g Toxoplasma gondii is a protozoan parasite that can damage the human brain and eyes. There are no curative medicines. Herein, we describe our discovery of N-benzoyl-2-hydroxybenzamides as a class of compounds effective in the low nanomolar range against T. gondii in vitro and in vivo. Our lead compound, QQ-437, displays robust activity against the parasite and could be useful as a new scaffold for development of novel and improved inhibitors of T. gondii. Our genome-wide investigations reveal a specific mechanism of resistance to N-benzoyl-2-hydroxybenzamides mediated by adaptin-3␤, a large protein from the secretory protein complex. N-Benzoyl-2-hydroxybenzamide-resistant clones have alterations of their secretory pathway, which traffics proteins to micronemes, rhoptries, dense granules, and acidocalcisomes/plant-like vacuole (PLVs). N-Benzoyl-2-hydroxybenzamide treatment also alters micronemes, rhoptries, the contents of dense granules, and, most markedly, acidocalcisomes/PLVs. Furthermore, QQ-437 is active against chloroquine-resistant Plasmodium falciparum. Our studies reveal a novel class of compounds that disrupts a unique secretory pathway of T. gondii, with the potential to be used as scaffolds in the search for improved compounds to treat the devastating diseases caused by apicomplexan parasites.T oxoplasma gondii is an apicomplexan, intracellular parasite that infects one third to one half of the world's population. It can cause eye and brain disease and death, and the presence of infection has been correlated with a variety of neurologic illnesses. Moreover, it is the most frequent cause of infectious uveitis worldwide. Disease can be especially severe in immunocompromised persons and in those infected congenitally (28).There is no perfect treatment for T. gondii infection in humans, as the few available medicines are limited by their side effects and target only the rapidly proliferating tachyzoite form of the parasite. Pyrimethamine and sulfadiazine, which are effective against the tachyzoite form, are currently used to treat active disease. However, treatment with these medicines can be associated with toxicity and hypersensitivity (29), and they do not eradicate the bradyzoite form of the parasite, which remains latent. There are few secondary medicines, and some of them have a delayed mechanism of killing the tachyzoites. No medicines have been reported to be effective against the latent, encysted bradyzoite stage. T. gondii remains in a person's body throughout life, leading to a risk for recurrence of active infection. Novel, effective, and nontoxic antiToxoplasma agents are urgently needed. Herein, we present a series of experiments to identify new lead compounds effective against T. gondii and to begin to understand how they act on this parasite. MATERIALS AND METHODSParasites and cell culture. Confluent monolayers of human foreskin fibroblasts (HFF) were maintained in Iscove's modified Dulbecco's medium supplemented with 10% fetal bovine serum, 1% Glutamax, and 1% penicillin-streptomycin-amp...

The protozoan parasite responsible for malaria affects over 500 million people each year. Current antimalarials have experienced decreased efficacy due to the development of drug-resistant strains of Plasmodium spp., resulting in a critical need for the discovery of new antimalarials. Hemozoin, a crystalline by-product of heme detoxification that is necessary for parasite survival, serves as an important drug target. The quinoline antimalarials, including amodiaquine and chloroquine, act by inhibiting the formation of hemozoin. The formation of this crystal does not occur spontaneously, and recent evidence suggests crystallization occurs in the presence of neutral lipid particles located in the acidic digestive vacuole of the parasite. To mimic these conditions, the lipophilic detergent NP-40 has previously been shown to successfully mediate the formation of ␤-hematin, synthetic hemozoin. Here, an NP-40 detergent-based assay was successfully adapted for use as a high-throughput screen to identify inhibitors of ␤-hematin formation. The resulting assay exhibited a favorable Z of 0.82 and maximal drift of less than 4%. The assay was used in a pilot screen of 38,400 diverse compounds at a screening concentration of 19.3 M, resulting in the identification of 161 previously unreported ␤-hematin inhibitors. Of these, 48 also exhibited >90% inhibition of parasitemia in a Plasmodium falciparum whole-cell assay at a screening concentration of 23 M. Eight of these compounds were identified to have nanomolar 50% inhibitory concentration values near that of chloroquine in this assay.

Toxoplasma gondii(T. gondii) is an apicomplexan parasite that can cause eye disease, brain disease, and death, especially in congenitally infected and immune-compromised people. Novel medicines effective against both active and latent forms of the parasite are greatly needed. The current study focused on the discovery of such medicines by exploring a family of potential inhibitors whose anti-apicomplexan activity has not been previously reported. Initial screening efforts revealed that niclosamide, a drug approved for anthelmintic use, possessed promising activity in vitro against T. gondii. This observation inspired the evaluation of the activity of a series of salicylanilides and derivatives. Several inhibitors with activities in the nanomolar range with no appreciable in vitro toxicity to human cells were identified. An initial structure-activity relationship was explored. Four compounds were selected for evaluation in an in vivo model of infection, and two derivatives with potentially enhanced pharmacological parameters demonstrated the best activity profiles.

Through our focused effort to discover new and effective agents against toxoplasmosis, a structure-based drug design approach was utilized to develop a series of potent inhibitors of the enoyl-acyl carrier protein (ACP) reductase (ENR) enzyme in Toxoplasma gondii (TgENR). Modifications to positions 5 and 4′ of the well-known ENR inhibitor triclosan afforded a series of 29 new analogs. Among the resulting compounds, many showed high potency and improved physicochemical properties in comparison with the lead. The most potent compounds 16a and 16c have IC50 values of 250 nM against Toxoplasma gondii tachyzoites without apparent toxicity to the host cells. Their IC50 values against the recombinant TgENR were 43 and 26 nM, respectively. Additionally, 11 other analogs in this series had IC50 values ranging from 17 to 130 nM in the enzyme-based assay. With respect to their excellent in vitro activity as well as improved drug-like properties, the lead compounds 16a and 16c are deemed to be an excellent starting point for the development of new medicines to effectively treat Toxoplasma gondii infections.

Triclosan is a potent inhibitor of Toxoplasma gondii enoyl reductase (TgENR), which is an essential enzyme for parasite survival. In view of triclosan’s poor druggability, which limits its therapeutic use, a new set of B-ring modified analogs were designed to optimize its physico-chemical properties. These derivatives were synthesized and evaluated by in vitro assay and TgENR enzyme assay. Some analogs display improved solubility, permeability and a comparable MIC50 value to that of triclosan. Modeling of these inhibitors revealed the same overall binding mode with the enzyme as triclosan, but the Bring modifications have additional interactions with the strongly conserved Asn130.