Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stagePlasmodium falciparumandCryptosporidium parvumin cell-culture studies. Target deconvolution inP. falciparumhas shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of bothPfKRS1 andC. parvumKRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90= 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology betweenPfKRS1 andCpKRS. This series of compounds inhibitCpKRS andC. parvumandCryptosporidium hominisin culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds forPfKRS1 andCpKRS vs. (human)HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

The evolution of the digestive system in the Order Orthoptera is disclosed from the study of the morphophysiology of the digestive process in its major taxa. This paper deals with a cricket representing the less known suborder Ensifera. Most amylase and trypsin activities occur in crop and caeca, respectively. Maltase and aminopeptidase are found in soluble and membrane-bound forms in caeca, with aminopeptidase also occurring in ventriculus. Amaranth was orally fed to Gryllodes sigillatus adults or injected into their haemolymph. The experiments were performed with starving and feeding insects with identical results. Following feeding of the dye the luminal side of the most anterior ventriculus (and in lesser amounts the midgut caeca) became heavily stained. In injected insects, the haemal side of the most posterior ventriculus was stained. This suggested that the anterior ventriculus is the main site of water absorption (the caeca is a secondary one), whereas the posterior ventriculus secretes water into the gut. Thus, a putative counter-current flux of fluid from posterior to anterior ventriculus may propel digestive enzyme recycling. This was confirmed by the finding that digestive enzymes are excreted at a low rate. The fine structure of midgut caeca and ventriculus cells revealed that they have morphological features that may be related to their involvement in secretion (movement from cell to lumen) and absorption (movement from lumen to cell) of fluids. Furthermore, morphological data showed that both merocrine and apocrine secretory mechanisms occur in midgut cells. The results showed that cricket digestion differs from that in grasshopper in having: (1) more membrane-bound digestive enzymes; (2) protein digestion slightly displaced toward the ventriculus; (3) midgut fluxes, and hence digestive enzyme recycling, in both starved and fed insects.

With increasing drug resistance in tuberculosis (TB) patient populations, there is an urgent need for new drugs. Ideally, new agents should work through novel targets so that they are unencumbered by preexisting clinical resistance to current treatments. Benzofuran 1 was identified as a potential lead for TB inhibiting a novel target, the thioesterase domain of Pks13. Although, having promising activity against Mycobacterium tuberculosis, its main liability was inhibition of the hERG cardiac ion channel. This article describes the optimization of the series toward a preclinical candidate. Despite improvements in the hERG liability in vitro, when new compounds were assessed in ex vivo cardiotoxicity models, they still induced cardiac irregularities. Further series development was stopped because of concerns around an insufficient safety window. However, the demonstration of in vivo activity for multiple series members further validates Pks13 as an attractive novel target for antitubercular drugs and supports development of alternative chemotypes.

Enhanced mitochondrial generation of oxidants, including hydrogen peroxide (H2O2), is related to a large number of pathological conditions, including diet-induced obesity and steatohepatosis. Indeed, we have previously shown that high fat diets increase the generation of H2O2 in liver mitochondria energized by activated fatty acids. Here, we further study fatty-acid induced H2O2 release in liver mitochondria, and determine the characteristics that regulate it. We find that this production of H2O2 is independent of mitochondrial inner membrane integrity and insensitive to purine nucleotides. On the other hand, palmitate-induced H2O2 production is strongly enhanced by high fat diets and is pH-sensitive, with a peak at a matrix pH of ~8.5. Using recombinantly expressed human very long chain acyl-CoA dehydrogenase, we are able to demonstrate that palmitate-induced H2O2 release may be ascribed to the activity of this enzyme alone, acting as an oxidase. Our results add to a number of findings indicating that sources outside of the electron transport chain can generate significant, physiopathologically relevant, amounts of oxidants in mitochondria.