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.

ObjectivesDrugs for Neglected Diseases initiative (DNDi) has identified three chemical lead series, the nitroimidazoles, benzoxaboroles and aminopyrazoles, as innovative treatments for visceral leishmaniasis. The leads discovered using phenotypic screening, were optimised following disease- and compound-specific criteria. Several leads of each series were progressed and preclinical drug candidates have been nominated. Here we evaluate the efficacy of the lead compounds of each of these three chemical classes in in vitro and in vivo models of cutaneous leishmaniasis.MethodsThe in vitro activity of fifty-five compounds was evaluated against the intracellular amastigotes of L. major, L. aethiopica, L. amazonensis, L. panamensis, L. mexicana and L. tropica. The drugs demonstrating potent activity (EC50 < 5 μM) against at least 4 of 6 species were subsequently evaluated in vivo in different L. major – BALB/c mouse models using a 5 or 10-day treatment with either the oral or topical formulations. Efficacy was expressed as lesion size (measured daily using callipers), parasite load (by quantitative PCR – DNA) and bioluminescence signal reduction relative to the untreated controls.ResultsThe selected drug compounds (3 nitroimidazoles, 1 benzoxaborole and 3 aminopyrazoles) showed consistent and potent activity across a range of Leishmania species that are known to cause CL with EC50 values ranging from 0.29 to 18.3 μM. In all cases, this potent in vitro antileishmanial activity translated into high levels of efficacy with a linear dose-response against murine CL. When administered at 50 mg/kg/day, DNDI-0690 (nitroimidazole), DNDI-1047 (aminopyrazole) and DNDI-6148 (benzoxaborole) all resulted in a significant lesion size reduction (no visible nodule) and an approximate 2-log-fold reduction of the parasite load as measured by qPCR compared to the untreated control.ConclusionsThe lead compounds DNDI-0690, DNDI-1047 and DNDI-6148 showed excellent activity across a range of Leishmania species in vitro and against L. major in mice. These compounds offer novel potential drugs for the treatment of CL.

Tropical protozoal infections are a significant cause of morbidity and mortality worldwide; four in particular (human African trypanosomiasis (HAT), Chagas disease, cutaneous leishmaniasis, and malaria) have an estimated combined burden of over 87 million disability-adjusted life years. New drugs are needed for each of these diseases. Building on the previous identification of NEU-617 (1) as a potent and nontoxic inhibitor of proliferation for the HAT pathogen (Trypanosoma brucei), we have now tested this class of analogs against other protozoal species: T. cruzi (Chagas disease), Leishmania major (cutaneous leishmaniasis), and Plasmodium falciparum (malaria). Based on hits identified in this screening campaign, we describe the preparation of several replacements for the quinazoline scaffold and report these inhibitors’ biological activities against these parasites. In doing this, we have identified several potent proliferation inhibitors for each pathogen, such as 4-((3-chloro-4-((3-fluorobenzyl)oxy)phenyl)amino)-6-(4-((4-methyl-1,4-diazepan-1-yl)sulfonyl)phenyl)quinoline-3-carbonitrile (NEU-924, 83) for T. cruzi and N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-7-(4-((4-methyl-1,4-diazepan-1-yl)sulfonyl)phenyl)cinnolin-4-amine (NEU-1017, 68) for L. major and P. falciparum.

Cutaneous leishmaniasis remains ignored in therapeutic drug discovery programs worldwide. This is mainly because cutaneous leishmaniasis is frequently a disease of impoverished populations in countries where funds are limited for research and patient care. However, the health burden of individuals in endemic areas mandates readily available, effective, and safe treatments. Of the existing cutaneous leishmaniasis therapeutics, many are growth inhibitory to Leishmania parasites, potentially creating dormant parasite reservoirs that can be activated when host immunity is compromised, enabling the reemergence of cutaneous leishmaniasis lesions or worse spread of Leishmania parasites to other body sites. To accelerate the identification and development of novel cutaneous leishmaniasis therapeutics, we designed an integrated in vitro and in vivo screening platform that incorporated multiple Leishmania life cycles and species and probed a focused library of pharmaceutically active compounds. The objective of this phenotypic drug discovery platform was the identification and prioritization of bona fide cytotoxic chemotypes toward Leishmania parasites. We identified the Food and Drug Administration-approved drug auranofin, a known inhibitor of Leishmania promastigote growth, as a potent cytotoxic anti-leishmanial agent and inducer of apoptotic-like death in promastigotes. Significantly, the anti-leishmanial activity of auranofin transferred to cell-based amastigote assays as well as in vivo murine models. With appropriate future investigation, these data may provide the foundation for potential exploitation of gold(I)-based complexes as chemical tools or the basis of therapeutics for leishmaniasis. Thus, auranofin may represent a prototype drug that can be used to identify signaling pathways within the parasite and host cell critical for parasite growth and survival.

Hesperadin, an established human Aurora B inhibitor, was tested against cultures of Trypanosoma brucei, Leishmania major, and Plasmodium falciparum, and was identified to be a potent proliferation inhibitor. A series of analogs was designed and tested to establish the initial structure-activity relationships for each parasite. In this study, we identified multiple non-toxic compounds with high potency against T. brucei and P. falciparum with good selectivity. These compounds may represent an opportunity for continued optimization.

A kinase-targeting cell-based high-throughput screen (HTS) against Trypanosoma brucei was recently reported, and this screening set included the Published Kinase Inhibitor Set (PKIS). From the PKIS was identified 53 compounds with pEC50 ≥ 6. Utilizing the published data available for the PKIS, a statistical analysis of these active antiparasitic compounds was performed, allowing identification of a set of human kinases having inhibitors that show a high likelihood for blocking T. brucei cellular proliferation in vitro. This observation was confirmed by testing other established inhibitors of these human kinases and by mining past screening campaigns at GlaxoSmithKline. Overall, although the parasite targets of action are not known, inhibitors of this set of human kinases displayed an enhanced hit rate relative to a random kinase-targeting HTS campaign, suggesting that repurposing efforts should focus primarily on inhibitors of these specific human kinases. We therefore term this statistical analysis-driven approach “preferred lead repurposing”.

Malaria remains one of the deadliest diseases in the world today. Novel chemoprophylactic and chemotherapeutic antimalarials are needed to support the renewed eradication agenda. We have discovered a novel antimalarial acridone chemotype with dual stage activity against both liver stage and blood stage malaria. Several lead compounds generated from structural optimization of a large library of novel acridones exhibit efficacy in the following systems: 1) Picomolar inhibition of in vitro Plasmodium falciparum blood stage growth against multi-drug resistant parasites; 2) Curative efficacy after oral administration in erythrocytic P. yoelii murine malaria model; 3) Prevention of in vitro P. berghei sporozoite-induced development in human hepatocytes; and 4) Protection of in vivo P. berghei sporozoite-induced infection in mice. This study offers the first account of liver stage antimalarial activity in acridone chemotype. Details of the design, chemistry, structure-activity relationships, safety, metabolic/pharmacokinetic studies, and mechanistic investigation are presented herein.