Novel Toxoplasma gondii inhibitor chemotypes

Abstract

We profiled three novel T. gondii inhibitors identified from an antimalarial phenotypic high throughput screen (HTS) campaign: styryl 4-oxo-1,3-benzoxazin-4-one KG3, tetrahydrobenzo[b]pyran KG7, and benzoquinone hydrazone KG8. These compounds inhibit T. gondii in vitro with IC₅₀ values ranging from 0.3 to 2μM, comparable to that of 0.25 to 1.5μM for the control drug pyrimethamine. KG3 had no measurable cytotoxicity against five mammalian cell lines, whereas KG7 and KG8 inhibited the growth of 2 of 5 cell lines with KG8 being the least selective for T. gondii. None of the compounds were mutagenic in an Ames assay. Experimental gLogD_7.4 and calculated PSA values for the three compounds were well within the ranges predicted to be favorable for good ADME, even though each compound had relatively low aqueous solubility. All three compounds were metabolically unstable, especially KG3 and KG7. Multiple IP doses of 5mg/kg KG7 and KG8 increased survival in a T. gondii mouse model. Despite their liabilities, we suggest that these compounds are useful starting points for chemical prospecting, scaffold-hopping, and optimization.

Keywords: Anti-parasitics; Drug discovery; Lead compounds; Plasmodium falciparum; Toxoplasma gondii.

Figure 1. Chemical structures

Styryl 4-oxo-1,3-benzoxazin-4-one (KG3), Tetrahydrobenzo[b]pyran (KG7), and Benzoquinone hydrazone (KG8).

Figure 2. Host cell pre-treatment and extracellular tachyzoite exposure results in reduced growth

HFF cells were pretreated with the indicated compound at 10 μM for 24 h (pre-treatment). Cells were then washed with media and infected with 2,000 RH-dTom tachyzoites. Isolated RH-dTom tachyzoites were also treated with respective compound at 10 μM for 4 h (post-treatment) and were then allowed to infect HFF cells in a 96 well plate at 2,000 tachyzoites/mL. Fluorescent plate readings were taken at day 4 post-infection for both experiments. p value <0.05(*); p value <0.001(**)

Figure 3. In vivo Survival Following Compound Treatment

CFW mice were treated with compounds at respective amounts twice per day for 10 d. Survival was quantified over 30 d. Survival was plotted as a Kaplan-Meier curve with a calculated p value <0.05 (*)