Chagas disease, leishmaniasis, and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually1. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drug(s) modulating the activity of a conserved parasite target2. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases.

SummaryWith renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited.

A novel Saccharomyces cerevisiae mutant, unable to grow in the presence of 12.5 mM EGTA, was isolated by replica plating. The phenotype of the mutant is caused by a single amino acid change (Gly'49 to Arg) leu2, his3, ade2, trpl, ura3), leu2, his3, ade2, trpl, ura3), E20 (MA Ta, masi), and CRB1-5A (MATa, leu2, his3, his4-4Q1, trpl, ura3-52, HOLJ-1, masl-ts) were used. EGTA-sensitive mutants were generated by exposing yeast cells to ethyl methanesulfonate (EMS) as described (14) and replica plating on YPD plates (pH 6.0) containing 50 mM Mes and 12.5 mM EGTA. The mutants denoted as csp (chelator-sensitive phenotype) were back-crossed to the wild-type strain as described elsewhere (15)

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We have previously shown that mutations in the Saccharomyces cerevisiae BSD2 gene suppress oxidative damage in cells lacking superoxide dismutase and also lead to hyperaccumulation of copper ions. We demonstrate here that bsd2 mutant cells additionally accumulate high levels of cadmium and cobalt. By biochemical fractionation and immunofluorescence microscopy, BSD2 exhibited localization to the endoplasmic reticulum, suggesting that BSD2 acts at a distance to inhibit metal uptake from the growth medium. This BSD2 control of ion transport occurs independently of the CTR1 and FET4 metal transport systems. Genetic suppressor analysis revealed that hyperaccumulation of copper and cadmium in bsd2 mutants is mediated through SMF1, previously shown to encode a plasma membrane transporter for manganese. A nonsense mutation removing the carboxyl-terminal hydrophobic domain of SMF1 was found to mimic a smf1 gene deletion by eliminating the copper and cadmium toxicity of bsd2 mutants and also by precluding the bsd2 suppression of superoxide dismutase deficiency. However, inactivation of SMF1 did not eliminate the elevated cobalt levels in bsd2 mutants. Instead, this cobalt accumulation was found to be specifically mediated through the SMF1 homologue, SMF2. Hence, BSD2 prevents metal hyperaccumulation by exerting negative control over the SMF1 and SMF2 metal transport systems.Transition metals such as copper and manganese serve as essential cofactors for a variety of enzymatic reactions and play important structural and functional roles in cell metabolism. However, these same ions can be toxic when present at elevated levels. One mechanism of toxicity is believed to involve the metal-catalyzed generation of hydroxyl radicals in the Fenton reaction (1). Nonessential metals such as cadmium have no known biological function and are highly toxic at relatively low concentrations. To balance the stimulatory and inhibitory effects of essential ions and to counteract the toxicity of nonessential metals, all organisms possess homeostatic mechanisms that properly control the cellular accumulation, distribution, and detoxification of metals.The bakers' yeast Saccharomyces cerevisiae provides an ideal system in which to study the factors controlling metal homeostasis. A number of heavy metal transport proteins have been identified in yeast that function in transporting and delivering the ions to cellular targets. For example, the plasma membrane protein CTR1 is required for high affinity copper uptake (2). Copper uptake is also controlled by the S. cerevisiae CTR3 gene, although this gene is inactivated in most laboratory strains of yeast through insertion of a transposable element (3). In addition to these pathways for the high affinity uptake of copper, a low affinity divalent metal transporter encoded by the FET4 gene has been identified in yeast (4). FET4 exhibits specificity for a subset of divalent metal ions including cobalt, cadmium, and iron (4). Another gene that directly participates in metal ion uptake is SMF1. SMF1 was ori...