Novel lactate dehydrogenase inhibitors with in vivo efficacy against Cryptosporidium parvum

Abstract

Cryptosporidium parvum is a highly prevalent zoonotic and anthroponotic protozoan parasite that causes a diarrheal syndrome in children and neonatal livestock, culminating in growth retardation and mortalities. Despite the high prevalence of C . parvum , there are no fully effective and safe drugs for treating infections, and there is no vaccine. We have previously reported that the bacterial-like C . parvum lactate dehydrogenase (CpLDH) enzyme is essential for survival, virulence and growth of C . parvum in vitro and in vivo . In the present study, we screened compound libraries and identified inhibitors against the enzymatic activity of recombinant CpLDH protein in vitro . We tested the inhibitors for anti- Cryptosporidium effect using in vitro infection assays of HCT-8 cells monolayers and identified compounds NSC158011 and NSC10447 that inhibited the proliferation of intracellular C . parvum in vitro , with IC 50 values of 14.88 and 72.65 μM, respectively. At doses tolerable in mice, we found that both NSC158011 and NSC10447 consistently significantly reduced the shedding of C . parvum oocysts in infected immunocompromised mice’s feces, and prevented intestinal villous atrophy as well as mucosal erosion due to C . parvum . Together, our findings have unveiled promising anti- Cryptosporidium drug candidates that can be explored further for the development of the much needed novel therapeutic agents against C . parvum infections.

Novel acyl carbamates and acyl / diacyl ureas show in vitro efficacy against Toxoplasma gondii and Cryptosporidium parvum

“…1 ). Paromomycin at 800 μM was used as the inhibitor control and all experimental data were normalized with DMSO-treated wells (0% inhibition) and inhibitor control (100% inhibition) wells as previously reported ( Li et al, 2019 ). On the other hand, we found 4 compounds (JS-1-09, JS-2-20, JS-2-35 and JS-2-49) that had significant inhibitory effect against the growth of T. gondii in vitro at 20 μM final concentration ( Fig.…”

“…Among all the compounds tested, only GMG-1-09 had in vitro efficacy against C. parvum with an anti- Cryptosporidium IC 50 concentration that was 14-fold lower than its cytotoxicity IC 50 concentration in a human intestinal cell line (HCT-8). We used paromomycin as a positive control that has an anti- Cryptosporidium IC 50 of 450 μM ( Li et al, 2019 ) and has been shown to be non-toxic to human cells at concentrations higher than 1000 μM ( Gargala et al, 2000 ; Downey et al, 2008 ). Comparatively, compound GMG-1-09 was efficacious against C. parvum at a much lower concentration (IC 50 = 32.34 μM) than paromomycin.…”

“…Even if T. gondii and C. parvum are both apicomplexan coccidian protozoans, they differ considerably at metabolic level, which in turn could explain, to some extent, the observed difference in antiprotozoal activity of the tested compounds. Based on the completed and annotated genome sequence of Cryptosporidium , it is evident that, while the parasite lacks genes for conventional molecular drug targets found in other important protozoan parasites, it possesses several genes encoding plant-like and bacterial-like enzymes that catalyze potentially essential biosynthetic and metabolic pathways ( Li et al, 2019 ; Abrahamsen et al, 2004 ). Therefore, it is possible that the compounds that we found to have activity against T. gondii but not C. parvum , could be targeting specific essential metabolic pathways that are present in T. gondii but absent in C. parvum .…”

“…Immediately, test compounds (reconstituted in DMSO) were added to one set of wells at 20 μM final concentration. Paromomycin reconstituted in distilled sterile water was added to a separate set of wells as a positive control at 800 μM final concentration ( Li et al, 2019 ). To negative control wells, volumes of DMSO equivalent to the volumes of test compounds used, were added.…”

Milk Replacer Supplementation Ameliorates Growth Performance and Rumen Microbiota of Early-Weaning Yimeng Black Goats

“…Ruminococcus can produce acetic acid, formic acid, and a small amount of lactic acid (42). Previous research has indicated that short-chain fatty acid including formic acid, acetic acid play a critical role in regulating the balance of gut microbiota and maintaining morphology and function of intestinal epithelial cells (43,44). Moreover, the short-chain fatty acid regulates energy intake by the brain-gut axis (45).…”