Validation of Babesia proteasome as a drug target

Abstract

Babesiosis is a tick-transmitted zoonosis caused by apicomplexan parasites of the genus Babesia. Treatment of this emerging malaria-related disease has relied on antimalarial drugs and antibiotics. The proteasome of Plasmodium, the causative agent of malaria, has recently been validated as a target for anti-malarial drug development and therefore, in this study, we investigated the effect of epoxyketone (carfilzomib, ONX-0914 and epoxomicin) and boronic acid (bortezomib and ixazomib) proteasome inhibitors on the growth and survival of Babesia. Testing the compounds against Babesia divergens ex vivo revealed suppressive effects on parasite growth with activity that was higher than the cytotoxic effects on a non-transformed mouse macrophage cell line. Furthermore, we showed that the most-effective compound, carfilzomib, significantly reduces parasite multiplication in a Babesia microti infected mouse model without noticeable adverse effects. In addition, treatment with carfilzomib lead to an ex vivo and in vivo decrease in proteasome activity and accumulation of polyubiquitinated proteins compared to untreated control. Overall, our results demonstrate that the Babesia proteasome is a valid target for drug development and warrants the design of potent and selective B. divergens proteasome inhibitors for the treatment of babesiosis.

Keywords: Babesia; Carfilzomib; Cytotoxicity; Epoxyketone; Proteasome.

Graphical abstract

Fig. 1

Treatment of B. divergens ex vivo cultures: red blood cells infection. (A) Microscopy image of B. divergens ex vivo culture treated with 100 nM carfilzomib. (B) Microscopy image of B. divergens ex vivo culture treated with 0.008% DMSO. Smears were stained using DiffQuik staining set. Experimental conditions: starting parasitemia 2%, medium changed in 12 h intervals, total cultivation 48 h. Green dots indicate the parasitized red blood cells. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Treatment of B. divergens ex vivo cultures with peptide epoxyketone inhibitors. Structure and IC₅₀ determination of (A) carfilzomib, (B) epoxomicin, (C) ONX-0914. Data represent means of three independent biological replicates and the error bars indicate standard deviations. Smears were stained using DiffQuik staining set, parasitemia was counted at 1000 RBCs. Statistical analysis was performed in R using ANOVA (Kolmogorov-Smirnov test and the Bartlett test passed): * = p < 0.05, *** = p < 0.001 (compared to the DMSO treated culture). Experimental conditions: starting parasitemia 2% (dotted line), medium with inhibitory compounds exchanged in 12 h intervals, total cultivation duration 48 h. DC: DMSO treated culture (0.008%). UC: untreated culture. IC₅₀: half maximal inhibitory concentration. RBCs: red blood cells.

Fig. 3

Treatment of B. divergens ex vivo cultures with peptide boronic acid inhibitors. Structure and IC₅₀ determination of (A) bortezomib and (B) ixazomib. Results represent means of three independent biological replicates, error bars indicate standard deviations. Smears were stained using DiffQuik staining set, parasitemia was counted at 1000 RBCs. Statistical analysis was performed in R using ANOVA (Kolmogorov Smirnov test and the Bartlett test passed): * = p < 0.05, *** = p < 0.001 (compared to the DMSO treated culture). Experimental conditions: starting parasitemia 2% (dotted line), medium with inhibitory compounds exchanged in 12 h intervals, total cultivation duration 48 h. DC: DMSO treated culture (0.008%). UC: untreated culture. IC₅₀: half maximal inhibitory concentration. RBCs: red blood cells.

Fig. 4

Detection and inhibition of B. divergens proteasome β5 subunit activity. IC₅₀ values of all tested proteasome inhibitors on B. divergens crude cell lysates. Parasite lysates were obtained from ex vivo cultures and analysed using the fluorogenic peptidyl substrate, Suc-LLVY-AMC. Results represent the means of three independent replicates, error bars indicate standard deviations. IC₅₀ values were analysed using GraphPad Prism software (version 5).

Fig. 5

Cell viability assay: carfilzomib treatment of macrophage cell line. (A) Results represent means of three biological replicates for (i) carfilzomib (200 nM) and (ii) control (medium + 0.004% DMSO) experiments; PMJ2R cells (medium changed every 12 h, total cultivation 48 h). Error bars indicate standard deviation. Statistical analysis was performed in R using unpaired t-test (Kolmogorov-Smirnov test and the Bartlett test passed). (B) Dot plot results of flow cytometry analysis of carfilzomib treated cells: dead cells (red color) stained by EthD (measured in PE channel, emission 620 nm) and live cells (green color) stained by Calcein (FITC channel, emission 517 nm). DC: 0.004% DMSO treatment control. EthD: ethidium homodimer. PE: phycoerythrin. FITC: fluorescein isothiocyanate. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Treatment of B. microti infected mice. (A) The growth curve of B. microti in mice treated with either carfilzomib (0.75 mg/kg) or PBS (control). Treatment (intraperitoneal application) was performed daily from 1 DPI up to 7 DPI (blue line in the graph). (B) Growth curve of B. microti pre-treated with either 400 nM carfilzomib for 1 h or vehicle control prior to injection into mice. Respective values of AUC (Area Under Curve) and parasitemia values were compared using the two-sample (independent) t-test; AUC and parasitemia values differed significantly: * = p < 0.05, ** = p < 0.01. DPI: days post infection. (C) Inhibition of proteasome chymotrypsin-like activity of B. microti isolated from mice on 7 DPI. The infected animals were treated with carfilzomib at 5 and 6 DPI. Activity was detected in crude cell lysates using Suc-LLVY-AMC fluorescent peptidyl substrate using equal amounts of parasite proteins (visualized at coomassie gel, Fig. 6D). Results represent the means of technical triplicates, error bars indicate standard deviations. Respective values were compared using the two sample t-test: ** = p < 0.01. (D) Detection of poly-ubiquitinated proteins in in vivo inhibited B. microti proteasome on 7 DPI in B. microti crude cell lysate obtained from mice previously treated on 5 and 6 DPI. Coomassie stained SDS PAGE visualizes separated protein loads of treated and untreated parasite lysates prior electro-blotting to PVDF membrane. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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