Insights into antitrypanosomal drug mode-of-action from cytology-based profiling

Abstract

Chemotherapy continues to have a major impact on reducing the burden of disease caused by trypanosomatids. Unfortunately though, the mode-of-action (MoA) of antitrypanosomal drugs typically remains unclear or only partially characterised. This is the case for four of five current drugs used to treat Human African Trypanosomiasis (HAT); eflornithine is a specific inhibitor of ornithine decarboxylase. Here, we used a panel of T. brucei cellular assays to probe the MoA of the current HAT drugs. The assays included DNA-staining followed by microscopy and quantitative image analysis, or flow cytometry; terminal dUTP nick end labelling to monitor mitochondrial (kinetoplast) DNA replication; antibody-based detection of sites of nuclear DNA damage; and fluorescent dye-staining of mitochondria or lysosomes. We found that melarsoprol inhibited mitosis; nifurtimox reduced mitochondrial protein abundance; pentamidine triggered progressive loss of kinetoplast DNA and disruption of mitochondrial membrane potential; and suramin inhibited cytokinesis. Thus, current antitrypanosomal drugs perturb distinct and specific cellular compartments, structures or cell cycle phases. Further exploiting the findings, we show that putative mitogen-activated protein-kinases contribute to the melarsoprol-induced mitotic defect, reminiscent of the mitotic arrest associated signalling cascade triggered by arsenicals in mammalian cells, used to treat leukaemia. Thus, cytology-based profiling can rapidly yield novel insight into antitrypanosomal drug MoA.

A Parasite Coat Protein Binds Suramin to Confer Drug Resistance

“…While the binding of suramin to VSGsur is seen to be required for drug resistance, it is still unclear by what mechanism VSGsur is producing this effect. Genetic studies coupled with examination of the trypanosome's single lysosome suggest that suramin accumulates in this compartment, while other evidence links suramin toxicity to effects on the glycosome and impairment of cytokinesis 19,20 . Our internalization data decouple suramin uptake via the LDL endocytic pathway from the VSGsur-related mechanism of resistance.…”

“…The specific mechanism behind the trypanolytic activity of suramin remains unresolved, although studies have implicated effects on the glycosome and impairment of cytokinesis 19,20 . A model for the internalization of suramin has been proposed in which the drug enters through receptor-mediated endocytosis involving two distinct pathways: (1) low-density lipoproteins (LDLs) with possible involvement of other serum proteins and (2) the invariant surface glycoprotein 75 (ISG75) receptor of the pathogen [21][22][23] .…”

ATrypanosoma bruceiORFeome-based Gain-of-Function Library reveals novel genes associated with melarsoprol resistance

“…Downstream from T(SH) 2 , we saw increased levels of trypanothione reductase (TR, Tb927.10.10390 ), a trypanothione dependent peroxidase ( Tb927.7.1140 ), and ribonucleotide reductase (RR, Tb927.11.7840 ), in some survivor cultures. The generation of dNTPs by the action of RR has known effects on kDNA replication (43) and melarsoprol treatment was recently shown to stall cell cycle (44), which might suggest inhibition of DNA synthesis as a mechanism of cell killing. Collectively these findings may further support the trypanothione pathway as a primary target of melarsoprol-based cell killing.…”

“…Connections between T(SH) 2 and mitochondrial redox reactions have been widely established (31) and the anti-Trypanosomatid drugs pentamidine and nifurtimox have been linked to specific mitochondrial functions (44, 47). Yet, the data arising from the GoF screen described herein represent the first genetic links between melarsoprol and the mitochondria.…”

New Drugs for Human African Trypanosomiasis: A Twenty First Century Success Story

“…NTR1 has been proposed to act physiologically as an NADH dehydrogenase involved in reducing ubiquinone to ubiquinol [81], hence its essentiality even in bloodstream form T. brucei that requires ubiquinone-based electron transport in its mitochondrial alternative oxidase system [82]. In nifurtimox-treated T. brucei, loss of mitochondrial membrane potential and other mitochondrial morphology defects were noted [83]. Other nitroheterocycles, including fexinidazole, may also exert their effects on the mitochondrion, where the drug's activation occurs.…”