Activity Comparison of Epigenetic Modulators against the Hemoprotozoan Parasites Babesia divergens and Plasmodium falciparum

Abstract

Babesiosis is a tick-borne parasitic disease of humans and livestock that has dramatically increased in frequency and geographical range over the past few decades. Infection of cattle often causes large economic losses, and human infection can be fatal in immunocompromised patients. Unlike for malaria, another disease caused by hemoprotozoan parasites, limited treatment options exist for Babesia infections. As epigenetic regulation is a promising target for new antiparasitic drugs, we screened 324 epigenetic inhibitors against Babesia divergens blood stages and identified 75 (23%) and 17 (5%) compounds that displayed ≥90% inhibition at 10 and 1 μM, respectively, including over a dozen compounds with activity in the low nanomolar range. We observed differential activity of some inhibitor classes against Babesia divergens and Plasmodium falciparum parasites and identified pairs of compounds with a high difference in activity despite a high similarity in chemical structure, highlighting new insights into the development of epigenetic inhibitors as antiparasitic drugs.

Keywords: Babesia; Plasmodium; epigenetic; parasites; small molecule screen.

Figure 1.

Evolutionary conservation of epigenetic modifying enzymes in piroplasmid parasites. Comparison of epigenetic modifying enzyme orthologues among P. falciparum, B. microti, T. equi, T. annulata, C. felix, B. bovis, and B. microti with a representation of the evolutionary relationship among these species.

Figure 2.

Activity of epigenetic inhibitors against Babesia divergens. (A) 125 compounds with ≥50% inhibition at 10 μM. Heat map of mean percent inhibition at 10 and 1 μM compared to solvent-treated controls (n = 3). Compounds are grouped based on the reported epigenetic process affected in higher eukaryotes: Histone deacetylation (HDAC), histone acetylation (HAT), histone methylation (KMT), histone demethylases (HDM), DNA methylation (DNMT), and “Other”. (B) Dose response analysis for 17 compounds with submicromolar EC50 values (n = 2) with corresponding HepG2 inhibition at 1 μM.

Figure 3.

Differential activity of epigenetic inhibitors against B. divergens and P. falciparum. (A) Scatterplot comparing percent inhibition at 1 μM against B. divergens and P. falciparum. Compound names are indicated for compounds with more than twofold difference in activity (dotted lines) and more than 50% inhibition at 1 μM against one species (dashed lines). An enlarged scatterplot with labeled compound names is displayed for compounds with ≥75% at 1 μM against both species. (B) Heat map of compounds with at least 50% inhibition at 1 μM against one species, ordered by the delta activity (% Pf inhibition – % Bd inhibition) and grouped by proposed target category.

Figure 4.

Activity cliff analysis. (A) Scatterplot of 19 activity cliff pairs with >50% delta activity and >80% structural similarity, grouped by species. Compound pairs that display an activity cliff in both species are indicated in matching colors. (B, C) Examples of activity cliff pairs with respective chemical structures and in vitro activity at 1 μM.

Figure 5.

Structure–activity relationships of diaminoquinazoline KMT inhibitors. (A) Compound pairs with >60% similarity and >50% delta activity at 1 μM. (B) Structure–activity relationship against Babesia divergens. (C) Changes in chemical structure that confer differential activity against both species.