In Vitro and In Vivo Antimalarial Activity of LZ1, a Peptide Derived from Snake Cathelicidin

Abstract

Antimalarial drug resistance is an enormous global threat. Recently, antimicrobial peptides (AMPs) are emerging as a new source of antimalarials. In this study, an AMP LZ1 derived from snake cathelicidin was identified with antimalarial activity. In the in vitro antiplasmodial assay, LZ1 showed strong suppression of blood stage Plasmodium falciparum (P. falciparum) with an IC50 value of 3.045 μM. In the in vivo antiplasmodial assay, LZ1 exerted a significant antimalarial activity against Plasmodium berghei (P. berghei) in a dose- and a time- dependent manner. In addition, LZ1 exhibited anti-inflammatory effects and attenuated liver-function impairment during P. berghei infection. Furthermore, by employing inhibitors against glycolysis and oxidative phosphorylation in erythrocytes, LZ1 specifically inhibited adenosine triphosphate (ATP) production in parasite-infected erythrocyte by selectively inhibiting the pyruvate kinase activity. In conclusion, the present study demonstrates that LZ1 is a potential candidate for novel antimalarials development.

Keywords: ATP; antimicrobial peptide; cytokine; malaria; pyruvate kinase.

Figure 1

In vitro antimalarial activity of LZ1. In vitro antimalarial activity of LZ1 against the asexual blood stage of Plasmodium falciparum (P. falciparum). Data are presented as mean ± SEM. *** p < 0.001.

Figure 2

In vivo antimalarial activity of LZ1 against Plasmodium berghei (P. berghei). (A) Schematic of the four day suppression test. (B) Schematic of Rain’s test. (C) Parasitemia of different group was shown in the four day suppression test. (D) Parasitemia of a different group was shown in Rain’s test. (E) Survival rates were determined in Rain’s test. NS, normal saline. CQ, chloroquine. Data are presented as mean ± SEM (n = 6 mice per group). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Effects of LZ1 and CQ on serum cytokines concentration of infected mice in four day suppression test. The concentrations of serum interleukin (IL)-6 (A), tumor necrosis factor (TNF)-α (B), interferon (IFN)-γ (C), and IL-10 (D) of mice were measured. CK and NS represent uninfected untreated and infected untreated mice, respectively. LZ1 represents infected mice treated with LZ1 at 12 mg·kg⁻¹ body weight. CQ presents infected mice treated with chloroquine at 25 mg·kg⁻¹ body weight. Data are presented as mean ± SEM (n = 6 mice per group). ** p < 0.01, *** p < 0.001.

Figure 4

The liver function test of infected mice with LZ1 treatment in the four day suppression test. The concentrations of serum alanine transaminase (ALT) (A), aspartate transaminase (AST) (B), and bilirubin (BIL) (C) of mice were measured. CK and NS represent uninfected untreated and infected untreated mice, respectively. LZ1 represents infected mice treated with LZ1 at 12 mg·kg⁻¹ body weight. CQ represents infected mice treated with chloroquine at 25 mg·kg⁻¹ body weight. Data are presented as mean ± SEM (n = 6 mice per group). * p < 0.05, ** p < 0.01.

Figure 5

LZ1 inhibits the pyruvate kinase activity and adenosine triphosphate (ATP) synthesis during plasmodium infection. (A) Schematic of glycolysis pathway. Metabolic enzymes are bolded. PK, pyruvate kinase. (B) LZ1 inhibits the pyruvate kinase activity of infected erythrocyte. (C) 2-deoxyglucose (2-DG) induces ATP reduction in uninfected erythrocyte. (D) 2-DG induces ATP reduction in infected erythrocyte. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, ^# p< 0.05, ^aa p < 0.01, ^aaa p < 0.001.