Highly Potential Antiplasmodial Restricted Peptides

Accepted Article

Received Date : 13-Jan-2014 Revised Date : 01-Apr-2014 Accepted Date : 29-Apr-2014 Article type

: Research Article

Corresponding author e-mail id: [email protected] Highly potential anti-plasmodial restricted peptides Marcelo Der Torossian Torresa, Adriana Farias Silvaa, Flávio Lopes Alvesb, Margareth Lara Capurroc, b a, Antonio de Miranda , Vani Xavier Oliveira Junior *

a

Universidade Federal do ABC, Centro de Ciências Naturais e Humanas, Santo André, SP, Brazil.

b

Universidade Federal de São Paulo, Departamento de Biofísica, São Paulo, SP, Brazil.

c

Universidade de São Paulo, Instituto de Ciências Biomédicas II, São Paulo, SP, Brazil.

Corresponding author: Vani Xavier Oliveira Junior Rua Santa Adélia, 166, Santo André, SP, Brazil, 09210-170 Tel.: + 55 11 4996-0194; fax: + 55 11 4996-0150 E-mail:[email protected]

Abstract

Malaria is an infectious disease responsible for approximately one million deaths annually. The antimalarial effects of angiotensin II and its analogs against Plasmodium gallinaceum and P. falciparum have recently been reported. To evaluate anti-plasmodial activity, we synthesized five angiotensin II restricted analogs that containing disulfide bridges. To accomplish this, peptides containing two inserted amino acid residues (cysteine), were synthesized by the Fmoc solid phase method, purified by liquid chromatography, and characterized by mass spectrometry. Conformational studies were performed by circular dichroism. The results indicated that two of the analogs had higher anti-plasmodium activity (92% and 98% activity) than angiotensin II (88% activity), measured by fluorescence microscopy. Results showed that the insertion position must be selected, to preserve the hydrophobic interactions between the nonpolar residues, as this affects anti-plasmodial activity. The circular dichroism studies suggested that the active analogs as well as the native angiotensin II adopt a β-turn conformation in different solutions. This approach provided insight for understanding the

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effects of restricting the ring size and position on the bioactivity of angiotensin II and provides a new direction for the design of potential chemotherapeutic agents. Keywords: Angiotensin II; SAR; Disulfide Bridge; Plasmodium; Malaria. 1. Introduction Malaria is a tropical disease that affects humans in approximately one hundred countries around the world. It is present mainly in Africa, Asia, and Central and South America therefore approximately three billion people are at risk. The disease is caused by a protozoan of the genus Plasmodium and is transmitted to humans by Anopheles female mosquitoes (1). Five of the 150 species of Plasmodium catalogued infect humans (1). They are P. falciparum, P. vivax, P. malariae, P. ovale and P. Knowlesi (2-4). There are approximately one million deaths annually(5) and the most likely to contract the disease are children and pregnant women (6).

The use of peptides to prevent or treat malaria is increasing with the advent of possible antimalarial candidates belonging to the peptide class of molecules (7-9). Among them, the most efficacious peptides are generally characterized by the presence of aside-chain-to-side-chain covalent bond. The most common kinds of restrictions present in natural and synthetic bioactive peptides are the lactam (10) and disulfide bridges (11).

The introduction of a scaffold in a synthetic peptide can result in a highly constrained structure with defined limits on the conformations (12), which is important to most of the biological activities.

Maciel et al. tested some natural peptides in vitro and in vivo and correlated their ability to alter the development of the avian parasite P. gallinaceum. Angiotensin II (AII) showed significant antiplasmodial activity (88% reduction in the population of plasmodium). However, AII (Asp-Arg-Val-TyrIle-His-Pro-Phe) is a well known hormone (13) of the renin-angiotensin-aldosterone system, responsible for regulating blood pressure (14). Due to its effects on blood pressure, AII cannot be used as an antimalarial drug (13).Therefore, several AII analogs, including restricted peptides, were synthesized and tested in blood pressure (15) and antimalarial assays (16). The results showed that some restricted analogs promoted a decrease of approximately 75% of the sporozoites population and did not exhibit effects on blood pressure (15).

Several authors (17-19) suggest that the mechanism of action of antimicrobial peptides depends on a combination of hydrophobic and electrostatic effects (20). Most peptides are attracted to the charged surface of bacterial membranes, where they undergo conformational changes. However, the mechanism by which peptides interact with bacterial membranes has not been elucidated (17, 21).

In this work, new constrained AII analogs were synthesized to provide a better understanding of the anti-plasmodial effects of the disulfide bridge restriction. The relevance of the ring position in the structural properties of the peptides was analyzed. Disulfide bridge i-(i+2), i-(i+3), i-(i+4) and i-(i+9)

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peptides were synthesized, and their activities against mature P. gallinaceum sporozoites were determined by fluorescence microscopy. Finally, the effects of the medium on the peptide structure were evaluated by circular dichroism (CD) in phosphate-buffered saline (PBS) and solutions with sodium dodecylsulfate (SDS), 2,2,2-trifluoroethanol (TFE) or methanol.

2. Experimental Procedures 2.1 Solid-phase peptide synthesis (SPPS) Peptides were synthesized manually (22) using the fluoromethyloxycarbonyl (Fmoc) strategy (23) and Wang (24) resins, with a substitution degree around 0.5 mmolg-1. Deprotection steps were carried out by treatment with 20% 4-Methylpiperidine (4-MePip) in dimethylformamide (DMF) for 40 minutes; the reaction was monitored by the colorimetric Kaiser test (25), where an aliquot of the peptidyl-resin was analyzed with a solution of ninhydrin, phenol and pyridine, confirming the presence of free amino groups in solution (effective deprotection) with purple coloring.Amino acid coupling steps were accomplished by treating the deprotected amino acyl-resin with a 2.5-fold molar excess of the Fmocprotected amino acid, activated by DIC/HOBt, for two hours at room temperature in DCM/DMF, this solvent system is used to obtain a relevant swelling of the solid support, leading to optimal conditions of coupling by permeating of the coupling reagents and amino acids in the resin pores. The Kaiser test was performed again, but now indicating no presence of free amino groups in solution (effective coupling) with yellow coloring, if it indicated free amino groups (purple coloring), recoupling steps are needed. . To obtain a rapid acylation of the amino function (peptidyl-resin) and have a limiting effect on possible side reactions and racemization, the DIC and HOBt were used to active the carboxylic group of sequencial amino acid residue. Recouplings were performed for one hour, when needed, using a 2.5-fold excess of TBTU in the presence of DIPEA in DCM/NMP (1:1, v/v). Each step is followed by a washing procedure with DMF, methanol and DCM to alternately change the degree of resin swelling, favoring the elimination of excess reagents and byproducts(26).

2.2 Cleavage, deprotection and cyclization Dry-protected peptidyl-resin was exposed to TFA/water/anisole (95:2.5:2.5, v/v/v) for two hours at room temperature. The crude deprotected peptides were precipitated with anhydrous diethyl ether, filtered from the ether-soluble products, extracted from the resin with 60% acetonitrile (ACN) in water and lyophilized. The disulfide bridges were formed by first solubilizing the crude peptides (1 gL-1) in an 80% acetic -1

acid solution containing 0.04 molL iodine (27).After 40 minutes the reaction was extracted with water and diethyl ether. The resulting solution was lyophilized.

2.3 Peptide Purification and Analysis The crude lyophilized peptides were then purified by preparative reverse-phase high-performance liquid chromatography (RP-HPLC) in 0.1% trifluoroacetic acid (TFA)/60% ACN (A/B) on a Delta Prep 600 (Waters Associates). Briefly, the peptides were loaded onto a Phenomenex C18 (21.2 mm × 250

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mm, 15 µm particles, 300 Å pores) column at a flow rate of 10.0 mLmin-1 and eluted using a linear gradient (0.33% B/min slope) of TFA/ACN with detection at 220 nm. Selected fractions containing the purified peptides were pooled and lyophilized. Purified peptides were characterized by liquidchromatography electrospray-ionization mass spectrometry (LC/ESI-MS) (Table 1). LC/ESI-MS data were obtained on a Model ZMD mass spectrometer (Micromass) coupled to a Model 2690 HPLC system (Waters Alliance) using a Phenomenex Gemini C18 column (2.0 mm × 150 mm, 3.0 µm particles, 110 Å pores). Solvent A was 0.1% TFA in water, and solvent B was 60% ACN in solvent A. Elution with a 5–95% B gradient was performed over 30 min and peptides were detected at 220 nm. Mass measurements were performed in a positive mode with the following conditions: mass range -1

between 500 to 2000 m/z, nitrogen gas flow rate at 4.1 Lh , capillary voltage at 2.3 kV, cone voltage at 32 V, extractor voltage at 8 V, source heater at 100 °C, solvent heater at 400 °C, ion energy at 1.0 V and a multiplier at 800 V.

2.4 Circular Dichroism Spectroscopy All CD experiments were performed on a J-810 Circular Dichroism Spectropolarimeter (Jasco). FarUV (190–250 nm) CD spectra were recorded after four accumulations at 20 °C using a 0.5 mm path length quartz cell between 260 and 195 nm at 50 nmmin-1 with a band width of 0.5 nm. All peptides -1

-1

were analyzed in the following four solutions: 15 mmolL PBS (pH = 7.4), 10 mmolL SDS in PBS, 50% TFE in PBS and 50% methanol in PBS. The peptide concentration was roughly 10-4 molL-1. The CD spectra for the SDS, TFE and methanol solutions were subtracted from that for PBS, and a Fourier transform filter (FFT) filter was applied to minimize background effects.

2.5 Mosquito rearing and maintenance of the parasite life cycle The Aedes aegypti RED strain is highly susceptible to P. gallinaceum (28) and was used in all experiments. Mosquitoes were reared using standard laboratory procedures (29). An aliquot of frozen chicken blood infected with the P. gallinaceum strain 8A was obtained from A. Krettli (René Rachou Institute of Research, FIOCRUZ, MG, Brazil). This sample was used to inoculate and establish initial infections in chickens. All subsequent infections of chickens and mosquitoes were accomplished by allowing the mosquitoes to feed on the chickens.

The experiments followed the current guidelines for the care and use of laboratory animals as well as the ethical guidelines for investigations, and these experiments were pre-approved by the Animal Care Committee of the Universidade de São Paulo – number 133.

2.6 Effects of AII analogs on salivary gland-derived Plasmodium gallinaceum sporozoites Nine thousand P. gallinaceum mature sporozoites were recovered from the salivary glands of Aedes -1

-1

aegypti and incubated with 40 μmolL digitonin (positive control), 60 μmolL AII analogs (test), or PBS (negative control) at 37ºC for one hour (13, 16). Cell membrane integrity was then monitored using an Observer Axio Vision A.1 inverted fluorescence microscope (Carl Zeiss) coupled to an AxioCam HR digital camera (Zeiss) with 1300 x 1030 pixels resolution and 8-bit quantization after addition of 1 μL

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of the ethidium bromide aqueous solution (200 μmolL-1). Images were obtained using a 40X objective lens and a green filter effect in red. The spectral range was set with excitation at 524 nm in the visible spectrum in order to produce orange-red fluorescence centered at 605 nm. Processing was completed by Axio 4.7 software.

2.7 Contractile response assays - stimulations with AII and its analogs The pharmacological experiment followed the current guidelines for the care and use of laboratory animals as well as the ethical guidelines for investigations, and these experiments were pre-approved by the Animal Care Committee of the Universidade Federal de São Paulo –number 2013/479357. The bioassay experiment was carried out in duplicate and generated equivalent results.

Experiments were carried out with C57BL/6J mice from the Centro de Desenvolvimento de Modelos Experimentais da Universidade Federal de São Paulo (CEDEME-UNIFESP). Stomach fundus was isolated from each mouse, divided in two strips along the longitudinal muscle, and mounted into 5 mL organ baths containing modified Krebs-Ringer solution (144 mmolL-1 NaCl, 5 mmolL-1 KCl, 1.1 mmolL1

-1

-1

-1

-1

MgSO4, 25 mmolL NaHCO3, 1.1 mmolL NaH2PO4, 1.25 mmolL CaCl and 5.5 mmolL glucose at

37 °C (pH 7.4)) and were continuously carboxygenated (95% O2/5% CO2) (30). Contractile responses to the stimulations with AII and its analogs (10-6 molL-1) were measured with a TRI201 tension transducer (PanLab) through an amplifier (Powerlab 4/30). Data was collected through Labchart Pro V7 software. The resting tension was maintained at 0.5 g and the tissues were left to equilibrate for 90 min. The bath solution was frequently changed. The maximal effect was obtained by comparing -6

-1

contractile responses induced by carbachol (10 molL ). The specificity test of the analogs to the AT1 receptor was not verified by muscle tissue incubation with Losartan because the stimulation with the analogs was not significant (15). Data analysis was performed using GraphPad-Prism 5.0 software.

2.8 Statistical analysis The Kruskal-Wallis (31) and Tukey’s (32) multiple comparison tests were used to assess the statistical significance of the differences between the control and the analogue-treated groups.

3. Results Based on the anti-plasmodial activity reported by Maciel (13) and Chamlian (16), we synthesized five AII restricted analogs that contained disulfide bridge cyclizations by inserting two Cys residues in the native sequence of the hormone.

The analogs were designed in view of the higher anti-plasmodial activity obtained for lactam bridge restricted AII analogs, which presented no pressor activity. The constraint was added on the Nterminal extremity, described by Chamlian (16) as the most important region to be constrained in this class of peptides. We also synthesized an analog that presented an i-(i+9) disulfide bridge restricting the entire native sequence of AII.

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All DIC- and HOBt-mediated peptides were synthesized on a Wang resin using solid phase method and the Fmoc strategy. Orthogonal protection of the side chains of the bridgehead elements was employed to allow the formation of the disulfide bridge after the peptide was cleaved from the resin. -1

Cyclization was performed by dissolving the peptide in 80% acetic acid containing 0.04 molL of iodine (Fig 1). Formation of the disulfide bridge by this method is more probable in smaller ring sizes, but in this case formation of the i-(i+9) disulfide bridge was accomplished with no difficulties, and despite its increased flexibility, the enlargement of the ring did not detract from the expected bioactivity. Peptides were cleaved and deprotected, then purified by RP-HPLC, resulting in a chromatographic purity greater than 98% in all cases. Mass characterization by LC/MS-ESI(+) confirmed that the purified peptides agreed with expected theoretical values (Table 1).

The anti-plasmodial activity in vitro was analyzed by a sporozoitic inactivation assay using fluorescence microscopy after one hour of incubation of each peptide and control. Results were reported as percent fluorescent sporozoites (Fig 2). Assays were performed in triplicate using three different mosquito batches (n = 9).

Results obtained in the contractile response assays showed that, out of the five analogs tested, none presented significant contractile activity compared to AII and carbachol (Fig 3).

The effect of disulfide bridge insertion in the conformational behavior of the synthesized peptides was analyzed using CD. They were acquired in PBS, SDS, TFE/PBS (1:1 v/v) and methanol/PBS (1:1 v/v) (Fig 4). The PBS buffer was chosen in order to preserve conformational integrity of the peptides and due to low absorbance in the 260 and 195 nm region, besides that, its range is in the region of interest (pH 7.4). The surfactant (SDS) was used to simulate peptide-membrane interaction (33), it is well known that it induces α-helix conformation in short peptides and it does not absorb in the same spectrum range of peptides (34, 35). The TFE solvent is widely used in studies of peptides since it promotes the formation of α-helix (36) and helix stability (37). The methanol is known as a β-type inductor (38).

4. Discussion The AII molecule has a high degree of flexibility, creating difficulty in assigning it to a single bioactive conformation. It is well established that, in solution, this hormone readily interconverts between several conformations and so that, at equilibrium, it exists in several different structural forms (39).

One method of probing the receptor-bound conformation of a ligand in the favorable intermolecular and membrane-peptide interactions is through the use of a conformational restriction (40, 41).Structural changes in the molecule present analogs in which one or more conformations are excluded. Miranda and Juliano (42) reported the preparation and biological activity assays of some AII restricted analogs having a Cys moiety at diverse locations. In addition, they observed the preferential folded conformation in the pH range 4.0 to 9.0 and found that the biological activities of all cyclic

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analogs were insignificant compared to AII in terms of pressor activity. Replacement of some of the residues may be critical for both the pressor (43, 44) and the anti-plasmodial activities. We chose to insert the Cys residue because of the promising work by Chamlian (16), where a lactam bridge near the N-terminus of AII resulted in increased antimalarial activity. We had the additional goal of taking the primary sequence of AII and the role of each amino acid residue into account in the synthesis of the peptides. According to some authors (43-46), the positions of the residues on the AII molecule determined the degree of pressor activity because of the stabilization of bioactive conformations anchoring, and interactions with the hormone-receptor. Results obtained using techniques such as NMR, CD spectroscopy and X-ray diffraction (47-51), in addition to theoretical calculations, suggest that the preferential a compact folded structure is adopted. This is thought to be a result of the proximity of its amino and carboxy charged terminal extremities (51, 52) and the interaction between 4

8

6

the Tyr and Phe aromatic side chains (53, 54), mediated by the presence of the His side chain. The 7

folded conformation may occur because of the rigidity of Pro residue, the hydrophobic steric effects of Val3, or the Ile residue stabilizing this conformation. Val3 is thought to be responsible for guiding the Tyr4 side chain through steric hindrance (55), allowing the interaction of the Arg2 guanidine side chain with the hydroxyl group of Tyr4 and contributing to the stabilization of the turned conformation in native AII (56). If considering the interaction with membranes, the most important residues to be evaluated are: Phe8 because of its hydrophobic 2

character and positively charged guanidinium group of Arg residue contributes to the peptide1

membrane charge-charge interactions. The Asp residue contributes less to the conformation when compared to aliphatic residues because its side chain is positioned external to the hydrophobic cluster 1

(51). Nevertheless, a significant interaction occurs between the positive charge of the Asp amino group and the negative charge of the carboxyl-terminus present in Phe8 which assists in formation of β-turn conformation of these peptides (55). β-turns are a type of secondary structuration, in which a tight loop is formed when the carbonyl oxygen of one residue forms a hydrogen bond with the amide proton of an amino acid, approximately, three residues down the chain(57).In addition, the negatively 1

2

charged Asp carboxy side chain interacts with the positive guanidyl group present on the Arg side chain, and is responsible for directing these amino acid side chains(58). Considering all these related interactions and the importance of the residue side chain in the native molecule’s primary sequence, it was possible to review previous syntheses and propose modifications such as those produced in this work. The native peptide was bioactive with respect to the AT1 receptor, probably because the insertion of the disulfide bridge caused conformational changes that provided better peptidemembrane interactions and no contractile response. Thus, this approach leads to a better understanding of the proposed sequences and their effects on the anti-plasmodial activity.

The anti-plasmodial activity obtained by AII analogs were compared to positive control (digitonin, a steroid glycoside which increases the permeability of different types of cells to inorganic ions, metabolites, enzymes and peptides, it offers a major advantage over other commonly used surfactants, such as Triton® and glycerol, because the latter are relatively nonspecific in their effects

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on different types of cell membranes (59)) and to negative control (PBS solution) and AII, some of them restricted to that present for lactam bridge scaffolds. Among them, three restricted analogs of AII synthesized by Chamlian (16), presented considerable anti-plasmodial activity (VC-5, VC-17 and VC19; see Table 1). All were constricted at or near the N-terminal extremity. The design of new analogs that contained i-(i+2), i-(i+3),i-(i+4) disulfide was based upon them.

Comparisons between lactam and disulfide bridges are not uncommon (60-63). Both present a conformational restriction effect, and depending on the insertion point and the size of the constriction, some conformations are favored over others. Restrictors have different physicochemical properties such as rigidity, stability, and interaction with the main chain functional groups (64).

Considering this, we designed new disulfide bridge restricted peptides based on previous studies of anti-plasmodial activity of lactam bridge constricted AII analogs (13, 16). We expected improved plasmodial inactivity, approximately 80%, with no pressor activity. The structure of the peptides were found to have π*-π* interactions between the Tyr4 and the Phe8 aromatic side chains (44, 45), that is mediated by the His6 side chain in all cases. This might be important to the interaction of these analogs with parasite membranes. Additionally, the unrestricted C-terminal extremity preserves the hydrophobic cluster region of the peptide (Tyr4-Ile5-His6) and the 7

presence of a Pro residue favors the tendency of these peptides to take on the β-turn conformation.

Anti-plasmodial assay results indicated that the most important structural pattern affecting antiplasmodial activity is the hydrophobic cluster (51), which encourages the folded conformations. All the peptides synthesized in this work contained the disulfide bridge at N-terminal extremity, except for one (analog 5). The Cys-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-Cys contained restriction along all of the peptide residues. The anti-plasmodial activity of all the analogs was compared to positive control – digitonin, which has presented 98.5% of activity.

Use of this kind of restriction proved to be important because, in all cases, the anti-plasmodial activity was higher when compared to most active analogs with the lactam bridge at the same N-terminal extremity or in another parts of the molecule, including the linear AII analogs (16).

The new analog based on VC-5 (analog 1) possessed a smaller ring (11 atoms) in the same position 3

(insertions restricting the Val residue). This peptide was sensitive to changing the restriction from a lactam to a disulfide bridge. The position of the amide bond in the lactam ring likely caused a different type of interaction with the side chains or others main chain groups, in addition to the fact that the Val residue changed the hydrophobic balance of interactions. This was responsible for stabilizing the molecule in the β-turn conformation, providing the Arg2 and Tyr4 side chain interactions (56).

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The new analogs based on VC-17 and VC-19 (analogs 2 and 3) possessed 14 atom rings. The analogs VC-17 and 2 presented the Asp and Arg amino acid residues as part of the lactam and disulfide bridge, respectively. Analog 2 achieved the most impressive result (98% of anti-plasmodial activity) obtained along the studies with this class of restricted peptides. This may be due to the maintenance of both the positively charged terminal amine and the intramolecular hydrophobic interactions between Val3-Tyr4. Comparing this, with the similar analogs VC-19 and 3, it is possible to 2

3

verify that the restrictions are in the same region, but they present Arg and Val residues as part of the bridge. In this case, the activity was maintained even with the exchange of the bridge. The change in conformational restriction may have affected the guidance of the Tyr4 side chain and the hydrophobic interactions in the molecule; but the rigidity may not have provided marked changes in the conformation, since the antimalarial activity did not decreased. All the analogs synthesized in this work tended to take on the β-turn conformation when analyzed, similar to AII and some of its bioactive analogs, suggesting that this conformation(57, 65)is one most frequently adopted by this class of peptides in aqueous organic solvents (PBS, MeOH/PBS and TFE/PBS solutions) and in the presence of SDS. It can be noticed by a common negative minima around 208 nm and 220 nm, the dislocation of this minima is due to the introduction of a disulfide bond in the original sequence(57).

After analyzing the biological results of the new analogs synthesized, we proposed that it is important to preserve the hydrophobic intramolecular interactions of the aromatic side chain containing residues as well as the interactions presented by Arg2/Tyr4 (mediated by the position of the Val side chain) to maintain anti-plasmodial activity. In addition, we felt that there were interactions between nonpolar residues (Ile5 and Phe8), and that the Ile5 residue promotes further organization of His6 and Tyr4 residues. This was also proposed by Fermandjian et al. (45, 55), that proved the guidance of these side chains due to their interactions. In this case, the effect of steric hindrance probably favors the interaction of the molecule with the sporozoite membrane through guiding the side chains to the formation of a hydrophobic cluster.

When the restraint is due to the Val residue outside of the disulfide ring, preserving the original sequence of nonpolar residues is of utmost importance to maintain the interaction of the molecule with the sporozoite membrane and increase anti-plasmodial activity. Peptides containing this type of disulfide bridge with spatial orientation conducive to the formation of the hydrophobic cluster conserved (Cys-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-Cys) showed 92% activity. The most active peptide synthesized (Cys-Asp-Arg-Cys-Val-Tyr-Ile-His-Pro-Phe) showed 98% of activity and had the following features: hydrophobic cluster preservation and maintenance of the distance between the nonpolar residues and the interaction between the nonpolar residues in the peptide chain. These analogs presented equipotent anti-plasmodial activity when compared to the digitonin (positive control – 98.5%).

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Contractile response assays showed that all the analogs presented insignificant contractile activity by their interaction with the AT1 receptor. These results are in opposition to those obtained previously by Spear et al. (27) in which some disulfide-bridge-containing analogs showed pressor activity. However, according to Regoli et al. (46) the substitution of certain residues in the primary sequence of AII caused a decrease on the pressor activity, as was observed in this work.

The importance of each residue of the native sequence of AII with the AT1 receptor is described by Tzakos et al. (43). Considering this and the maintenance of the β-turn conformational tendency among the analogs synthesized, it is possible that the constrictions imposed were not mimicked by 1

disulfide bridge cyclization. Therefore, the conformational restrictions harm the anchoring since Asp , Arg2, His6, and Phe8 residues were affected by the introduction of the disulfide bridge as well as the 4

8

activation of the AT1 receptor, which is mediated by Tyr and Phe residues (10). The Val residue that is part of the introduced ring is cited by Tzakos (43) as being responsible for the stabilization of the bioactive conformation of the native peptide. The conformational restrictions on the analogs were responsible for increasing the distances between the amino acid residues that are important in facilitating the biological activity of AII (i.e., Asp1, Arg2, Tyr4, His6, and Phe8), inducing new conformational changes in the AII molecule that preclude recognition and binding to the AII AT1 receptor, according to Tzakos et al.

5. Conclusions Five restricted AII analogs with an i-(i+2), i-(i+3), i-(i+4) and i-(i+9) disulfide bridge scaffold were synthesized. Among those, two peptides exhibited high anti-plasmodium activity (92% for Cys-Asp-ArgVal-Tyr-Ile-His-Pro-Phe-Cys and 98% for Cys-Asp-Arg-Cys-Val-Tyr-Ile-His-Pro-Phe), as indicated by

fluorescence microscopy, and all of them exceeded the limit of 80% activity reached by the lactam bridge constrained analogs previously synthesized. CD spectra results suggest that the β-turn conformation is the most frequent conformation adopted by peptides against P. gallinaceum in aqueous organic solvents and in the presence of SDS.

Contractile response assays showed that AII disulfide bridge analogs did not exhibit significant contractile activity. The constraints imposed by the side chains of the residues in the original sequence of the peptide are not mimicked by disulfide bridge cyclization of the insertions imposed to the new designed analogs.

These results allow us to point out the effectiveness of the strategy of using peptides to combat the parasite that causes avian malaria, enabling these compounds to be tested against human malaria.

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Acknowledgments This research was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, VXO #2011/10823-9, MDTT #2011/15083-3, AFS #2011/11448-2).

Figures Figure 1.

Figure 2.

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Figure 4.

1- DRCVCYIHPF

2- CDRCVYIHPF

3

0 -3

0

-6 -3

-1 3

2

[θ] X 10 deg.cm .dmol

Accepted Article

Figure 3.

3- DCRVCYIHPF

4- CDRVCYIHPF

2

1

0 0 -2 -4

-1 5- CDRVYIHPFC

200

210

230

240

250

λ (nm)

0

PBS (15mM) SDS (10mM) MeOH 50% (v/v) TFE 50% (v/v)

-2

-4

220

200

210

220

230

240

250

260

λ (nm)

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260

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Figure Caption Figure 1. General synthetic method for disulfide bridged peptides.

Figure 2. Effects of AII analogs on membrane permeability expressed as the percent of fluorescent mature sporozoites (mean ± standard deviation, N = 9). Letters indicate those results not significantly different from each other at the p < 0.05 level. Positive control group (+): digitonin/PBS; negative control group (–): PBS. The most active analog (2) was based on the VC-17 analog that presented 67% of activity (16).

Figure 3. Effects of peptides in contractile responses by muscle tissue incubation compared to carbachol (CCh) activity. The AT1 receptor recognition was analyzed via pre-incubation with Losartan (mean ± standard deviation, N = 2).

Figure 4. CD spectra were recorded after four accumulations at 20°C using a 0.5 mm path length quartz cell between 260 and 195 nm at 50 nmmin-1 with a band width of 0.5 nm. All peptides were -1

-1

analyzed in the following four solutions: 15 mmolL PBS, 10 mmolL SDS/PBS, 50% TFE/PBS, and -4

-1

50% MeOH/PBS. The peptide concentration was approximately 10 molL .

Table Table 1.

Entry

Sequence

HPLC Puritya

Calculated

Observed

Mass

Massb

AII

Asp-Arg-Val-Tyr-Ile-His-Pro-Phe

VC-5

Asp-Arg-Asp-Val-Lys-Tyr-Ile-His-Pro-Phe

VC-17

Asp-Asp-Arg-Lys-Val-Tyr-Ile-His-Pro-Phe

VC-19

Asp-Asp-Arg-Val-Lys-Tyr-Ile-His-Pro-Phe

1

Asp-Arg-Cys-Val-Cys-Tyr-Ile-His-Pro-Phe

99%

1249.7

1250

2

Cys-Asp-Arg-Cys-Val-Tyr-Ile-His-Pro-Phe

99%

1249.7

1249

3

Asp-Cys-Arg-Val-Cys-Tyr-Ile-His-Pro-Phe

99%

1249.7

1249

4

Cys-Asp-Arg-Val-Cys-Tyr-Ile-His-Pro-Phe

98%

1249.7

1250

5

Cys-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-Cys

98%

1249.7

1250

Table Legend Table 1. Purities as determined by RP-HPLC and characterized by LC/MS-ESI. a

HPLC profiles were obtained under the following conditions: Column Supelcosil C18 (4.6 x 150 mm),

60 Å, 5 μm; Solvent System: A (0.1% TFA/H2O) and B (0.1% TFA in 60% ACN/H2O); Gradient: 5–

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95% B in 30 minutes; Flow: 1.0 mL/min; λ=220 nm; Injection Volume: 50 μL and Sample Concentration: 1.0 mg/mL. b

Masses were determined by LC/ESI-MS using a Micromass instrument (model ZMD) coupled to a

Waters Alliance (model 2690) system. Mass measurements were performed in a positive mode with the following parameters: mass range between 500 and 2000 m/z; nitrogen gas flow: 4.1 L/h; capillary: 2.3 kV; cone voltage: 32 V; extractor: 8 V; source heater: 100 °C; solvent heater: 400 °C; ion energy: 1.0 V and multiplier: 800 V.

References

1. 2. 3.

4. 5.

6.

7.

8.

9. 10.

11. 12. 13.

14. 15.

Neves, D. P., Melo, A. L., Linardi, P. M., and Vitor, R. W. A. (2005) Parasitologia Humana, 11 ed., Atheneu, São Paulo. Garcia, L. S. (2010) Malaria, Clin. Lab. Med. 30, 93-+. Tang, T. H. T., Salas, A., Ali-Tammam, M., Martinez, M. D., Lanza, M., Arroyo, E., and Rubio, J. M. (2010) First case of detection of Plasmodium knowlesi in Spain by Real Time PCR in a traveller from Southeast Asia, Malar. J. 9. Teixeira, C., Gomes, J. R. B., and Gomes, P. (2011) Falcipains, Plasmodium falciparum Cysteine Proteases as Key Drug Targets Against Malaria, Curr. Med. Chem. 18, 1555-1572. Akaddar, A., Doderer-Lang, C., Marzahn, M. R., Delalande, F., Mousli, M., Helle, K., Van Dorsselaer, A., Aunis, D., Dunn, B. M., Metz-Boutigue, M. H., and Candolfi, E. (2010) Catestatin, an endogenous Chromogranin A-derived peptide, inhibits in vitro growth of Plasmodium falciparum, Cell. Mol. Life Sci. 67, 1005-1015. Peter, G., Manuel, A. L., and Shetty, A. (2011) Study comparing the clinical profile of complicated cases of Plasmodium falciparum malaria among adults and children, Asian Pacific Journal of Tropical Disease 1, 35-37. Auvin-Guette, C., Baraguey, C., Blond, A., Xavier, H. S., Pousset, J. L., and Bodo, B. (1999) Pohlianins A, B and C, cyclic peptides from the latex of Jatropha pohliana ssp molissima, Tetrahedron 55, 11495-11510. Devappa, R. K., Makkar, H. P. S., and Becker, K. (2010) Nutritional, Biochemical, and Pharmaceutical Potential of Proteins and Peptides from Jatropha: Review, J. Agric. Food Chem. 58, 6543-6555. Kaur, K., Jain, M., Kaur, T., and Jain, R. (2009) Antimalarials from nature, Bioorg. Med. Chem. 17, 3229-3256. Torossian Torres, M., Silva, A., Alves, F., Capurro, M., Miranda, A., and Oliveira Junior, V. (2014) The Importance of Ring Size and Position for the Antiplasmodial Activity of Angiotensin II Restricted Analogs, International Journal of Peptide Research and Therapeutics online version. Hruby, V. J. (1982) Conformational restrictions of biologically-active peptides via amino-acid side-chain groups, Life Sciences 31, 189-199. Mosberg, H. I. (1999) Complementarity of delta opioid ligand pharmacophore and receptor models, Biopolymers 51, 426-439. Maciel, C., de Oliveira, V. X., Fazio, M. A., Nacif-Pimenta, R., Miranda, A., Pimenta, P. F. P., and Capurro, M. L. (2008) Anti-Plasmodium Activity of Angiotensin II and Related Synthetic Peptides, PLoS One 3. Vijayaraghavan, K., and Deedwania, P. (2011) Renin-Angiotensin-Aldosterone Blockade for Cardiovascular Disease Prevention, Cardiol. Clin. 29, 137-+. Oliveira, V. X., Jr., Fazio, M. A., Silva, A. F., Campana, P. T., Pesquero, J. B., Santos, E. L., Costa-Neto, C. M., and Miranda, A. (2011) Biological and conformational evaluation of angiotensin II lactam bridge containing analogues, Regul Pept 172, 1-7.

This article is protected by copyright. All rights reserved.

Accepted Article

16.

17. 18.

19.

20. 21.

22. 23. 24.

25. 26.

27.

28. 29.

30.

31. 32. 33.

34.

Chamlian, M., Bastos, E. L., Maciel, C., Capurro, M. L., Miranda, A., Silva, A. F., Torres, M. D. T., and Oliveira, V. X. (2013) A study of the anti-plasmodium activity of angiotensin II analogs, Journal of Peptide Science 19, 575-580. Bechinger, B., and Lohner, K. (2006) Detergent-like actions of linear amphipathic cationic antimicrobial peptides, Biochim. Biophys. Acta-Biomembr. 1758, 1529-1539. Pukala, T. L., Bowie, J. H., Maselli, V. M., Musgrave, I. F., and Tyler, M. J. (2006) Host-defence peptides from the glandular secretions of amphibians: structure and activity, Natural Product Reports 23, 368-393. Resende, J. M., Moraes, C. M., Prates, M. V., Cesar, A., Almeida, F. C. L., Mundim, N., Valente, A. P., Bemquerer, M. P., Pilo-Veloso, D., and Bechinger, B. (2008) Solution NMR structures of the antimicrobial peptides phylloseptin-1,-2, and-3 and biological activity: The role of charges and hydrogen bonding interactions in stabilizing helix conformations, Peptides 29, 1633-1644. Sanderson, J. M. (2005) Peptide lipid interactions: insights and perspectives, Org. Biomol. Chem. 3, 201-212. Bechinger, B. (1999) The structure, dynamics and orientation of antimicrobial peptides in membranes by multidimensional solid-state NMR spectroscopy, Biochim. Biophys. ActaBiomembr. 1462, 157-183. Merrifield, R. B. (1963) Solid phase peptide synthesis .1. Synthesis of tetrapeptide, J. Am. Chem. Soc. 85, 2149-&. Fields, G. B., and Noble, R. L. (1990) Solid-phase peptide-synthesis utilizing 9fluorenylmethyloxycarbonyl amino-acids, Int. J. Pept. Protein Res. 35, 161-214. Wang, S. S. (1973) p-alkoxybenzyl alcohol resin and p-alkoxybenzyloxycarbonylhydrazide resin for solid-phase synthesis of protected peptide fragments, J. Am. Chem. Soc. 95, 13281333. Kaiser, E., Colescot.Rl, Bossinge.Cd, and Cook, P. I. (1970) Color test for detection of free terminal amino groups in solid-phase synthesis of peptides, Anal. Biochem. 34, 595-&. Malavolta, L., Oliveira, E., Cilli, E. M., and Nakaie, C. R. (2002) Solvation of polymers as model for solvent effect investigation: proposition of a novel polarity scale, Tetrahedron 58, 43834394. Spear, K. L., Brown, M. S., Reinhard, E. J., McMahon, E. G., Olins, G. M., Palomo, M. A., and Patton, D. R. (1990) Conformational restriction of angiotensin-II - Cyclic analogs having high potency, J. Med. Chem. 33, 1935-1940. Thathy, V., Severson, D. W., and Christensen, B. M. (1994) Reinterpretation of the genetics of susceptibility of aedes-aegypti to plasmodium-gallinaceum, J. Parasitol. 80, 705-712. Munstermann, L. E., and Conn, J. E. (1997) Systematics of mosquito disease vectors (Diptera, Culicidae): Impact of molecular biology and cladistic analysis, Annu. Rev. Entomol. 42, 351369. Barbosa, A., Felipe, S. A., Pesquero, J. B., Paiva, A. C. M., and Shimuta, S. I. (2006) Disruption of the kinin B-1 receptor gene affects potentiating effect of captopril on BK-induced contraction in mice stomach fundus, Peptides 27, 3377-3382. Kruskal, W. H., and Wallis, W. A. (1952) Use of ranks in one-criterion variance analysis, J. Am. Stat. Assoc. 47, 583-621. Cicchett, D. V., Collins, D., and Latvis, V. (1972) PROGRAM FOR COMPARING GROUP MEANS - ONE-WAY ANOVA AND TUKEY MULTIPLE-RANGE TESTS, Behav. Sci. 17, 399-&. Wallace, B. A., and Mao, D. (1984) Circular-dichroism analyses of membrane-proteins - An examination of differential light-scattering and absorption flattening effects in large membrane-vesicles and membrane sheets, Anal. Biochem. 142, 317-328. Grottesi, A., Sette, M., Palamara, A. T., Rotilio, G., Garaci, E., and Paci, M. (1998) The conformation of peptide thymosin alpha 1 in solution and in a membrane-like environment

This article is protected by copyright. All rights reserved.

Accepted Article

35. 36. 37.

38.

39. 40.

41. 42. 43.

44. 45.

46. 47.

48. 49. 50. 51.

52. 53.

by circular dichroism and NMR spectroscopy. A possible model for its interaction with the lymphocyte membrane, Peptides 19, 1731-1738. Mattice, W. L., Riser, J. M., and Clark, D. S. (1976) Conformational properties of complexes formed by proteins and sodium dodecyl-sulfate, Biochemistry 15, 4264-4272. Buck, M. (1998) Trifluoroethanol and colleagues: cosolvents come of age. Recent studies with peptides and proteins, Q. Rev. Biophys. 31, 297-355. Starzyk, A., Barber-Armstrong, W., Sridharan, M., and Decatur, S. M. (2005) Spectroscopic evidence for backbone desolvation of helical peptides by 2,2,2-trifluoroethanol: An isotopeedited FTIR study, Biochemistry 44, 369-376. Mahalakshmi, R., Shanmugam, G., Polavarapu, P. L., and Balaram, P. (2005) Circular dichroism of designed peptide helices and beta-hairpins: Analysis of Trp- and Tyr-rich peptides, Chembiochem 6, 2152-2158. Premilat, S., and Maigret, B. (1979) Conformations of angiotensin-II and enkephalin related to NMR, Biochem. Biophys. Res. Commun. 91, 534-539. Hruby, V. J. (1984) Design of Peptide Superagonists and Antagonists - Conformational and Dynamic Considerations, In Conformationally Directed Drug Design - Peptides and Nucleic Acids as Templates or Targets (Vida, J. A., and Gordon, M., Eds.), pp 9-27, Bristol-Myers Company, New York. Schiller, P. W., and Nguyen, T. M. D. (1984) Activity profiles of novel side-chain to side-chain cyclized opioid peptide analogs, Neuropeptides 5, 165-168. Miranda, A., and Juliano, L. (1988) Conformationally restricted analogs of angiotensin-II Titration and biological-activity, Brazilian J. Med. Biol. Res. 21, 903-914. Tzakos, A. G., Gerothanassis, I. P., and Troganis, A. N. (2004) On the structural basis of the hypertensive properties of angiotensin II: A solved mystery or a controversial issue?, Curr. Top. Med. Chem. 4, 431-444. Bumpus, F. M., and Khosla, M. C. (1977) Hypertension - Physiopathology and Treatment, McGraw-Hill, New York. Fermandjian, S., Sakarellos, C., Piriou, F., Juy, M., Toma, F., Thanh, H. L., Lintner, K., Khosla, M. C., Smeby, R. R., and Bumpus, F. M. (1983) The key role of residue 5 in angiotensin-II, Biopolymers 22, 227-231. Regoli, D., Park, W. K., and Rioux, F. (1974) Pharmacology of angiotensin, Pharmacol. Rev. 26, 69-123. Carpenter, K. A., Wilkes, B. C., and Schiller, P. W. (1998) The octapeptide angiotensin II adopts a well-defined structure in a phospholipid environment, Eur. J. Biochem. 251, 448453. Cho, N. J., and Asher, S. A. (1996) UV resonance Raman and absorption studies of angiotensin II conformation in lipid environments, Biospectroscopy 2, 71-82. Cushman, J. A., Mishra, P. K., Bothnerby, A. A., and Khosla, M. S. (1992) Conformations in solution of angiotensin-II, and its 1-7 and 1-6 fragments, Biopolymers 32, 1163-1171. Pellegrini, M., and Mierke, D. F. (1999) Structural characterization of peptide hormone/receptor interactions by NMR spectroscopy, Biopolymers 51, 208-220. Tzakos, A. G., Bonvin, A., Troganis, A., Cordopatis, P., Amzel, M. L., Gerothanassis, I. P., and van Nuland, N. A. J. (2003) On the molecular basis of the recognition of angiotensin II (AII) NMR structure of AII in solution compared with the X-ray structure of AII bound to the mAb Fab131, Eur. J. Biochem. 270, 849-860. Juliano, L., and Paiva, A. C. M. (1974) Conformation of angiotensin-II in aqueous-solution Titration of several peptide analogs and homologs, Biochemistry 13, 2445-2450. Kuter, D., Chibale, K., and Egan, T. J. (2011) Linear free energy relationships predict coordination and pi-stacking interactions of small molecules with ferriprotoporphyrin IX, J. Inorg. Biochem. 105, 684-692.

This article is protected by copyright. All rights reserved.

Accepted Article

54. 55.

56. 57. 58.

59.

60.

61.

62.

63.

64.

65.

Moll, G. N., Konings, W. N., and Driessen, A. J. M. (1999) Bacteriocins: mechanism of membrane insertion and pore formation, Antonie Van Leeuwenhoek 76, 185-198. Piriou, F., Lintner, K., Fermandjian, S., Fromageot, P., Khosla, M. C., Smeby, R. R., and Bumpus, F. M. (1980) Amino-acid side-chain conformation in angiotensin-II and analogs Correlated results of circular-dichroism and H-1 nuclear magnetic-resonance, Proceedings of the National Academy of Sciences of the United States of America-Biological Sciences 77, 8286. Kyte, J., and Doolittle, R. F. (1982) A simple method for displaying the hydropathic character of a protein, J. Mol. Biol. 157, 105-132. Fasman, G. D. (1996) Circular Dichroism and the Conformational Analysis of Biomolecules, 1 ed., Plenum Press, New York. Printz, M. P., Williams, H. P., and Craig, L. C. (1972) Evidence for presence of hydrogenbonded secondary structure in angiotensin-II in aqueous-solution, Proc. Natl. Acad. Sci. U. S. A. 69, 378-&. Fiskum, G., Craig, S. W., Decker, G. L., and Lehninger, A. L. (1980) The cytoskeleton of digitonin-treated rat hepatocytes, Proceedings of the National Academy of Sciences of the United States of America-Biological Sciences 77, 3430-3434. Abraham, W. M., Ahmed, A., Cortes, A., Spinella, M. J., Malik, A. B., and Andersen, T. T. (1993) A specific endothelin-1 antagonist blocks inhaled endothelin-1-induced bronchoconstriction in sheep, Journal of Applied Physiology 74, 2537-2542. Hargittai, B., Sole, N. A., Groebe, D. R., Abramson, S. N., and Barany, G. (2000) Chemical syntheses and biological activities of lactam analogues of alpha-conotoxin SI, J. Med. Chem. 43, 4787-4792. Limal, D., Briand, J. P., Dalbon, P., and Jolivet, M. (1998) Solid-phase synthesis and on-resin cyclization of a disulfide bond peptide and lactam analogues corresponding to the major antigenic site of HIV gp41 protein, Journal of Peptide Research 52, 121-129. Thurieau, C., Janiak, P., Krantic, S., Guyard, C., Pillon, A., Kucharczyk, N., Vilaine, J. P., and Fauchere, J. L. (1995) A new somatostatin analog with optimized ring size inhibits neointima formation induced by balloon injury in rats without altering growth-hormone release, European Journal of Medicinal Chemistry 30, 115-122. Grieco, P., Carotenuto, A., Patacchini, R., Maggi, C. A., Novellino, E., and Rovero, P. (2002) Design, synthesis, conformational analysis, and biological studies of urotensin-II lactam analogues, Bioorg. Med. Chem. 10, 3731-3739. Greef, W. J. D., Dullaart, J., and Zeilmaker, G. H. (1976) Serum follicle-stimulating-hormone, luteinizing-hormone and progesterone concentrations in pseudopregnant rats treated with medroxyprogesterone acetate, J. Endocrinol. 70, 465-471.

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