federal de São Paulo - UNIFESP/EPM (License number 738690/2013).

2.2. Materials The venom of Crotalus durissus terrificus was extracted from snakes maintained at the Faculdade de Medicina de Ribeirão Preto (FMRP) serpentarium, Universidade de São Paulo. All chemicals and solvents were purchased from Sigma (Deisenhofen, 6

Germany or St. Louis, MO, USA). Human plasma and erythrocytes were obtained from health volunteer donors and written informed consent was obtained from all participants recruited. All procedures were strictly conducted according to the principles expressed in the Helsinki Declaration.

2.3. Preparation and biochemical characterization of crotamine Purification of native crotamine from snake venom was performed essentially as described elsewhere [29]. Briefly, six hundred milligrams of crude dried venom were dissolved in 5 mL of 0.25 M ammonium formate buffer pH 3.5, and the bulk of crotoxin, the major venom component, was eliminated by slow speed centrifugation as a heavy precipitate that formed upon slow addition of 20 mL of cold water to the solution. Tris-base 1 M was then added dropwise to the supernatant to raise the pH to 8.8 and the solution was applied to a CM-Sepharose FF (1.5 x 4.5 cm; GE Healthcare, Buckinghamshire, UK) column, equilibrated with 0.04 M Tris-HC1 buffer pH 8.8, containing 0.064 M NaCl. After washing the column with 100 mL of equilibrating solution, crotamine was recovered as a narrow protein peak by raising the NaCl concentration of the eluting solution to 0.64 M. The material was thoroughly dialyzed against water (benzoylated membrane, cut-off MW = 3,000) and lyophilized. Amino acid analysis after acid hydrolysis of a sample (4 N MeSO3H + 0.1% tryptamine; 24 h at 115oC) indicated a yield of 72 mg (14.7 mol) of crotamine and trace amounts of Thr, Ala and Val (purity  98%). The purity of crotamine were further confirmed by analytical C18 reversed phase HPLC, using linear gradient of 10-30% acetonitrile containing 0.1% TFA, and the molecular masses were verified by a mass by liquid chromatography-mass spectrometry using a LCMS-2010 EV equipped with an electronspray ionization (ESI)-probe (Shimadzu, Tokyo, Japan), as previously described 7

[29]. Pure crotamine was then labeled with the Cy3-fluorescent dye as previously described using the Fluorolink Cyanine 3 (Cy3) reactive dye (GE Healthcare, Little Chalfont, UK) [32,34].

2.4. Parasites Plasmodium falciparum chloroquine-resistant strain 3D7 was cultured in culture bottles using RPMI 1640 medium (Atená Biotecnologia, Campinas, SP, Brazil), which was supplemented with 10% of inactivated human plasma (human plasma and erythrocytes were obtained from health volunteer donors) as previously described [35]. Parasitemia was verified by Giemsa-stained smears. The parasites were isolated from the infected erythrocytes (iRBCs) by selective lysis using 10 mg/mL saponin in PBS buffer (composed by 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM NaH2PO4, and 1 mM CaCl2), followed by centrifugation at 2000 × g for 10 min at 4oC. Then, the isolated parasites were washed twice in PBS buffer to remove the red cell membranes.

2.5. Confocal microscopy P. falciparum culture was incubated with Cy3-crotamine (10 µM) for 1 h, at room temperature. In the last 10 min of incubation, DAPI (0.01 mg/mL) was added, followed by washing with PBS buffer. Cells were resuspended in the same buffer and plated on a microscopy chamber previously treated for 1 h with L-polylysine (1 mg/mL). The data acquisition was performed in a Leica TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) using an oil immersion 63  objective. The dyes parameters used were λEX 545 nm and λEM at 590-620 nm for Cy3-crotamine, and λEX 405 nm/λEM 420-470 nm for DAPI fluorescence. 8

Image acquisition of H+ mobilization from acidic compartments was observed with lysosomotropic fluorochrome acridine orange (AO) loaded iRBCs. For this, 5 µM of AO was incubated with iRBC during 30 min. After three washes with PBS buffer, iRBCs were resuspended in PBS buffer, and the effects on fluorescence of AO in the presence of 20 µM of crotamine were monitored. The metachromatic dye AO accumulates mainly in the acidic compartments, preferentially in lysosomes and nucleus. AO shows different emission wavelengths, red or green fluorescence, depending of the dye concentration, when excited by blue light (488 nm). Therefore, the AO acquisition parameters were λEX 488 nm (argon laser), and λEM (green) 500-530 nm and λEM (red)  560 nm. Lysosomal/endosomal compartments accumulate higher concentration of AO, and consequently these compartments are labeled in red. In the opposite side, nucleus and cytoplasm accumulates lower dye concentration emitting green light. Analysis of merged green and red emission lights can be used to observe simultaneously concentrated and non-concentrated compartments (uptake method). The software-based analysis allowed measurements in the selected cells (region of interest), as a function of time.

2.6. Flow Cytometry Culture of iRBC containing synchronous parasites at ring stage (2.0% hematocrit and 2.5% parasitemia) was added to a 96-wells microplate. Crotamine at five different concentrations (from 1.25 µM to 20 µM) were added on iRBC culture. As control, culture of red blood cells (RBC) and iRBC culture without treatment were used. The plate was placed in an incubator set at 37oC for 48 h. Next the medium was removed and the cells were fixed with 200 µL of 2 % formaldehyde in PBS (v/v) for 24 h at room temperature. A solution with 0.1 % Triton X-100 in PBS (v/v), containing 1 nM 9

YOYO-1 (Invitrogen/Life Technologies, Grand Island, NY, USA) was added before the data acquisition. Parasitemia was measured in a flow cytometer FACS Calibur (BD Biosciences, Franklin Lakes, New Jersey, USA), with DNA stain YOYO-1 as a marker for cell survival. The number of fluorescent events in 10,000 cells represents de percentage of parasitemia. The IC50 values were obtained using a non-linear doseresponse curve fitting analysis via Graph Pad Prism v.5.0 software (San Diego, CA, USA).

2.7. Spectrofluorometer measurements Isolated parasites (107 cells mL-1) were incubated with PBS buffer in 500 µL cuvette at 37oC to measure the intracellular proton mobilization. Cells were loaded with 5 µM of AO and the uptake of fluorescent marker by parasite was monitored. After complete internalization of AO in the food vacuole of the parasite, crotamine was added in steps for each concentration (1.25, 5 and 20 µM). The fluorescence was measured continuously in a Shimadzu RF-5301 PC spectrofluorometer at λEX 495nm and λEM 530 nm. The control with chloroquine was performed in the same conditions [16].

2.8. Statistical analysis Statistical analyses were performed using Prism5® by one-way ANOVA and Bonferroni’s post test. The results are from three independent experiments performed on different days .A p value < 0.05 was considered statistically significant.

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3. Results 3.1. Localization of crotamine by confocal microscopy After 1 h of incubation with 10 µM of Cy3-crotamine, the toxin was visualized within the parasites inside the infected red blood cells (iRBCs) (Figure 1, supplemental figure 1A). It is of note that crotamine was not internalized by uninfected RBC, demonstrating the selectivity for iRBC. Staining with DAPI also allowed demonstrating the localization of Cy3crotamine in the parasite nucleus, although part of the labeled crotamine remained attached to the lipid cell membrane of the iRBCs (Figure 1A).

3.2. Toxicity of crotamine against parasites The potential toxicity of crotamine against Plasmodium falciparum parasites was assessed in culture, and we observed that crotamine affected the parasite development and displayed a very potent anti-plasmodial activity, with an IC50 value of 1.87 µM (Figure 1B). A potential anti-leishmanial activity was also described for crotamine by others, but with no mention or discussion on the molecular mechanism of action [36].

3.2. Effects of crotamine on acidic compartments The H+ homeostasis in isolated P. falciparum and iRBC was explored in the presence of crotamine to verify the eventual disruption of lysosome ion maintenance, as should be expected based on our previous studies on crotamine cytotoxic activity [26,27]. Figure 2A shows that crotamine promotes an increase in the uptake of the lysosomotropic fluorochrome Acridine Orange (AO) in subcellular compartments in isolated parasites, in a dose-dependent manner. As control, the same experiment was performed with chloroquine (a weakly basic molecule), which promotes the extrusion of 11

AO from the acidic compartments to the cytosol, presumably as a result of alkalinization of the organelle lumen [37]. Our results suggest that crotamine acts causing lysosomal destabilization and by interfering in the organelle and parasite cell membrane structure (not alkalinization), as previously observed in other models [38]. Therefore, crotamine acts by a different manner of that described for chloroquine. The H+ homeostasis destabilization by crotamine was also confirmed by confocal microscopy (Figure 2B and 2C). We visualized an AO mobilization from acid compartment (fluorescence channel, red) and parasite cytosol (fluorescence channel, green). Fluorescence reduction was observed in both cases, indicating changes in membrane structure. Thus, our data indicates that crotamine interferes with H+ homeostasis in P. falciparum parasites.

4. Discussion Aiming to explore the mechanism of crotamine anti-plasmodial activity, we first verified the internalization of crotamine by infected red blood cells (iRBCs). Incubation with fluorescently labeled crotamine (Cy3-crotamine) showed the selectivity for iRBCs, as well as no internalization was observed in uninfected RBCs (Figure 1). This result is in good agreement with our previous findings showing no hemolytic activity for crotamine against human erythrocytes for concentrations up to 100 µM [27], and the importance of the negative charge on membranes for the selective affinity and activities of native crotamine [34]. The selectivity for the iRBCs may therefore be explained by the extensive host cell remodeling mediated by parasites, aiming to provide nutrients from serum for their survival and export proteins and lipids to iRBC cytoplasm and membrane [39]. As described by others, these alterations may affect the trafficking

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routes (vesicles, channels and parasitophorous vacuole membrane extensions) and membrane charge composition (proteins and phospholipids) [39-41]. A number of small amphipathic peptides from natural source possess both the antimicrobial and cytotoxic effects [27], but very few of them also have cell-penetrating properties as described for crotamine [42]. In addition, to our knowledge, among known amphipathic peptides none of them can form complex with DNA molecules and carry cargos into the cells as crotamine does [30,32,33]. We believe that this gives to crotamine a unique advantage, as the anti-plasmodial activity described for crotamine here could also be further potentiated by its combination with lethal genes specific for malaria parasites aiming therapeutic intervention [43,44]. Crotamine was shown to completely disrupt mammalian cells lysosomes, proving its effect on negatively charged vesicles of mammalian cells [31]. This allowed us to suggest that crotamine effectively acts on the alteration of vesicle internal pH, as a consequence of its abundant content of Lys and Arg residues and the resulting high net charge (8+) [24,25]. Regulation of the internal pH is important for the survival of parasites, as this sets the environment for the functioning of several intracellular enzymes crucial for parasite devolpment. Confocal microscopy studies allowed confirming that crotamine interferes with H+ homeostasis in P. falciparum parasites as visualized by AO mobilization (fluorescence reduction) from acid compartment (fluorescence channel, red) and parasite cytosol (fluorescence channel, green) suggestive of changes in membrane structure, supporting its potential as a candidate for a novel antimalarial drug or molecular template. It is important to emphasize that the presence of crotamine in cell nucleus and its binding to chromosomal DNA were also demonstrated by us in several cultured

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mammalian cells [27], which is in line with the localization of Cy3-crotamine in the parasite nucleus observed here (Figure 1, supplemental figure 1).

5. Conclusion Taken together, we have demonstrated here that crotamine is selectively internalized by parasite-infected erythrocytes, with co-localization with DAPI. The dose-dependent anti-plasmodial activity of crotamine was also shown here for the first time, and our data suggests the potential involvement of disruption of H+ homeostasis in P. falciparum parasites in the mechanism of action for the crotamine antimalarial activity. Our data presented here, allow us to suggest crotamine, as well as derived peptidomimetics, as a potential molecule for the inhibition of malaria infection, and as a powerful tool for the Plasmodium cell biology studies at the molecular and cellular level. Aiming to innovative strategies for antimalarial therapy, crotamine activity described here can potentially be further improved by the combination of crotamine with therapeutic drugs and/or genes, as crotamine was also shown to be an efficient transport vector [30,32]. Although in vivo animal model experiments with crotamine would be now decisive for encouraging further studies for antimalarial activity, the viability of employment of crotamine in therapy was already demonstrated by us in vivo, at least for the antitumoral activity [29,30]. In addition, the potential of targeting specifically infected erythrocytes versus uninfected ones and also the ability to disrupt the acidic compartments provide a great value for crotamine as a useful new tool compound for the Plasmodium cell biology studies.

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6. Acknowledgments This work was supported by the Brazilian Agencies: Fundação de Amparo Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). We also thank the Multiuser Multiphoton Confocal Microscopy Laboratory (INFAR-UNIFESP) for the access to confocal microscopy devices.

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References 1. 2. 3. 4. 5.

6.

7.

8. 9.

10.

11.

12.

13.

14. 15.

16.

17.

WHO World malaria report 2013. Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, et al. Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 2009; 361: 455-67. Sibley CH. Infectious diseases. Understanding artemisinin resistance. Science 2015; 347: 373-4. Visser BJ, van Vugt M, Grobusch MP. Malaria: an update on current chemotherapy. Expert Opin Pharmacother 2014; 15(15): 2219-54. Goncalves LA, Cravo P, Ferreira MU. Emerging Plasmodium vivax resistance to chloroquine in South America: an overview. Mem Inst Oswaldo Cruz 2014; 109(5): 534-9. Fidock DA, Rosenthal PJ, Croft SL, Brun R, Nwaka S. Antimalarial drug discovery: Efficacy models for compound screening. Nat Rev Drug Discov 2004; 3(6): 509-520. Baumeister S, Winterberg M, Przyborski JM, Lingelbach K. The malaria parasite Plasmodium falciparum: cell biological peculiarities and nutritional consequences. Protoplasma 2010; 240(1-4): 3-12. Rosenthal PJ. Hydrolysis of erythrocyte proteins by proteases of malaria parasites. Curr Opin Hematol 2002; 9(2): 140-5. Gazarini ML, Thomas AP, Pozzan T, Garcia CR. Calcium signaling in a low calcium environment: how the intracellular malaria parasite solves the problem. J Cell Biol 2003; 161(1): 103-10. Tokumasu F, Crivat G, Ackerman H, Hwang J, Wellems TE. Inward cholesterol gradient of the membrane system in P. falciparum-infected erythrocytes involves a dilution effect from parasite-produced lipids. Biol Open 2014; 3(6): 529-41. Richard D, Kats LM, Langer C, Black CG, Mitri K, Boddey JA, et al. Identification of rhoptry trafficking determinants and evidence for a novel sorting mechanism in the malaria parasite Plasmodium falciparum. PLoS Pathog 2009; 5(3): p. e1000328. Bagnaresi P, Barros NM, Assis DM, Melo PM, Fonseca RG, Juliano MA, et al. Intracellular proteolysis of kininogen by malaria parasites promotes release of active kinins. Malar J 2012; 11: 156. Melo PM, Bagnaresi P, Paschoalin T, Hirata IY, Gazarini ML, Carmona AK. Plasmodium falciparum proteases hydrolyze plasminogen, generating angiostatin-like fragments. Mol Biochem Parasitol 2014; 193: 45-54. Martin RE, Ginsburg H, Kirk K, Membrane transport proteins of the malaria parasite. Mol Microbiol 2009; 74(3): 519-28. Staines HM, Ashmore S, Felgate H, Moore J, Powell T, Ellory JC. Solute transport via the new permeability pathways in Plasmodium falciparum-infected human red blood cells is not consistent with a simple single-channel model. Blood 2006; 108(9): 3187-94. Gazarini ML, Sigolo CAO, Markus RP, Thomas AP, Garcia CRS. Antimalarial drugs disrupt ion homeostasis in malarial parasites. Mem Inst Oswaldo Cruz 2007; 102(3): 329-34. Miranda K, de Souza W, Plattner H, Hentschel J, Kawazoe U, Fang J, et al. Acidocalcisomes in Apicomplexan parasites. Exp Parasitol 2008; 118(1): 2-9. 16

18.

19.

20.

21.

22. 23

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

Farias SL, Gazarini ML, Melo RL, Hirata IY, Juliano MA, Juliano L, Garcia CR. Cysteine-protease activity elicited by Ca2+ stimulus in Plasmodium. Mol Biochem Parasitol 2005; 141(1): 71-79. Drew ME, Banerjee R, Uffman EW, Gilbertson S, Rosenthal PJ, Goldberg DE. Plasmodium food vacuole plasmepsins are activated by falcipains. J Biol Chem 2008; 283(19): 12870-6. Sijwali PS, Rosenthal PJ. Gene disruption confirms a critical role for the cysteine protease falcipain-2 in hemoglobin hydrolysis by Plasmodium falciparum. Proc Natl Acad Sci U S A 2004; 101(13): 4384-4389. Sullivan DJ Jr., Gluzman IY, Russell DG, Goldberg DE. On the molecular mechanism of chloroquine's antimalarial action. Proc Natl Acad Sci U S A 1996; 93(21): 11865-11870. Wernsdorfer WH. The Development and Spread of Drug-Resistant Malaria. Parasitol Today 1991; 7(11): 297-303. Rádis-Baptista G, Oguiura N, Hayashi MA, Camargo ME, Grego KF, Oliveira EB, Yamane T. Nucleotide sequence of crotamine isoform precursors from a single South American rattlesnake (Crotalus durissus terrificus). Toxicon 1999; 37(7): 973-984. Fadel V, Bettendorff P, Herrmann T, de Azevedo WF Jr, Oliveira EB, Yamane T, et al. Automated NMR structure determination and disulfide bond identification of the myotoxin crotamine from Crotalus durissus terrificus. Toxicon 2005; 46(7): 759-767. Nicastro G, Franzoni L, de Chiara C, Mancin AC, Giglio JR, Spisni A. Solution structure of crotamine, a Na+ channel affecting toxin from Crotalus durissus terrificus venom. Eur J Biochem 2003; 270(9): 1969-1979. Pereira A, Kerkis A, Hayashi MA, Pereira A, Silva FS, Oliveira EB. Crotamine toxicity and efficacy in mouse models of melanoma. Expert Opin Investig Drugs 2011; 20(9): 1189-1200. Yamane ES, Bizerra FC, Oliveira EB, Moreira JT, Rajabi M, Nunes GL, et al. Unraveling the antifungal activity of a South American rattlesnake toxin crotamine. Biochimie 2013; 95(2): 231-240. Kerkis I, Hayashi MA, Prieto da Silva AR, Pereira A, De As Junior PL, Zaharenko AJ, et al. State ofthe art in the studies on crotamine, a cell penetrating peptide from South American rattlesnake. Biomed Rest Int 2014. Hayashi MA, Oliveira EB, Kerkis I, Karpel RL. Crotamine: a novel cellpenetrating polypeptide nanocarrier with potential anti-cancer and biotechnological applications. Methods Mol Biol 2012; 906: 337-352. Nascimento FD, Hayashi MA, Kerkis A, Oliveira V, Oliveira EB, RadisBaptista G, et al. Crotamine mediates gene delivery into cells through the binding to heparan sulfate proteoglycans. J Biol Chem 2007; 282(29): 2134921360. Hayashi MA, Nascimento FD, Kerkis A, Oliveira V, Oliveira EB, Pereira A, et al. Cytotoxic effects of crotamine are mediated through lysosomal membrane permeabilization. Toxicon 2008; 52(3): 508-517. Chen PC, Hayashi MA, Oliveira EB, Karpel RL. DNA-interactive properties of crotamine, a cell-penetrating polypeptide and a potential drug carrier 2012; PLoS One. 7(11): p. e48913. Nascimento FD, Sancey L, Pereira A, Rome C, Oliveira V, Oliveira EB, et al. The natural cell-penetrating peptide crotamine targets tumor tissue in vivo and 17

34.

35. 36.

37.

38. 39. 40.

41. 42.

43.

44.

triggers a lethal calcium-dependent pathway in cultured cells. Mol Pharm 2012; 9(2): 211-221. Costa BA, Sanches L, Gomide AB, Bizerra F, Dal Mas C, Oliveira EB, et al. Interaction of the rattlesnake toxin crotamine with model membranes. J Phys Chem B 2014; 118(20): 5471-5479. Trager W, Jensen JB, Human malaria parasites in continuous culture. Science 1976; 193(4254): 673-675. Passero LF, Tomokane TY, Corbett CE, Laurenti MD, Toyama MH. Comparative studies of the anti-leishmanial activity of three Crotalus durissus ssp. venoms. Parasitol Res 2007; 101(5): 1365-1371. Gazarini ML, Sigolo CA, Markus RP, Thomas AP, Garcia CR. Antimalarial drugs disruption homeostasis in malarial parasites. Mem Inst Oswaldo Cruz 2007; 102(3): 329-334. Schlesinger PH, Krogstad DJ, Herwaldt BL. Antimalarial Agents - Mechanisms of Action. Antimicrob Agents Chemother 1988; 32(6): 793-798. Goldberg DE, Cowman AF. Moving in and renovating: exporting proteins from Plasmodium into host erythrocytes. Nat Rev Microbiol 2010; 8(9): 617-621. Cooke BM, Lingelbach K, Bannister LH, Tilley L. Protein trafficking in Plasmodium falciparum-infected red blood cells. Trends Parasitol 2004; 20(12): 581-589. Maier AG, Cooke BM, Cowman AF, Tilley L. Malaria parasite proteins that remodel the host erythrocyte. Nat Rev Microbiol 2009; 7(5): 341-354. Kerkis I, Hayashi MA, Prieto da Silva AR, Pereira A, De As Junior PL, et al. Crotamine is a novel cell-penetrating protein from the venom of rattlesnake Crotalus durissus terrificus. FASEB J 2004; 18(12): 1407-1409. Banerjee T, Jaijyan DK, Surolia N, Singh AP, Surolia A. Apicoplast triose phosphate transporter (TPT) gene knockout is lethal for Plasmodium. Mol Biochem Parasitol 2012; 186(1): 44-50. Lee SJ, Seo E, Cho Y. Proposal for a new therapy for drug-resistant malaria using Plasmodium synthetic lethality inference. Int J Parasitol Drugs Drug Resist 2013; 3: 119-128.

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Figure 1. Uptake of crotamine by parasite-infected RBCs and effect on P. falciparum survival in vitro. (A) Plasmodium falciparum culture was incubated with Cy3-crotamine (10 µM) for 1 h, at room temperature. In the last 10 min of incubation, DAPI (0.01 mg/mL) was added, followed by washing with PBS buffer. Cells were resuspended in the same buffer and plated on a microscopy chamber. The dye parameters used were λEX 545 nm and λEm at 590-620 nm for Cy3-crotamine (red channel), and λEX 405 nm/λEm 420-470 nm for DAPI fluorescence (blue). Scale bar = 10 µm. (B) Crotamine was added to 20, 10, 5, 2.25 and 1.25 µM in 200 µL of P. falciparum culture (iRBC). The plate was incubated for 48 h at 37oC. YOYO-1 (1 nM) was used as the cell nucleus dye. RBC and iRBC without treatment was used as controls performed in the same conditions. The data were acquired via flow cytometry (FACS Calibur, BD Biosciences). The results are from three independent experiments performed in triplicate on different days. The combined data were compared by paired one-way ANOVA and Boferroni’s post test * p< 0.05.

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Figure 2. Effect of chloroquine and crotamine on intracellular acridine orange (AO) mobilization from acidic compartments of isolated P. falciparum parasites. (A) The lysosomotropic fluorochrome AO (5 µM) were added in isolated parasites (107 cells mL-1) solution. Different concentrations of crotamine (1.25, 5, and 20 µM) were added during AO fluorescence acquisition in spectrofluorometer cuvette. Fluorescence intensities (arbitrary fluorescence units - AFU) represent at least three different cell preparations. Crotamine (gray line) and chloroquine (black line). (B) The alterations of acridine orange (AO) fluorescence was performed in confocal microscopy. iRBC was loaded with 5 µM of AO during 30 min. After addition of 20 µM of crotamine, the effects on fluorescence of AO were monitored. (a-f) Confocal images: (a) Brightfield (BF); (b) Merged; (c) basal AO red channel; (d) basal AO green channel; (e-f) AO red and green channels after crotamine addition; (C) Histogram of AO fluorescence (red channel) with mean  SD (N = 3). The data were analyzed by paired t-test * p < 0.05.

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