Angiotensin II-derived constrained peptides with antiplasmodial activity and suppressed vasoconstriction

Abstract

Angiotensin II (Ang II) is a natural mammalian hormone that has been described to exhibit antiplasmodial activity therefore constituting a promising alternative for the treatment of malaria. Despite its promise, the development of Ang II as an antimalarial is limited by its potent induction of vasoconstriction and its rapid degradation within minutes. Here, we used peptide design to perform targeted chemical modifications to Ang II to generate conformationally restricted (disulfide-crosslinked) peptide derivatives with suppressed vasoconstrictor activity and increased stability. Designed constrained peptides were synthesized chemically and then tested for antiplasmodial activity. Two lead constrained peptides were identified (i.e., peptides 1 and 2), each composed of 10 amino acid residues. These peptides exhibited very promising activity in both our Plasmodium gallinaceum (>80%) and Plasmodium falciparum (>40%) models, an activity that was equivalent to that of Ang II, and led to complete suppression of vasoconstriction. In addition, peptide 5 exhibited selective activity towards the pre-erythrocytic stage (98% of activity against P. gallinaceum), thus suggesting that it may be possible to design peptides that target specific stages of the malaria life cycle. The Ang II derived stable scaffolds presented here may provide the basis for development of a new generation of peptide-based drugs for the treatment of malaria.

Figure 1

Circular dichroism spectra of constrained peptides. Spectra were recorded after four accumulations at 20 °C using a 0.5 mm path-length quartz cell between 260 nm and 195 nm at 50 nm/min with a band width of 0.5 nm. All peptides were analyzed in the following four solutions: 15 mM PBS, 10 mM SDS/PBS, 50% TFE/PBS, and 50% MeOH/PBS. The peptide concentration was approximately 10⁻⁴ mol L⁻¹.

Figure 2

Antiplasmodial activity of designed synthetic peptides. (A) Effect of peptides on sporozoite membrane permeability expressed as % fluorescent mature sporozoites (mean ± standard deviation, n = 9). Letters indicate those results not significantly different from each other at p < 0.05 level. Positive control group (+): digitonin/PBS; negative control group (−): PBS. The most active peptides were 1, 2, 4 and 5 that presented 92, 91, 95 and 98% antiplasmodial activity, respectively. (B) Effect of peptides in new ring formation. The percentage of rings was determined after 24 h incubation of erythrocyte cultures infected with 2–3% schizonts in the absence (control) or presence of 10⁻⁸ mol L⁻¹ of peptides. Double asterisk statistically significant compared with control value p < 0.01. Triple asterisk statistically significant compared with control value p < 0.001. Dark grey shading indicates that the result is statistically significant compared with control (mean ± standard deviation, n = 2). Data corresponding to ring form % in the presence of Ang II was retrieved from Saraiva et al.. Ring formation inhibition (IC₅₀ values) mediated by designed peptides 1 (C) and 2 (D). Peptides were diluted to seven concentrations [(10⁻⁴; 10⁻⁶; 10⁻⁸; 10⁻⁹; 10⁻¹⁰; 10⁻¹²; 10⁻¹⁴) mol L⁻¹] leading to an inhibitory range between 6–54.5%. Data have been normalized due to differences between controls of each assay. The IC₅₀ data were analyzed by GraphPad Prism analysis. Parameters: non-linear regression; log (inhibitor) vs response equation was chosen and least square (ordinary) fit method was applied (mean ± standard deviation, n = 2).

Figure 3

Hemolytic assay of human red blood cells. Red blood cells were treated with distilled water which causes lysis of red blood cells; uninfected erythrocytes were kept in the same conditions used in the invasion assay with a control and each peptide tested (10⁻⁸ mol L⁻¹), at 37 °C for 24 h. After incubation, the supernatant was collected, clarified at 900 g/8 min and the hemoglobin content was detected in a spectrophotometer at 530 nm. **** denotes statistical significance of group compared with distilled water value p < 0.05. Light grey shading indicates that the result is not statistically significant compared with control (mean ± standard deviation, n = 3).

Figure 4

Effect of Ang II derived peptides in contractile responses by muscle tissue incubation compared to carbachol (CCh) activity. Triple asterisk statistically significant compared with control value p < 0.05 (mean ± standard deviation, n = 2).

Figure 5

Stability of Ang II derivatives in human serum. Aliquots of peptides in the presence of human serum were taken over time (from 0 to 6 h) and were analyzed by LC-MS/ESI. The values shown correspond to % of area under the peak normalized according to the initial concentration of each peptide. All experiments were performed in duplicate.