Antimalarial Activity of Ultra-Short Peptides

Ultra-short peptides 1-9 were designed and synthesized with phenylalanine, ornithine and proline amino acid residues and their effect on antimalarial activity was analyzed. On the basis of the IC50 data for these compounds, the effects of nature, polarity, and amino acid sequence on Plasmodium berghei schizont cultures were analyzed too. Tetrapeptides Phe-Orn-Phe-Orn (4) and Lys-Phe-Phe-Orn (5) showed a very important activity with IC50 values of 3.31 and 2.57 μM, respectively. These two tetrapeptides are candidates for subsequent in vivo assays and SARS investigations.


Introduction
Malaria is one of the most lethal and widespread infectious parasite diseases in the world, affecting approximately 300 million people annually, mainly from developing countries like Central-and South-America, Asia, and Sub-Saharan Africa [1,2]. According to the World Health Organization, the number of clinical cases has reached between 300 and 500 million people per year [3]. About 1.1-2.7 million people in tropical and subtropical regions die of malaria every year [1,[4][5][6]. In humans, it is caused by protozoan parasites from four Plasmodium species: P. falciparum, P. vivax, P. malariae and P. ovale. The most serious infections among these species are caused by P. falciparum [7,8]. In recent years, the social impact of malaria has increased due, to the lower abundance and high cost of artemisinin and related products (their effective distribution to combat malaria in economically disadvantaged regions could require an annual global subsidy of $300-500 million) [9], and to the emergence of resistant strains of P. falciparum and P. vivax to chloroquine, mefloquine, pyrimethamine, and to immunoprophylactic methods (such as vaccines) [10]. For these reasons, there is an increasing need for new chemical pharmacophores which may prove effective therapeutic antimalarial agents.

Results and Discussion
Cationic (including arginine, lysine, ornithine and histidine residues, for example) and bulky (like proline, phenylalanine, valine, isoleucine and tryptophan) side chains are essential for antimicrobial activity in peptides [32] because they can confer amphiphatic character (simultaneous lipophilicity and hydrophilicity properties). In this paper, we analyzed the influence of the amphiphatic character of ultra-short peptides (including four or less amino acids residues) on antimalarial activity.
Peptides 1-9 were synthesized in solid phase using Fmoc strategy and were designed to investigate the nature, polarity, and amino acid sequence on Plasmodium berghei schizont cultures. Our main purpose was to explore their potential antimalarial activity. On this context, peptides 1-4 were prepared to investigate the effect of N-terminal amino acid, while sequences in compounds 3 and 5 allowed us to analyze the effect of a change on the second amino acid residue on the antimalarial activity. With this same purpose, antimalarial activity of compound 2 was compared with its retropeptide sequence at compound 6. Tetrapeptides 1-5 were synthesized to study the activity of peptides including Xaa-Xbb-Phe-Orn sequence, while the tetrapeptides 7 and 8, and compound 9 were prepared to analyze the influence of the presence of proline residue, and tripeptide nature on antimalarial activity, respectively. Compounds 1-9 were evaluated in vitro for chemosuppression during 16 h with P. berghei schizont cultures. The results of the biological assay against this parasite blood stage are indicated in Table 1.
Peptide 1 showed moderate antimalarial activity, with an IC 50 = 32.35 μM. Substitution of a valine residue for isoleucine leads to a more active compound (peptide 2), showing an IC 50 = 10.00 μM, suggesting that polarity of the amino acid at the N-terminal residue is important for the antimalarial activity. In this case, substitution of a hydrogen in valine for a methyl group (isoleucine) confers more lipophilicity to the peptide and results in diminished antimalarial activity. Probably, the chiral center in the isoleucine residue is another reason for this decreased activity. The retropeptide of compound 2 (peptide 6) was inactive, indicating that amino acid sequence is very important for the activity.  (6) 26.58 >200 Lys-Val-Pro-Orn (7) 27.39 >200 Lys-Val-Phe-Pro (8) 72.78 51.28 Phe-Orn-Val (9) 55.95 120. IC 50 data for compounds 4 and 5 corroborate that N-terminal residue nature is very important for antimalarial activity, and that when a polar residue (like lysine or ornithine) is substituted by a less polar residue (like phenylalanine) antimalarial activity is increased. In our hands, peptides possessing the proline residue (compounds 7 and 8) and tripeptide 9 were inactive. These results are in accordance with the findings of other authors who have suggested that the presence of non polar amino acid residues (providing lipophilic anchors) dramatically increases the biological activity, because those residues might be helping their penetration (disruption) through plasma membrane [25,32].

Conclusions
Tetrapeptides 1-5 including the sequence Xaa-Xbb-Phe-Orn showed antimalarial activity during in vitro schizont development assays, which significantly increased when a second phenylalanine amino acid (aromatic residue) was present, such as in compounds 4 and 5. These compounds showed the highest antimalarial activity, with IC 50 = 3.31 and 2.57 µM, respectively, and will be candidates for subsequent in vivo assays. These results showed that compounds 4 and 5 include interesting amino acid sequences for future SAR investigations. Fmoc-Ile-OH, Fmoc-Pro-OH, and HOBt were obtained from ANASPEC, and they were all used without further purification.

Cell culture and antimalarial activity measurements
Chloroquine-sensitive cultured Plasmodium berghei schizonts (ANKA 2.34) were used to assess the antimalarial activity of compounds 1-9. Mature P. berghei schizonts were prepared as described by Thathy and Menard [34]. Peptides 1-9 were dissolved in PBS containing DMSO 0.1% and 5% ethanol. Parasites were incubated for 16 h at 37 °C as described. Chloroquine (C6628 Sigma Chloroquine diphosphate salt solid, ≥98% purity) was used as positive control. Samples of 0.5 mL of each culture were taken to prepare smears and used to count numbers of schizonts in 2,000 erythrocytes. The IC 50 values of each compound were determined using concentrations of 5, 50, 100, and 200 µM, according to Khalid et al. [35], and by extrapolation from the concentration response curve. The IC 50 value represents the drug concentration producing a 50% reduction in the number of P. berghei schizonts (compared to drug-free control cultures). For each assay, each drug dilution was analyzed in duplicate, and the results were averaged in each case. Based on NMR spectroscopic and HPLC chromatographic analyses, all compounds were at least of 98% purity.

General synthesis procedure for tri-and tetrapeptides 1-9
Solid phase synthesis of the peptides 1-9 [Ile-Lys-Phe-Orn (1) The synthetic cycle was repeated to assemble the resin-bond protected peptide. The peptides were cleaved from the resin with simultaneous side-chain deprotection by acidolysis with 3% TFA/CH 2 Cl 2 (1:1, 2 × 10 mL) containing 5% anisole at rt for 1 h. Cold diethyl ether (10 mL) was added and the precipitated peptide was filtered through a fine sintered funnel. The crude peptides were subjected first to FCC, and then purified to chromatographic homogeneity in the range of 90 to 95% by reverse-phase high-pressure liquid chromatography, performed on a semipreparative Phenomenex Luna 5C18 column, 250 × 4.6 mm; mobile phase: 80% acetonitrile, 0.1% TFA; flow rate: 1.2 mL/min, 220 nm).
The purified peptides were subjected to amino acid analysis in order to confirm their composition.
Peptides were stored as a lyophilized powder at 220 °C. Prior to experimentation, fresh solutions were prepared in water, vortexed, sonicated, and centrifuged, and the supernatant was diluted in the appropriate medium.