Authors

Discussion

INTRODUCTION: Human African trypanosomiasis (HAT), caused by Trypanosoma brucei, leads to severe neurological dysfunction and is fatal if left untreated. Current chemotherapeutic options are limited and often associated with toxicity or emerging resistance. Pyrazolopyrimidines have recently emerged as a promising compound class with potent anti-trypanosomal activity. This study investigates the mode-of-action of two lead compounds, NPD-2975 and NPD-3519, against T. brucei.

METHODOLOGY: An inducible genome-wide RNA interference (RNAi) library was employed to identify genes modulating compound sensitivity. Candidate targets were validated in vitro. Comparative metabolomic profiling was used to uncover affected metabolic pathways, and Ligand Gaussian accelerated molecular dynamics simulations were performed to evaluate compound-target interactions.

RESULTS: Compound exposure resulted in multinucleated parasites, indicative of cytokinesis failure. RNAi screening implicated the leucine carboxy methyltransferase (lcmt) gene as a key mediator of compound activity. Metabolomic analysis revealed perturbations in S-adenosyl-L-methionine metabolism which is consistent with impaired methyltransferase function, while the L-glutamine pathway was also affected. Molecular dynamics simulations demonstrated that NPD-2975 destabilizes the interaction between LCMT and its co-factor PP2A, consistent with the observed cellular phenotype.

CONCLUSION: Our results indicate that NPD-2975 exerts it anti-trypanosomal activity by interfering with LCMT-PP2A regulation and associated metabolic pathways, leading to cytokinesis arrest and parasite death. These findings identify PP2A regulation as a critical vulnerability in T. brucei and establish pyrazolopyrimidines as compelling leads for next-generation HAT therapeutics.