With roughly 2 billion people infected, the neurotropic protozoan Toxoplasma gondii remains one of the most pervasive and infectious parasites. Toxoplasma infection is the second leading cause of death due to foodborne illness in the United States, causes severe disease in immunocompromised patients, and is correlated with several cognitive and neurological disorders. Currently, no therapies exist that are capable of eliminating the persistent infection in the central nervous system (CNS). In this study we report the identification of triazine nitrile inhibitors of Toxoplasma cathepsin L (TgCPL) from a high throughput screen and their subsequent optimization. Through rational design, we improved inhibitor potency to as low as 5 nM, identified pharmacophore features that can be exploited for isoform selectivity (up to 7-fold for TgCPL versus human isoform), and improved metabolic stability (t 1/2 > 60 min in mouse liver microsomes) guided by a metabolite ID study. We demonstrated that this class of compounds is capable of crossing the blood-brain barrier in mice (1:1 brain/plasma at 2 h). Importantly, we also show for the first time that treatment of T. gondii bradyzoite cysts in vitro with triazine nitrile inhibitors reduces parasite viability with efficacy equivalent to a TgCPL genetic knockout.

The neurotropic protozoan Toxoplasma gondii is the 2nd leading cause of death due to foodborne illness in the US, and has been designated as one of five neglected parasitic infections by the Center for Disease Control and Prevention. Currently, no treatment options exist for the chronic dormant-phase Toxoplasma infection in the central nervous system (CNS). T. gondii cathepsin L (TgCPL) has recently been implicated as a novel viable target for the treatment of chronic toxoplasmosis. In this study, we report the first body of SAR work aimed at developing potent inhibitors of TgCPL with selectivity vs the human cathepsin L. Starting from a known inhibitor of human cathepsin L, and guided by structure-based design, we were able to modulate the selectivity for Toxoplasma vs human CPL by nearly 50-fold while modifying physiochemical properties to be more favorable for metabolic stability and CNS penetrance. The overall potency of our inhibitors towards TgCPL was improved from 2 μM to as low as 110 nM and we successfully demonstrated that an optimized analog 18b is capable of crossing the BBB (0.5 brain/plasma). This work is an important first step toward development of a CNS-penetrant probe to validate TgCPL as a feasible target for the treatment of chronic toxoplasmosis.

The cystine/glutamate antiporter system x c − (Sx c − ) mediates the exchange of intracellular l-glutamate (l-Glu) with extracellular l-cystine (l-Cys 2 ). Both the import of l-Cys 2 and the export of l-Glu take on added significance in CNS cells, especially astrocytes. When the relative activity of Sx c − overwhelms the regulatory capacity of the EAATs, the efflux of l-Glu through the antiporter can be significant enough to trigger excitotoxic pathology, as is thought to occur in glioblastoma. This has prompted considerable interest in the pharmacological specificity of Sx c − and the development of inhibitors. The present study explores a series of analogues that are structurally related to sulfasalazine, a widely employed inhibitor of Sx c − . We identify a number of novel aryl-substituted amino-naphthylsulfonate analogues that inhibit Sx c − more potently than sulfasalazine. Interestingly, the inhibitors switch from a competitive to noncompetitive mechanism with increased length and lipophilic substitutions, a structure-activity relationship that was previously observed with aryl-substituted isoxazole. These results suggest that the two classes of inhibitors may interact with some of the same domains on the antiporter protein and that the substrate and inhibitor binding sites may be in close proximity to one another. Molecular modeling is used to explore this possibility.

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