Novel inhibitor of Plasmodium histone deacetylase that cures P. berghei-infected mice

Abstract

Histone deacetylases (HDAC) are potential targets for the development of new antimalarial drugs. The growth of Plasmodium falciparum and other apicomplexans can be suppressed in the presence of potent HDAC inhibitors in vitro and in vivo; however, in vivo parasite suppression is generally incomplete or reversible after the discontinuation of drug treatment. Furthermore, most established HDAC inhibitors concurrently show broad toxicities against parasites and human cells and high drug concentrations are required for effective antimalarial activity. Here, we report on HDAC inhibitors that are potent against P. falciparum at subnanomolar concentrations and that have high selectivities; the lead compounds have mean 50% inhibitory concentrations for the killing of the malaria parasite up to 950 times lower than those for the killing of mammalian cells. These potential drugs improved survival and completely and irreversibly suppressed parasitemia in P. berghei-infected mice.

FIG. 1.

Inhibition of PfHDAC activity. Trophozoite- and schizont-containing crude cell lysates were the sources of PfHDAC activity. Parasite lysates corresponding to 10⁶ parasites were mixed with the indicated concentrations of YC-II-88 or SAHA and used to measure the anti-PfHDAC activity by using a fluorimetric HDAC activity assay kit from BioMol, and the manufacturer's protocol was used, with slight modifications (14). TSA (1 μM) and Me₂SO were included as negative and positive controls, respectively. Assays were performed twice in triplicate. The standard errors of the means are presented as bars.

FIG. 2.

Screening of HDAC inhibitors for their antimalarial activities. Antimalarial activities were screened by a GIA (24), with modifications, by using P. falciparum strain 3D7. The levels of parasitemia were determined by FACS and were used to calculate the percent parasite growth inhibition. Assays were performed twice in triplicate, and the averages and the standard errors of the means are presented. Similar results were obtained with strain FCB1 (data not shown).

FIG. 3.

HDAC inhibitor-induced parasite inhibition is irreversible and is most significant in mature parasite stages. (a) A synchronized P. falciparum 3D7 culture was treated with the minimum concentrations of YC-II-88 (2.5 nM) and K.2 (1,000 nM) found to completely inhibit P. falciparum 3D7 continuously over 4 days (solid lines) or from 10 to 34 h postinvasion (dashed line). Parasite growth was assessed every 12 h for 4 days by FACS, and the degree of growth inhibition was compared to that for the controls (parasites treated with Me₂SO). The data are representative of those from three separate experiments. (b) Rings (1 to 16 h postinvasion), trophozoites (16 to 30 h), and schizonts (32 to 48 h) were treated with 1.25 nM YC-II-88 (the IC₅₀ for P. falciparum 3D7) or Me₂SO for 14 h. The assay was performed twice in triplicate. The standard errors of the means are presented as bars.

FIG. 4.

HDAC inhibitors irreversibly inhibit Plasmodium in vivo and increase the rate of survival. Five groups of mice (five mice per group) were infected with 10⁶ P. berghei cells intraperitoneally and were treated simultaneously with the indicated doses of YC-II-88 divided into two daily doses for 4 days. The percentage of parasite growth inhibition was plotted against the drug concentration (a). The mice were monitored for a total of 6 weeks or until they died; and the levels of parasitemia (b), hematocrit levels (c), and rates of survival (d) are shown. The standard errors of the means are presented as bars in panels a, b, and c. CTRL, control.