Two old drugs, NVP-AEW541 and GSK-J4, repurposed against the Toxoplasma gondii RH strain

Abstract

Background Toxoplasma gondii is a zoonotic pathogen that causes toxoplasmosis and leads to serious public health problems in developing countries. However, current clinical therapeutic drugs have some disadvantages, such as serious side effects, a long course of treatment and the emergence of drug-resistant strains. The urgent need to identify novel anti-Toxoplasma drugs has initiated the effective strategy of repurposing well-characterized drugs. As a principled screening for the identification of effective compounds against Toxoplasma gondii, in the current study, a collection of 666 compounds were screened for their ability to significantly inhibit Toxoplasma growth. Methods The inhibition of parasite growth was determined using a luminescence-based β-galactosidase activity assay. Meanwhile, the effect of compounds on the viability of host cells was measured using CCK8. To assess the inhibition of the selected compounds on discrete steps of the T. gondii lytic cycle, the invasion, intracellular proliferation and egress abilities were evaluated. Finally, a murine infection model of toxoplasmosis was used to monitor the protective efficacy of drugs against acute infection of a highly virulent RH strain. Results A total of 68 compounds demonstrated more than 70% parasite growth inhibition. After excluding compounds that impaired host cell viability, we further characterized two compounds, NVP-AEW541 and GSK-J4 HCl, which had IC50 values for parasite growth of 1.17 μM and 2.37 μM, respectively. In addition, both compounds showed low toxicity to the host cell. Furthermore, we demonstrated that NVP-AEW541 inhibits tachyzoite invasion, while GSK-J4 HCl inhibits intracellular tachyzoite proliferation by halting cell cycle progression from G1 to S phase. These findings prompted us to analyse the efficacy of the two compounds in vivo by using established mouse models of acute toxoplasmosis. In addition to prolonging the survival time of mice acutely infected with T. gondii, both compounds had a remarkable ability to reduce the parasite burden of tissues. Conclusions Our findings suggest that both NVP-AEW541 and GSK-J4 could be potentially repurposed as candidate drugs against T. gondii infection.

Systematic Analysis of Clemastine, a Candidate Apicomplexan Parasite-Selective Tubulin-Targeting Agent

“…To assess this, we gauged the therapeutic index by measuring compound EC 50 for Toxoplasma tachyzoites grown in vertebrate host cells versus the CC 50 for uninfected human foreskin fibroblast (HFF) cells. Plaque assays were an established plate-based method to assess compound efficacy on Toxoplasma lytic growth [ 14 , 25 , 26 , 27 , 28 ]. Importantly, this method captures effects of drugs that act early in replication (i.e., dinitroanilines), as well as drugs that have a delayed or cumulative effect that requires several rounds of host-cell invasion and replication before becoming apparent (i.e., clindamycin).…”

Toxoplasma TgAtg8-TgAtg3 Interaction Primarily Contributes to Apicoplast Inheritance and Parasite Growth in Tachyzoite

“…Standard plaque assays were performed using confluence HFFs in 24-well plates, infected with 100 tachyzoites treated with or without ATc for 7 days. The HFFs were fixed and stained as described previously ( 48 ). The plates were scanned, and the areas of plaques were analyzed using ImageJ.…”

Investigation of Antiparasitic Activity of Two Marine Natural Products, Estradiol Benzoate, and Octyl Gallate, on Toxoplasma gondii In Vitro

“…To determine inhibition of parasite growth, β-Gal activity assay was performed in the preliminary screening as described previously ( Liu et al, 2020 ). In brief, each compound was diluted fresh by using DMEM without phenol red to a final concentration of 10 μM and added to confluent HFFs in a 96-well half-area plate.…”

“…The plaque assay was performed as described previously ( Liu et al, 2020 ). Monolayers of HFF in 24-well plates containing 5 μM of either estradiol benzoate or octyl gallate in triplicate were infected with freshly released 100 tachyzoites.…”