Novel N -Benzoyl-2-Hydroxybenzamide Disrupts Unique Parasite Secretory Pathway

Abstract

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...

Molecular target validation, antimicrobial delivery, and potential treatment of Toxoplasma gondii infections

“…The principles of design and methods used previously were as described previously (1,9) and are presented in detail in SI Methods. Specific PPMO were designed to inhibit gene products of YFP (agctagatTCTAAAATGGTGAGCAAGGGCGAGc; PPMO sequence is bolded; starting codon is underlined; sequences before and after target genes, which are not included in PPMO designs, are indicted in lowercase letters); luciferase (gatggctGTCATGGAAGACGCCAAAAACATAaagaaa); DHFR (ctggGAAGATGCAGAAACCGGTGTCTGgtcgtc); ENR (aaatcgAAAATGGTTGGTTTCAAACTCCTCaccctc); AP2XI-3 (cgcgTCTGTTCCGTGCCGCGATGGAGTcgga).…”

A role for adaptor protein complex 1 in protein targeting to rhoptry organelles in Plasmodium falciparum

“…In fact, a recent screen for novel chemotherapeutic agents against Apicomplexa revealed that an inhibitor resulted in the complete absence of the lytic vacuole in T. gondii, which could be partially mitigated by mutations in AP-3β, suggesting that AP-3 may play a role in trafficking to this organelle [38]. These data have implications for post-Golgi trafficking, and specifically adaptor protein function, in organisms possessing multiple lysosome-related organelles.…”

Spiroindolone That Inhibits PfATPase4 Is a Potent, Cidal Inhibitor of Toxoplasma gondii Tachyzoites In Vitro and In Vivo

“…Once the most effective spiroindolone is identified, the mode of solubilization and administration can be optimized. A variety of models are available for such future studies (1,4,7,12,13).…”

“…ovel, improved medicines are needed to treat toxoplasmosis (1,2,3). One attractive approach to treat toxoplasmosis is to repurpose compounds effective against other, related apicomplexan parasites (4).…”

“…1A) was synthesized as described previously (5) by Reagents 4 Research. In vitro and in vivo assays were conducted as recently reported (1,6,7), with every experiment repeated at least twice (see the supplemental material). The significance of differences was determined using Student's t test, with a P value of Ͻ0.05 considered significant.…”