Lessons from a Single Amino Acid Substitution: Anticancer and Antibacterial Properties of Two Phospholipase A2-Derived Peptides

Abstract

The membrane-active nature of phospholipase A₂-derived peptides makes them potential candidates for antineoplastic and antibacterial therapies. Two short 13-mer C-terminal fragments taken from snake venom Lys49-PLA₂ toxins (p-AppK and p-Acl), differing by a leucine/phenylalanine substitution, were synthesized and their bioactivity was evaluated. Their capacity to interfere with the survival of Gram-positive and Gram-negative bacteria as well as with solid and liquid tumors was assessed in vitro. Toxicity to red blood cells was investigated via in silico and in vitro techniques. The mode of action was mainly studied by molecular dynamics simulations and membrane permeabilization assays. Briefly, both peptides have dual activity, i.e., they act against both bacteria, including multidrug-resistant strains and tumor cells. All tested bacteria were susceptible to both peptides, Pseudomonas aeruginosa being the most affected. RAMOS, K562, NB4, and CEM cells were the main leukemic targets of the peptides. In general, p-Acl showed more significant activity, suggesting that phenylalanine confers advantages to the antibacterial and antitumor mechanism, particularly for osteosarcoma lines (HOS and MG63). Peptide-based treatment increased the uptake of a DNA-intercalating dye by bacteria, suggesting membrane damage. Indeed, p-AppK and p-Acl did not disrupt erythrocyte membranes, in agreement with in silico predictions. The latter revealed that the peptides deform the membrane and increase its permeability by facilitating solvent penetration. This phenomenon is expected to catalyze the permeation of solutes that otherwise could not cross the hydrophobic membrane core. In conclusion, the present study highlights the role of a single amino acid substitution present in natural sequences towards the development of dual-action agents. In other words, dissecting and fine-tuning biomembrane remodeling proteins, such as snake venom phospholipase A₂ isoforms, is again demonstrated as a valuable source of therapeutic peptides.

Keywords: Agkistrodon; leucine; membrane; phenylalanine; venom peptides.

Figure 1

Evaluation of erythrocyte membrane disruption caused by the synthetic peptides. Red blood cells were incubated with 7 different concentrations of p-AppK (blue) and p-Acl (green). The percentage of hemolysis was measured in relation to the effect caused by a hemolytic surfactant, Triton X-100.

Figure 2

Dose-dependent inhibition of bacterial growth caused by the synthetic peptides using broth microdilution assay. (Left—(a)) p-AppK (green) and (Right—(b)) p-Acl (blue) reduced the bacterial viability (P. aeruginosa 31NM, P. aeruginosa ATCC, E. coli ATCC, S. aureus BEC 9393, and S. aureus rib1) after 24 h as a function of their concentrations. The growth inhibition was calculated considering the maximum optical density of the negative control as the reference. The experiments were performed in triplicate.

Figure 3

In vitro membrane-disruptive activity provoked by p-AppK (green) and p-Acl (blue). The membrane integrity evaluation of (A) P. aeruginosa ATCC and (B) S. aureus BEC9393 incubated with 100 µM peptides was determined as a function of fluorescent dye uptake.

Figure 4

In vitro cytotoxicity of p-AppK (green) and p-Acl (blue). (A) Leukemia cell lines and (B) solid tumors were exposed to a concentration of 100 µM of both peptides. A colorimetric assay determined the cell viability inhibition after 24 h of peptide treatment. Cells cultured in a growth medium without the peptides were considered a positive control with 100% viability. The positive control (reference antineoplastic drug) is represented in salmon. The data represent the mean ± SD.

Figure 5

p-Acl peptide interaction with the DOPS membrane bilayer (one-half of the membrane and water molecules were removed for better visualization; the inner hydrophobic core of the membrane is shown in grey; the positions of the phosphorus atoms in the headgroups are shown in salmon). Left (a): a large deformation in the position of the phospholipid headgroups is visible, with the phosphate moieties penetrating deeply into the membrane core, more pronounced in the upper leaflet; in addition, the penetration of water molecules deep into the membrane is visible, confirming that the peptide induces a membrane-permeabilization effect. Top-right (b): only membrane inner hydrophobic core and phosphorus positions are shown, for clarity; the deformation of the membrane headgroups towards the inner part of the membrane is evident. Lower-right (c): insertion of peptide p-Acl (stick model) into the membrane inner hydrophobic core, showing the peptide’s perfect structural fitness to span the whole width of the membrane.

Figure 6

Density of the main constituents of the system as a function of the bilayer normal. Top (a): the center of the bilayer is located at z = 0. Bottom (b): the number of water molecules whose oxygen atom is within 3.0 Å of any heteroatom of the p-Acl peptide.