Materials and chemical reagents were purchased from Sigma-Aldrich Company (USA). Compounds 1 to 4 were prepared according to our previous research [14]. The binding of ligands to heme and PfLDH were studied based on the molecular docking via AutoDock tool. The female BALB/C mice were purchased from Pasteur Institute of Iran. Chloroquine sensitive Plasmodium berghei (ANKA) strain was obtained from department of Medical Entomology, School of Public Health, Tehran University, Tehran, Iran.
Receptor structure preparation and generation of grid box for docking studies
Molecular docking is a worthwhile method to explore protein and ligand interactions at the molecular level [15]. The method was used to study the binding of ligands to heme and plasmodium falciparum L-lactate dehydrogenase (pLDH). The 3D structure of the L-lactate dehydrogenase and heme were taken from the Protein Data Bank (PDB ID 1LDG and 3P5Q, respectively) for molecular docking experiment. To this end, the receiver structure was prepared using the AutoDock toolkit. The missing atoms were added, residues were assigned, and the AutoGrid parameter file was adjusted via AutoDock tools [16]. The L-lactate dehydrogenase active site was selected as the ligand binding site [17]. The size of the docking grid for L-lactate dehydrogenase and heme were X= 32 A˚ Y=30 A˚ and Z=32 A˚ and X= 14 A˚ , Y=10 A˚ and Z=14 A˚, respectively. The grid spacing was adjusted on 1 Å.
Ligand Molecule Preparation
The ligand molecules, drawn by Marvin 19.10 (http://www.chemaxon.com) were subsequently optimized via HyperChem 8.0 software [18].
Molecular Docking Study
The ligand molecules were docked in the selected binding pocket via Smina AutoDock. Smina is a version of AutoDock Vina, focusing on the improvement of scoring and energy minimization [19]. Then, the crystal structure of receptors in complex with ligands was analyzed via LigPlot+ software [20].
In vivo anti-malarial assay
Acute toxicity of the compounds in mice
Before in vivo study, the toxicity of the compounds was assessed by Dixon’s up and down method in BALB/c mice with some modification [21]. In the first phase, 500 mg/kg of compounds administered intraperitoneally to two mice and observed for any signs of toxicity and mortality at 24 h. When a mouse died, the concentration was reduced to half, the test was repeated, and the alive mice were monitored for toxicity and mortality for 10 days. In the second phase, test was repeated with 5 mice, and if mice survived, the highest non-toxic dose would be determined for Rane and Peter’s tests (125 mg/kg).
Parasite inoculation
At first, two female BALB/C mice were infected intraperitoneally from frozen stock of CQ-sensitive P. berghei (ANKA) strain. Then, four and six mice were infected by continuous intraperitoneal passage. Next, the blood was diluted with PBS and finally the experimental mice were infected with an inoculum of 1.5 × 10⁷ of parasitized erythrocytes intraperitoneally.
Schizonticidal effect in early infection (Peter’s test)
The suppressive test was conducted in accordance with Peter’s method [8, 22]. The experimental mice were maintained under the standard conditions based on the international guidelines for ten days [23]. Seventy-five female BALB/C mice (weight 16-20 g) were grouped into fifteen groups. They were inoculated intra-peritoneally (i.p.) with 1.5˟107 infected erythrocytes of CQ-sensitive P. berghei in a saline (200 μl) on the first day (D0) of the test.
The test compounds were solubilized in a mixture of DMSO, sesame oil, and ethanol (a ratio of 1:2:0.05) pre-diluted in sesame oil for preparation of different dose (31.25, 62.5 and 125 mg/kg). The treatments began within 3 h post-inoculation of mice with the parasite (D0) and proceeded intraperitoneally for five days (D4).
Tail blood smear was taken, stained with 10% Giemsa in phosphate buffer (pH 7.2) on the fifth day (D4). The parasitaemia level was determined by counting the parasitized red blood cells on at least 2,000 red blood cells by microscope at 100 ×. The %suppression of parasitaemia was calculated by comparing the %parasitaemia between test mice and infected controls. The chloroquine diphosphate (25 mg/kg), oil and DMSO (12.5%) were applied as positive and negative controls, respectively. For all the groups, mortality was monitored daily and mean survival time was recorded to evaluate the efficacy of anti-malarial activity of the compounds. During the treatment, the body weight of each mouse was measured before the infection (D0) and after treatment (D4). Also, the internal organs (spleen, liver, and kidney) were evaluated on the seventh day of treatment after the dissection of the mice.
Schizontocidal activity in established infection (Rane’s test)
The curative test was conducted according to the method of Ryley and Peters (Rane’s test) [24].The animal housing, infecting, and dosing were done similar to Peter’s test. Nevertheless, the treatment began within 72 h post-inoculation of mice with the parasite (D2) to allow parasitemia to establish and then continue for four days (D7) intraperitoneally. The tail blood smears were taken, stained with 10% Giemsa in phosphate buffer (pH 7.2) on the 8th day (D7). The parasitaemia level and the %suppression of parasitaemia, mortality, mean survival time, and body weight were determined and recorded in line with Peter’s test.
Histopathological study of liver tissue
Histopathological studies were conducted based on the standard method [25]. After the dissection on seventh day after treatment, mice’s livers were fixed in 10% formalin solution. Then, the middle lobe of livers was severed and molds of fresh paraffin were made of tissue with a thickness of 5 µm. The sections were stained with hematoxylin–eosin (H&E) and analyzed by light microscope. The slides were examined under 40× objective lens and image saved as JPEG files. The sections were scored from 0–4 for sinusoidal congestion, vacuolization of hepatocyte cytoplasm, and parenchymal single cell necrosis as described by Suzuki et al. [26].
Statistical analysis
The data were analyzed using SPSS (IBM SPSS Statistics 22). Then one-way ANOVA was used to test statistical differences for three doses within a group, followed by Tukey’s test for multiple comparisons. P-values < 0.05 were considered significant in all tests.