A small-molecule inhibitor of the DNA recombinase Rad51 from Plasmodium falciparum synergizes with the antimalarial drugs artemisinin and chloroquine

Abstract

Malaria parasites repair DNA double-strand breaks (DSBs) primarily through homologous recombination (HR). Here, because the unrepaired DSBs lead to the death of the unicellular parasite Plasmodium falciparum, we investigated its recombinase, PfRad51, as a potential drug target. Undertaking an in silico screening approach, we identified a compound, B02, that docks to the predicted tertiary structure of PfRad51 with high affinity. B02 inhibited a drug-sensitive P. falciparum strain (3D7) and multidrug-resistant parasite (Dd2) in culture, with IC₅₀ values of 8 and 3 μm, respectively. We found that B02 is more potent against these P. falciparum strains than against mammalian cell lines. Our findings also revealed that the antimalarial activity of B02 synergizes with those of two first-line malaria drugs, artemisinin (ART) and chloroquine (CQ), lowering the IC₅₀ values of ART and CQ by 15- and 8-fold, respectively. Our results also provide mechanistic insights into the anti-parasitic activity of B02, indicating that it blocks the ATPase and strand-exchange activities of PfRad51 and abrogates the formation of PfRad51 foci on damaged DNA at chromosomal sites, probably by blocking homomeric interactions of PfRad51 proteins. The B02-mediated PfRad51 disruption led to the accumulation of unrepaired parasitic DNA and rendered parasites more sensitive to DNA-damaging agents, including ART. Our findings provide a rationale for targeting the Plasmodium DSB repair pathway in combination with ART. We propose that identification of a specific inhibitor of HR in Plasmodium may enable investigations of HR's role in Plasmodium biology, including generation of antigenic diversity.

Keywords: B02; DNA repair; PfRad51; artemisinin; comet assay; drug screening; homologous recombination; malaria; plasmodium; small-molecule inhibitor.

Figure 1.

Docking of B02 onto the predicted tertiary structure of PfRad51. Front view (A) and rear view (B) of the homology model of PfRad51. C, docked conformation of B02 at the Walker site. D, docked conformation of B02 at the dimerization site.

Figure 2.

B02 binds to PfRad51 and inhibits its ATP hydrolysis activity. A, quenching of intrinsic fluorescence of Trp-170 of PfRad51 upon B02 binding. Stern-Volmer plots showing the ratio of intrinsic fluorescence (F₀) and quenched fluorescence (F₁) at different concentrations of ligand binding are shown. The excitation was at 295 nm, and the emission was recorded at 332 nm. Data are the mean ± S.D. from three experiments. B, ssDNA-dependent ATPase activity of PfRad51 in the presence of various concentrations of drugs (as indicated on the x axis) as indicated. C, at 200 μm ATP concentration and 60 μm ssDNA, the activity of 1 μm PfRad51 is inhibited by B02 with an IC₅₀ value of 8.48 μm. Mean and standard errors from three independent experiments are plotted. D, three-strand exchange activity of PfRad51 is inhibited by B02 with an IC₅₀ value of 7.96 μm. Mean and standard errors from three independent experiments are plotted. LD, linear dsDNA (substrate); NC, nicked circular DNA (product).

Figure 3.

B02 inhibits the repair of damaged Plasmodium DNA. A, MMS sensitivity of parasites treated with B02 as determined by return–to–growth assay. Growth of parasites that are neither treated with B02 nor with MMS represents 100% survival. Error bars indicate S.D. (n = 3 experiments); asterisks indicate values significantly different from the control, as follows: **, p < 0.01; NS, not significant. B, comet assay visualization of the persistence of damaged DNA upon MMS treatment in the presence or absence of B02 (8 μm). Control represents undamaged DNA. Shortening of the comet length indicates the repair of damaged DNA during recovery time. C, quantitative measurement of comet tail length at different time points ranging from pre-DNA damage to post-DNA damage. The average tail length of comets at each time point (n >50) was calculated. The mean of the averages from six independent experiments is plotted. Asterisks indicate values significantly different from the control, as follows: ***, p < 0.001; **, p < 0.01; NS, not significant.

Figure 4.

B02 inhibits the formation of PfRad51 foci upon DNA damage. A, IFA displays PfRad51 foci (FITC) upon treatment with the DNA-damaging agent MMS (3rd row). Such foci are not visible in the control cells (untreated with MMS) neither in the presence (2nd row) nor in the absence of 8 μm B02 (1st row). MMS-induced PfRad51 foci formation are inhibited in the presence of 8 μm B02 (4th row). DAPI staining indicates the location of the parasite nucleus. B, quantitative analysis of PfRad51 foci formation. Percent of focus formation is defined as the number of infected RBC having PfRad51 foci (FITC-stained) of 100 infected RBC (DAPI-stained). The mean and the standard deviations from three independent experiments are plotted. Two-tailed t test was performed to obtain the statistical significance. The p value is indicated at the top. C, self-interaction of PfRad51 is inhibited in the presence of 8 μm B02. pGADC1 and PGBDUC1 are the parent plasmids encoding the GAL4 activation domain and DNA-binding domain, respectively. DNA fragments corresponding to the full-length WT PfRAD51 ORF were fused to the GAL4 activation domain in pGADC1 and fused to the DNA-binding domain in pGBDUC1. Two-hybrid interactions were tested with yeast strain PJ694A, which bears the ADE2 gene as one of the reporters. Yeast cells harboring both plasmids were patched on control plates (SC–Ura–Leu) as well as experimental plates (SC–Ura–Leu–Ade) to test for protein–protein interactions in the absence or in the presence of 8 μm B02 (as indicated at the bottom).

Figure 5.

B02 inhibits intra-erythrocytic development of P. falciparum. A, synchronous trophozoite stage 3D7 cultures were grown for 48 h in the presence of various concentrations of B02 (as marked on the x axis). After 48 h, percent of parasitemia of different cultures was counted. The mean and standard deviations from three parallel experiments are plotted. B, inhibition of parasite growth at various concentrations of B02 is plotted to obtain the IC₅₀ value. Growth of P. falciparum 3D7 strain was monitored by the Giemsa staining method. Parasite growth in the absence of B02 is considered as zero inhibition. Mean and standard deviations from three independent experiments are plotted. C and D, inhibition of growth of P. falciparum 3D7 and Dd2 as measured by SYBR Green I method. C, inset, depicts the standard curve of parasitemia versus SYBR Green I fluorescence intensity.

Figure 6.

Synergy of DHA and CQ with recombinase inhibitor B02. A and B, isobologram of DHA–B02 combination in 3D7 and Dd2 strains, respectively. C and D, isobologram of CQ–B02 combination in 3D7 and Dd2 strains, respectively. Fixed-ratio drug combination assays were performed. FIC, fraction inhibitory concentration. Each point represents the mean IC₅₀ of drug combination from three independent experiments. The solid line is plotted between the IC₅₀ values of each drug when used alone to emphasize the concave nature of the isobolograms.

Figure 7.

Model of parasite killing by the combination of ART and B02. ART is activated by Fe²⁺ source within the parasite to generate activated ART*, which creates DNA double-strand breaks and several other cellular damages (protein alkylations, membrane lipid peroxidation, etc.). DSBs are repaired by PfRad51-mediated HR pathway leading to survival of parasites. In the presence of B02, the parasitic HR mechanism is blocked resulting in unrepaired DSBs that eventually lead to parasite death. Similarly, the other kinds of cellular damages if remaining unrepaired also lead to parasite death. However, this second process of parasite killing is independent of B02 action.