Phloroglucinol as a Potential Candidate against Trypanosoma congolense Infection: Insights from In Vivo, In Vitro, Molecular Docking and Molecular Dynamic Simulation Analyses

Abstract

Sub-Saharan Africa is profoundly challenged with African Animal Trypanosomiasis and the available trypanocides are faced with drawbacks, necessitating the search for novel agents. Herein, the chemotherapeutic potential of phloroglucinol on T. congolense infection and its inhibitory effects on the partially purified T. congolense sialidase and phospholipase A₂ (PLA₂) were investigated. Treatment with phloroglucinol for 14 days significantly (p < 0.05) suppressed T. congolense proliferation, increased animal survival and ameliorated anemia induced by the parasite. Using biochemical and histopathological analyses, phloroglucinol was found to prevent renal damages and splenomegaly, besides its protection against T. congolense-associated increase in free serum sialic acids in infected animals. Moreover, the compound inhibited bloodstream T. congolense sialidase via mixed inhibition pattern with inhibition binding constant (Ki) of 0.181 µM, but a very low uncompetitive inhibitory effects against PLA₂ (Ki > 9000 µM) was recorded. Molecular docking studies revealed binding energies of -4.9 and -5.3 kcal/mol between phloroglucinol with modeled sialidase and PLA₂ respectively, while a 50 ns molecular dynamics simulation using GROMACS revealed the sialidase-phloroglucinol complex to be more compact and stable with higher free binding energy (-67.84 ± 0.50 kJ/mol) than PLA₂-phloroglucinol complex (-77.17 ± 0.52 kJ/mol), based on MM-PBSA analysis. The sialidase-phloroglucinol complex had a single hydrogen bond interaction with Ser453 while none was observed for the PLA₂-phloroglucinol complex. In conclusion, phloroglucinol showed moderate trypanostatic activity with great potential in ameliorating some of the parasite-induced pathologies and its anti-anemic effects might be linked to inhibition of sialidase rather than PLA₂.

Keywords: Trypanasoma congolense; anemia; molecular docking; molecular dynamics simulation; phloroglucinol; phospholipase A2; sialidase.

Figure 1

Structure of phloroglucinol.

Figure 2

Evaluation of parasitemia in T. congolense-infected rats treated with different doses of phloroglucinol. All data are presented as mean ± standard deviation of seven rats. Dunnett posthoc test was used to analyze the data following ONE-WAY ANOVA. IC was used as control. Values with different subscripts are considered statistically significant at p < 0.05. IC = infected control, ITPGN15 = Infected + 15 mg/kg BW PGN, ITPGN30 = Infected + 30 mg/kg PGN, ITDA = Infected + 3.5 mg/kg BW DA.

Figure 3

Cessation of mortality in T. congolense-infected rats treated with different doses of phloroglucinol. NC = Normal control, IC = Infected control, ITPGN15 = Infected + 15 mg/kg BW PGN, ITPGN30 = Infected + 30 mg/kg BW PGN, ITDA = Infected + 3.5 mg/kg BW DA.

Figure 4

Assessment of anemia progression in T. congolense-infected rats treated with different doses of phloroglucinol. All data are presented as mean ± standard deviation of seven rats. Data was analyzed using paired sample t. test within group. Bars with different alphabets are considered statistically significant at p < 0.05. NC = Normal control, IC = Infected control, ITPGN15 = Infected + 15 mg/kg BW PGN, ITPGN30 = Infected + 30 mg/kg BW PGN, ITDA = Infected + 3.5 mg/kg BW DA.

Figure 5

Liver histopathology of T. congolense-infected rats treated with different doses of phloroglucinol. H = Hepatocytes Normal, HN = Hepatocellular Necrosis, LH = Lymphocyte Hyperplasia. NC = Normal control, IC = Infected control, ITPGN15 = Infected + 15 mg/kg BW PGN, ITPGN30 = Infected + 30 mg/kg BW PGN, ITDA = Infected + 3.5 mg/kg BW DA.

Figure 6

Kidney histopathology of T. congolense-infected rats treated with different doses of phloroglucinol. T = Normal Tubules, G = Normal Glomerulus, GN = Glomerular Necrosis, TN = Tubular Necrosis. NC = Normal control, IC = Infected control, ITPGN15 = Infected + 15 mg/kg BW PGN, ITPGN30 = Infected + 30 mg/kg BW PGN, ITDA = Infected + 3.5 mg/kg BW DA.

Figure 7

Double reciprocal plot of T. congolense sialidase (a) and PLA₂ (b) inhibitions using phloroglucinol. All data are presented as mean ± standard deviation of three independent experiments. Ki = Inhibition constant.

Figure 8

Ramachandran plot of modelled trypanosomal sialidase (A) and PLA₂ (B) showing amino acid residue regions.

Figure 9

Homology models of trypanosomal sialidase (a) and PLA₂ (b) after 50 ns molecular dynamics simulation and 3D molecular docking interactions of phloroglucinol with the trypanosomal sialidase (c) and PLA₂ (d).

Figure 10

3D and 2D molecular interactions of phloroglucinol with trypanosomal sialidase (a) and (b) and PLA₂ (c) and (d) after 50 ns molecular dynamics simulation studies.

Figure 11

Molecular dynamics trajectory plots of unbound modelled trypanosomal sialidase and PLA₂ in complex with phloroglucinol. RMSD (a) and (d), RMSF (b) and (e), ROG (c) and (f).