Effectiveness of Two New Endochin-like Quinolones, ELQ-596 and ELQ-650, in Experimental Mouse Models of Human Babesiosis

Abstract

Endochin-like quinolones (ELQs) define a class of small molecule antimicrobials that target the mitochondrial electron transport chain of various human parasites by inhibiting their cytochrome bc₁ complexes. The compounds have shown potent activity against a wide range of protozoan parasites, including the intraerythrocytic parasites Plasmodium and Babesia, the agents of human malaria and babesiosis, respectively. First-generation ELQ compounds were previously found to reduce infection by Babesia microti and Babesia duncani in animal models of human babesiosis but achieved a radical cure only in combination with atovaquone and required further optimization to address pharmacological limitations. Here, we report the identification of two second-generation 3-biaryl ELQ compounds, ELQ-596 and ELQ-650, with potent antibabesial activity in vitro and favorable pharmacological properties. In particular, ELQ-598, a prodrug of ELQ-596, demonstrated high efficacy as an orally administered monotherapy at 10 mg/kg. The compound achieved radical cure in both the chronic model of B. microti-induced babesiosis in immunocompromised mice and the lethal infection model induced by B. duncani in immunocompetent mice. Given its high potency, favorable physicochemical properties, and low toxicity profile, ELQ-596 represents a promising drug for the treatment of human babesiosis.

Keywords: B. duncani; B. microti; Babesia; ELQ-596; endochin-like quinolones; human babesiosis; parasite.

Figure 1.. In vitro efficacy of active ELQs and corresponding prodrugs against B. duncani parasites.

(A) Chemical structures of prodrug ELQ-598 and corresponding active drug ELQ-596 used in this study. (B) Parasite growth inhibition curve of prodrug ELQ-598. (C) Parasite growth inhibition curve of active drug ELQ-596. (D) Chemical structures of prodrug ELQ-672 and corresponding active drug ELQ-650 used in this study. (E) Parasite growth inhibition curve of prodrug ELQ-672. (F) Parasite growth inhibition curve of active drug ELQ-650. IC₅₀ values for each compound were determined using the SYBR Green assay. The sigmoidal dose-response curves were plotted using GraphPad Prism and IC₅₀s are represented with mean (± SD) values. Each value in the curve is the average of two different experiments with biological triplicates.

Figure 2.. Efficacy of ELQ-598 and ELQ-672 monotherapy against B. duncani lethal infection mouse model.

(A-D) Depicted are parasitemia graphs from female C3H/HeJ mice which were intravenously infected with 10³ B. duncani-infected red blood cells. All animals were treated daily (DPI 3 – DPI 7) by oral gavage. Blood smears were made on stipulated days from each mouse and parasitemia was calculated by microscopic examination of the giemsa stained smears. Parasitemia curves with connecting lines from mice treated with the vehicle alone (PEG-400) (red) (a), ELQ-598 at 10 mg/kg (blue) (B), and ELQ-672 at 10 mg/kg (green) (C) are generated using GraphPad Prism. Survival rate of B. duncani-infected mice in the absence or following treatment with ELQ-598 or ELQ-672 derived using the Kaplan-Meier method and represented (D). ☨ indicates when an individual mouse was euthanized.

Figure 3.. Efficacy of ELQ-598 and ELQ-672 against B. microti-infected SCID mice.

In vivo efficacy data in B. microti-infected CB17-SCID mice following treatment with vehicle (PEG-400) alone or ELQ-598 or ELQ-672 dosed orally at a concentration of 10 mg/kg daily for 5 days. Parasitemia curves with connecting lines from the indicated times from the mice group infected with 10⁴ B. microti-iRBCs and subsequently treated with vehicle (red) (A), or ELQ-598 10mg/kg (blue) (B), or ELQ-672 10mg/kg (green) (C) are represented.

Figure 4.. Atovaquone + ELQ-672 combination displayed synergy in vitro and achieve cure in babesiosis mouse model.

(A-B) B. duncani parasites cultured in vitro in human red blood cells were treated with ten different concentrations of each drug by fixed ratio method. The isobologram presents the FIC₅₀ value for each combination as well as the mean FIC (∑FIC) values of atovaquone and ELQs active and prodrugs. A) Isobolograms with FIC₅₀ values for the combination of atovaquone+ELQ-596 (black) and atovaquone+ELQ-598 (brown) with an additive line and calculated mean ∑FIC values of 0.8 and 0.9, respectively. B) The FIC₅₀ values for the atovaquone+ELQ-650 (black) and atovaquone+ELQ-672 (aqua blue) combinations with mean ∑FIC values of 1 and 0.5, respectively. Each data point represents the average of two independent experiments with three technical triplicates. (C-D) In vivo efficacy of the atovaquone+ELQ combinations in mice. Percent parasitemia curves obtained from the atovaquone+ELQ-598 (brown) and atovaquone+ELQ-672 (aqua blue) prodrugs combination treatment (daily oral dose of 10+10 mg/kg for 5 days) in SCID mice (female, n=5) infected with B. microti at a dose of 10⁴ infected erythrocytes per inoculum. (E-F) In vivo efficacy of atovaquone+ELQ-598 (brown) and atovaquone+ELQ-672 (aqua blue) combination treatment (daily oral dose of 10+10 mg/kg for 5 days) in C3H/HeJ mice with 1×10³ of B. duncani-infected erythrocyte infection. Parasitemia from the vehicle (PEG-400)-treated C3H or SCID mice after infection is represented with red-colored connected lines. Parasitemia (%) was calculated by microscopic analysis of Giemsa-stained blood smear samples collected at different time points following infection and treatment (a minimum of 3000 red blood cells were counted per blood smear). (G) Survival (%) of B. duncani-infected C3H/HeJ mice represented in colors consistent with the foregoing. Survival rates were calculated using the Kaplan-Meier method.