Anti-Protozoan Activities of Polar Fish-Derived Polyalanine Synthetic Peptides

Abstract

Chagas disease, sleeping sickness and malaria are infectious diseases caused by protozoan parasites that kill millions of people worldwide. Here, we performed in vitro assays of Pa-MAP, Pa-MAP1.9, and Pa-MAP2 synthetic polyalanine peptides derived from the polar fish Pleuronectes americanus toward Trypanosoma cruzi, T. brucei gambiense and Plasmodium falciparum activities. We demonstrated that the peptides Pa-MAP1.9 and Pa-MAP2 were effective to inhibit T. brucei growth. In addition, structural analyses using molecular dynamics (MD) studies showed that Pa-MAP2 penetrates deeper into the membrane and interacts more with phospholipids than Pa-MAP1.9, corroborating the previous in vitro results showing that Pa-MAP1.9 acts within the cell, while Pa-MAP2 acts via membrane lysis. In conclusion, polyalanine Pa-MAP1.9 and Pa-MAP2 presented activity against bloodstream forms of T. b. gambiense, thus encouraging further studies on the application of these peptides as a treatment for sleeping sickness.

Keywords: antimicrobial peptide; antiplasmodial peptide; molecular dynamics; polyalanine; trypanocidal peptide.

Figure 1

Pa-MAP1.9 MD simulation in the membrane environment. (A) Structural snapshots during 200 ns of trajectory. (B) Tridimensional representation of Pa-MAP1.9 (green), highlighting (dotted square) the region where there is an interaction between the nitrogen NZ of Lys⁴, Lys⁷ and Lys¹¹ with the membrane lipids (orange) at 200 ns. (C) Mass density profile of Pa-MAP1.9, water and phospholipids during the last 5 ns of the simulation.

Figure 2

Root mean square deviation (A) for Pa-MAP1.9 (black) and Pa-MAP2 (gray). (B) Distance from the center of mass of Pa-MAP1.9 (black) and Pa-MAP2 (gray) during the simulation. (C) Root mean square fluctuation by residues of the peptides during MD. (D) Radius of gyration for Pa-MAP1.9 (black) and Pa-MAP2 (gray) during 200 ns of the simulation.

Figure 3

Evaluation of the molecular dynamics of Pa-MAP2. (A) Structures demonstrated during 200 ns of trajectory. (B) Tridimensional representation of the Pa-MAP2 (yellow), highlighting (dotted squares) the region where there is interaction between the nitrogen NZ of Lys², Lys⁹, Lys¹³, Lys¹⁶, Lys²⁰ and Leu¹ to membrane lipids (orange) at 200 ns. (C) Mass density profile of the Pa-MAP2 peptide, water and lipids during the last 5 ns of simulation.

Figure 4

Lipid area graph during molecular dynamics for Pa-MAP1.9(black) and Pa-MAP2 (gray) peptides.

Figure 5

Highlight of the interface region of the mass density profile for the peptides. The black arrow points where, for (A) Pa-MAP1.9 and (B) Pa-MAP2, the water density slightly increased, forming a shoulder-like, indicating permeabilization on the bilayer surface. The red curve shows the density of the peptides (approximation) in relation to the membrane.