Metabolomic Analysis of Trypanosoma Congolense and Leishmania Mexicana Treated with Selected Antiparasitic Drugs and in Silico Modelling of Potential Drug Targets

Abstract

Animal African Trypanosomiasis (AAT) is a devastating parasitic disease caused by a variety of haemoflagellate extracellular protozoa of the genus Trypanosoma. It is endemic in 37 of the 55 countries in Africa and is transmitted by the obligate blood feeder of the genus Glossina. It affects both domestic and wild animals causing huge economic losses and severe health problems. Chemotherapeutic and prophylactic agents such as Isometamidium chloride (ISM) are used to prevent the progression of AAT in endemic areas. These agents face limitations such as resistance, toxicity, low efficacy, and unknown mechanisms of action. Therefore, it is crucial to develop new, safe and effective trypanocides or optimize those in current use by understanding their mechanism of action (MOA) through untargeted metabolomics. In this study, the MOA of ISM was first investigated through statistical and metabolomic analysis and molecular docking. Statistical analysis was done through Principal Component Analysis (PCA), one-way Analysis of Variance (ANOVA), and Tukey’s HSD post hoc analysis, cluster, and fold change analysis. Significant perturbations were observed in glycolysis, acetate: succinate CoA transferase succinyl CoA synthetase (ASCT/SCS), and energy metabolites indicating that ISM may have an inhibitory effect on the glucose transporter. Molecular docking assays showed ISM interacting with the glucose binding site of the transporter. Isometamidium chloride interacted with amino acid residues known to be conserved in all sugar transporter members and to play an important role in transport of hexoses and formation of the exofacial substrate binding site. Secondly, to increase the drug arsenal against AAT, new or existing drugs used against biologically and biochemically similar kinetoplastids have been probed for their novel activity against Trypanosoma species. In this study, the MOA of novel anti-trypanosomatid compounds dubbed JYH and VMS were elucidated through the aforementioned analyses. The novel compounds are toxic to kinetoplastid protozoa with activity recorded against Leishmania mexicana. For one of the compounds (VMS), statistical and metabolomic analysis showed possible perturbations to T. congolense metabolism, particularly in energy metabolism, aromatic amino acid metabolism, and cellular redox. However, from the analyses, it was not possible to readily assign candidate target proteins in T. congolense. In L. mexicana, inhibition of the Coenzyme A biosynthesis pathway at dephospho-CoA kinase (DPCK) or phosphopantetheine adenylyltransferase (PPAT) enzymes was evident for the two novel compounds. Molecular docking assays showed a possible inhibition of dephospho-CoA kinase by the drugs interacting with the CoA binding domain where the native substrate dephospho-CoA binds. On phosphopantetheine adenylyl transferase the drugs seemed to interact with the active site where adenosine triphosphate (ATP) binds. This study identified potential drug targets for ISM, JYH and VMS. These findings provide new insights into the mechanisms of action of these drugs and suggest avenues for future drug development efforts.