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Alveolar echinococcosis (AE) is a severe zoonotic disease caused by the metacestode stage of the fox tapeworm Echinococcus multilocularis. We recently showed that E. multilocularis metacestode vesicles scavenge large amounts of L-threonine from the culture medium. This motivated us to study the effect of L-threonine on the parasite and how it is metabolized. We established a novel metacestode vesicle growth assay with an automated readout, which showed that L-threonine treatment led to significantly increased parasite growth. In addition, L-threonine increased the formation of novel metacestode vesicles from primary parasite cell cultures in contrast to the non-proteinogenic threonine analog 3-hydroxynorvaline. Tracing of [U-13C]-L-threonine and metabolites in metacestode vesicles and culture medium resulted in the detection of [U-13C]-labeling in aminoacetone and glycine, indicating that L-threonine was metabolized by threonine dehydrogenase (TDH). EmTDH-mediated threonine metabolism in the E. multilocularis metacestode stage was further confirmed by quantitative real-time PCR, which demonstrated high expression of emtdh in in vitro cultured metacestode vesicles and also in metacestode samples obtained from infected animals. EmTDH was enzymatically active in metacestode vesicle extracts. The compounds disulfiram, myricetin, quercetin, sanguinarine, and seven quinazoline carboxamides were evaluated for their ability to inhibit recombinantly expressed EmTDH. The most potent inhibitors, albeit not very strong or highly specific, were disulfiram, myricetin and sanguinarine. These compounds were subsequently tested for activity against E. multilocularis metacestode vesicles and primary parasite cells and only sanguinarine demonstrated significant in vitro activity. However, TDH is not its only cellular target, and it is also known to be highly toxic. Our findings suggest that additional targets of sanguinarine should be explored, and that it may serve as a foundation for developing more specific compounds against the parasite. Moreover, the EmTDH assay could be a valuable high-throughput, target-based platform for discovering novel anti-echinococcal compounds.

Background: African trypanosomiasis is a protozoan disease with huge socio-economic burden to sub-Saharan African exceeding US$4.6 annual loss. To mitigate the incidence of trypanosomal drug resistance, efforts are geared towards discovery of molecules, especially from natural products, with potential to inhibit important molecular target (trypanosome alternative oxidase, TAO) in trypanosomes that are critical to their survival. Method: Crude methanol extract of Anogeissus leiocarpa was subjected to in vitro bioassay-guided antitrypanosomal assay to identify the most active extract with trypanocidal activity. The most active extract was run on a column chromatography yielding five fractions, F1-F5. The fractions were assayed for inhibitory effect on TAO. The most promising TAO inhibitor was subjected to antitrypanosomal evaluation by trypanosome count, drug incubation infectivity test (DIIT) and in vivo studies. Gas chromatography-mass spectrometry (GC-MS) was used to identify and quantify phytochemical constituents of the potential TAO-inhibiting fraction. Results: Ethyl acetate extract (EtOAc) significantly (p

Microbial amino acid biosynthetic pathways are underexploited for the development of anti-bacterial agents. N-acetyl glutamate synthase (ArgA) catalyses the first committed step in L-arginine biosynthesis and is essential for M. tuberculosis growth. Here, we have purified and optimized assay conditions for the acetylation of l-glutamine by ArgA. Using the optimized conditions, high throughput screening was performed to identify ArgA inhibitors. We identified 2,5-Bis (2-chloro-4-guanidinophenyl) furan, a dicationic diaryl furan derivatives, as ArgA inhibitor, with a MIC 99 values of 1.56 μM against M. tuberculosis. The diaryl furan derivative displayed bactericidal killing against both M. bovis BCG and M. tuberculosis. Inhibition of ArgA by the lead compound resulted in transcriptional reprogramming and accumulation of reactive oxygen species. The lead compound and its derivatives showed micromolar binding with ArgA as observed in surface plasmon resonance and tryptophan quenching experiments. Computational and dynamic analysis revealed that these scaffolds share similar binding site residues with L-arginine, however, with slight variations in their interaction pattern. Partial restoration of growth upon supplementation of liquid cultures with either L-arginine or N-acetyl cysteine suggests a multi-target killing mechanism for the lead compound. Taken together, we have identified small molecule inhibitors against ArgA enzyme from M. tuberculosis., (Copyright © 2021. Published by Elsevier B.V.)