For over 50 years, metronidazole and other nitro compounds such as nitazoxanide have been used as a therapy of choice against giardiasis and more and more frequently, resistance formation has been observed. Model systems allowing studies on biochemical aspects of resistance formation to nitro drugs are, however, scarce since resistant strains are often unstable in culture. In order to fill this gap, we have generated a stable metronidazole- and nitazoxanide-resistant Giardia lamblia WBC6 clone, the strain C4. Previous studies on strain C4 and the corresponding wild-type strain WBC6 revealed marked differences in the transcriptomes of both strains. Here, we present a physiological comparison between trophozoites of both strains with respect to their ultrastructure, whole cell activities such as oxygen consumption and resazurin reduction assays, key enzyme activities, and several metabolic key parameters such as NAD(P)+/NAD(P)H and ADP/ATP ratios and FAD contents. We show that nitro compound-resistant C4 trophozoites exhibit lower nitroreductase activities, lower oxygen consumption and resazurin reduction rates, lower ornithine-carbamyl-transferase activity, reduced FAD and NADP(H) pool sizes and higher ADP/ATP ratios than wildtype trophozoites. The present results suggest that resistance formation against nitro compounds is correlated with metabolic adaptations resulting in a reduction of the activities of FAD-dependent oxidoreductases.

As there is a continuous need for novel anti-infectives, the present study aimed to fuse two modes of action into a novel 3-nitroimidazo[1,2-b]pyridazine scaffold to improve antiparasitic efficacy. For this purpose, we combined known structural elements of phosphodiesterase inhibitors, a target recently proposed for Trypanosoma brucei and Giardia lamblia, with a nitroimidazole scaffold to generate nitrosative stress. The compounds were evaluated in vitro against a panel of protozoal parasites, namely Giardia lamblia, Trypanosoma brucei, T. cruzi, Leishmania infantum and Plasmodium falciparum and for cytotoxicity on MRC-5 cells. Interestingly, selective sub-nanomolar activity was obtained against G. lamblia, and by testing several analogues with and without the nitro group, it was shown that the presence of a nitro group, but not PDE inhibition, is responsible for the low IC50 values of these novel compounds. Adding the favourable drug-like properties (low molecular weight, cLogP (1.2-4.1) and low polar surface area), the key compounds from the 3-nitroimidazo[1,2-b]pyridazine series can be considered as valuable hits for further anti-giardiasis drug exploration and development.

The metacestode stage of the fox tapeworm Echinococcus multilocularis causes the severe zoonotic disease alveolar echinococcosis. New treatment options are urgently needed. Disulfiram and dithiocarbamates were previously shown to exhibit activity against the trematode Schistosoma mansoni. As both parasites belong to the platyhelminths, here we investigated whether these compounds were also active against E. multilocularis metacestode vesicles in vitro. We used an in vitro drug-screening cascade for the identification of novel compounds against E. multilocularis metacestode vesicles with disulfiram and 51 dithiocarbamates. Five compounds showed activity against E. multilocularis metacestode vesicles after five days of drug incubation in a damage marker release assay. Structure-activity relationship analyses revealed that a S-2-hydroxy-5-nitro benzyl moiety was necessary for anti-echinococcal activity, as derivatives without this group had no effect on E. multilocularis metacestode vesicles. The five active compounds were further tested for potential cytotoxicity in mammalian cells. For two compounds with low toxicity (Schl-32.315 and Schl-33.652), IC50 values in metacestode vesicles and IC50 values in germinal layer cells were calculated. The compounds were not highly active on isolated GL cells with IC50 values of 27.0 ± 4.2 µM for Schl-32.315 and 24.7 ± 11.5 µM for Schl-33.652, respectively. Against metacestode vesicles, Schl-32.315 was not very active either with an IC50 value of 41.6 ± 3.2 µM, while Schl-33.652 showed a low IC50 of 4.3 ± 1 µM and should be further investigated in the future for its activity against alveolar echinococcosis.