ABSTRACT

As the malaria parasite becomes resistant to every drug that we develop, the identification and development of novel drug candidates are essential. Many studies have screened compounds designed to target the clinically important blood stages. However, if we are to shrink the malaria map, new drugs that block the transmission of the parasite are needed. Sporozoites are the infective stage of the malaria parasite, transmitted to the mammalian host as mosquitoes probe for blood. Sporozoite motility is critical to their ability to exit the inoculation site and establish infection, and drug-like compounds targeting motility are effective at blocking infection in the rodent malaria model. In this study, we established a moderate-throughput motility assay for sporozoites of the human malaria parasite Plasmodium falciparum, enabling us to screen the 400 drug-like compounds from the pathogen box provided by the Medicines for Malaria Venture for their activity. Compounds exhibiting inhibitory effects on P. falciparum sporozoite motility were further assessed for transmission-blocking activity and asexual-stage growth. Five compounds had a significant inhibitory effect on P. falciparum sporozoite motility in the nanomolar range. Using membrane feeding assays, we demonstrate that four of these compounds had inhibitory activity against the transmission of P. falciparum to the mosquito. Interestingly, of the four compounds with inhibitory activity against both transmission stages, three are known kinase inhibitors. Together with a previous study that found that several of these compounds could inhibit asexual blood-stage parasite growth, our findings provide new antimalarial drug candidates that have multistage activity.

INTRODUCTION

Malaria is caused by parasites of the genus Plasmodium, transmitted to humans by Anopheles mosquitoes. Plasmodium falciparum is responsible for the majority of malaria-induced deaths, with over 400,000 deaths and more than 200 million people affected annually (1). As P. falciparum is increasingly becoming resistant to artemisinin-based combination therapies (2), new drugs are essential. Drugs used to treat malaria target the asexual blood stages of the parasite, which are responsible for all clinical manifestations of malaria. The majority of these drugs have no impact on the transmission stages of the malaria parasite and in some cases have been found to increase transmission to the mosquito host (35). Recently, there has been some focus on developing drugs that target both blood stages and transmission stages, a goal that would enable us to work toward malaria elimination as we treat clinical malaria cases.
Malaria parasites cycle between mosquito and mammalian hosts. Infection in the mammalian host is initiated when mosquitoes inoculate sporozoites into the skin as they probe for blood. Sporozoites are actively motile, migrating through the dermis to enter the blood circulation (6), which carries them to the liver where they infect hepatocytes, producing thousands of hepatic merozoites that initiate the blood stage of infection. Some blood-stage parasites differentiate into gametocytes, which are responsible for transmission to the mosquito. Upon being ingested during blood feeding, fertilization occurs in the mosquito midgut and leads to the formation of ookinetes, which migrate across the midgut and develop into oocysts, where sporozoites develop and then exit to enter salivary glands to be inoculated into the next host.
Sporozoites move by gliding motility, a substrate-based form of locomotion that involves the rapid turnover of surface adhesion sites powered by a subpellicular actin-myosin motor (79). Invasive stages of apicomplexan parasites require motility to enter host cells; however, in contrast to other stages, sporozoites are inoculated far from their target organ and require motility to exit the dermal inoculation site (6, 10, 11). Although a few sporozoites find and enter blood vessels within minutes, the majority take longer (12), with motile sporozoites entering blood vessels up to 2 h after inoculation (10). Thus, the skin is where the malaria parasite is extracellular for the longest period of time in the mammalian host and where fast, sustained gliding motility is required. Indeed, mutant sporozoites with motility defects are significantly more attenuated after inoculation into the skin than after intravenous inoculation (8, 13, 14), and the skin is where antibodies targeting sporozoite motility have their greatest impact (15). These studies highlight the critical role of motility in the skin and the window of opportunity that this presents to target sporozoites.
Motility studies have been performed largely using the rodent malaria model (16, 17) since, to date, it has been difficult to perform live gliding assays with sporozoites of the human malaria parasite P. falciparum. In this study, we developed a moderate-throughput in vitro P. falciparum sporozoite motility assay and screened the 400 pathogen box compounds available from the Medicines for Malaria Venture (MMV), which demonstrate activity against a range of different pathogens, predominantly Mycobacterium and two groups of eukaryotic protists, the apicomplexans and kinetoplastids. Five compounds had inhibitory activity in the nanomolar range and were further screened for activity against gametocyte transmission to mosquitoes and blood-stage parasites to identify multistage-active compounds (Fig. 1A).
FIG 1
FIG 1 Pathogen box screening strategy and validation. (A) Pathogen box screening against different Plasmodium life cycle stages. Pathogen box compounds were initially screened for their impact on P. falciparum sporozoite motility, which is essential for establishing infection in a human host. Compounds with an inhibitory effect on sporozoite motility were further assessed against gametocyte-to-oocyst development to determine mosquito-transmission-blocking activity. Finally, compounds with a strong inhibitory effect on both transmission stages were assessed against asexual-stage parasite growth. (B) Strategy for screening of pathogen box compounds against P. falciparum sporozoite motility. Freshly isolated P. falciparum sporozoites were preincubated with pathogen box compounds at 1 μM for 30 min, added to plates, and allowed to glide for 1 h in the continued presence of the compound. Trails were visualized by immunofluorescence staining of the circumsporozoite protein (CSP) trails left behind on plates and quantified by high-content imaging of 25 positions in each well. (C to F) Validation of the P. falciparum motility assay. P. falciparum sporozoites were preincubated with 0.1 to 10 μg/mL of mAb 2A10 (C and D) or 0.1 to 10 μM cytochalasin D (E and F) for 30 min and added to wells for 1 h in the continued presence of the inhibitor. Sporozoites and trails were then stained for CSP, and quantification of the area occupied by fluorescence staining was performed. (C and E) Representative images of CSP-stained sporozoites and trails when sporozoites were preincubated with the indicated concentrations of mAb 2A10 (C) or cytochalasin D (E). Bars, 50 μm. (D and F) Quantification of the area occupied by CSP-stained sporozoites and trails in the indicated treatment groups. Imaging was performed on 25 positions per well, with each dot representing the area occupied by fluorescent trails in one image. Data were pooled from 3 independent experiments, and all conditions were compared to each other (****, P < 0.0001; *, P < 0.05; ns [not significant], P > 0.05 [by a Kruskal-Wallis test followed by Dunn’s test]). Red bars indicate the means.

RESULTS

Moderate-throughput Plasmodium falciparum sporozoite motility assay.

We began by establishing a motility assay for P. falciparum sporozoites. We found that if wells of a 96-well plate were coated with monoclonal antibody (mAb) 2A10, specific for the repeat region of the sporozoite’s major surface protein, the circumsporozoite protein (CSP), P. falciparum sporozoites would adhere to the wells and initiate motility. Unlike rodent malaria sporozoites, which will initiate gliding motility on uncoated glass slides, P. falciparum sporozoites appear to need the additional adhesive force provided by the binding of its major surface protein to an immobilized antibody in order to move in two-dimensional spaces. Sporozoites and the CSP trails that they leave behind as they move were visualized by staining for CSP using biotinylated mAb 2A10 followed by avidin conjugated to a fluorophore. Although we would not expect that mAb 2A10 bound to the well would induce the CSP reaction, the cross-linking of surface CSP that immobilizes sporozoites and leads to the shedding of the intact coat (18), scanning of control slides revealed no evidence of the CSP reaction in our motility assay (see Fig. S1A and B in the supplemental material). Plates were imaged with a high-content imaging system, and the area occupied by the trails was quantified using Cell Profiler software (Fig. 1B). This assay was validated by the quantification of motility in the presence of known inhibitors, soluble mAb 2A10 (19) and cytochalasin D, an actin polymerization inhibitor (20). Sporozoites were preincubated with 1 μM each compound and then allowed to move in wells of a glass-bottom plate for 1 h. Treatment with either mAb 2A10 or cytochalasin D inhibited sporozoite motility in a dose-dependent manner (Fig. 1C to F), indicating that this assay allows moderate-throughput measurement of sporozoite motility.

Screening of pathogen box compounds against Plasmodium falciparum sporozoite motility.

The pathogen box contains 400 drug-like molecules active against neglected diseases, including 125 compounds targeting the blood stages of malaria (https://www.mmv.org/mmv-open/pathogen-box/about-pathogen-box). Screening of the compounds at 1 μM in our sporozoite motility assay demonstrated that four compounds displayed ≥50% inhibition of motility (Fig. S2A, B, D, and E), and one compound showed somewhat less inhibition (Fig. S2C), although in a replicate, it had >50% inhibition, so we decided to include it in subsequent validation assays. We confirmed these results by retesting the active compounds in 3 biological replicates (Fig. 2A and B). The potency of each compound was then assessed by treating sporozoites with compounds serially diluted from 1 μM to 3.9 nM. MMV688703 and MMV030734 showed the highest potencies, with half-maximal inhibitory concentrations (IC50s) of 9.7 nM and 34.0 nM, respectively (Fig. 2C). The three remaining compounds, MMV688854, MMV687800, and MMV687807, had IC50 values of 157 nM, 95 nM, and 154 nM, respectively (Fig. 2C). Importantly, the hepatocyte cytotoxicity data provided by MMV showed that 4 of the 5 compounds did not have significant toxicity to HepG2 cells, while MMV687807 showed some toxicity to HepG2 cells (Table 1). We then determined whether these compounds were directly toxic to parasites, using a viability assay based on a live/dead dye that binds to free amines, resulting in dead cells becoming brightly fluorescent (Fig. 3A). Similar to the motility assay, sporozoites were preincubated with compounds at 1 μM and the live/dead dye for 30 min, followed by a 1 h incubation at 37°C. Sporozoites were then stained for CSP for visualization purposes. Sporozoites treated with any of the 5 motility-inhibiting compounds were >90% viable (Fig. 3B), suggesting that these compounds did not have a direct cytotoxic effect on sporozoites.
FIG 2
FIG 2 Five pathogen box compounds significantly reduce P. falciparum sporozoite motility. P. falciparum sporozoites were preincubated with the indicated compounds (MMV030734, MMV688854, MMV687800, MMV687807, and MMV688703) for 30 min and allowed to glide in the continued presence of the compound for 1 h. Following this, sporozoites and trails were stained for CSP, and the area occupied by the fluorescent sporozoites and trails was quantified by high-content imaging. (A) Representative images of CSP-stained sporozoites and trails in the presence of each of the 5 inhibitory compounds. Bars, 50 μm (B) Quantification of the area occupied by CSP-stained trails. Twenty-five images per well were analyzed, with each dot representing data from one image. Data were pooled from 3 independent experiments, and each inhibitor was compared to 1% BSA (****, P < 0.0001 [by a Kruskal-Wallis test followed by Dunn’s test]). Red bars indicate the means. (C) Dose-response of the inhibitory pathogen box compounds. P. falciparum sporozoites were incubated with the indicated serially diluted compound, and the area occupied by CSP-stained trails was quantified. Twenty-five images per well were analyzed, with each dot representing data from one image. Data were pooled from 2 independent experiments, and serially diluted compounds were compared to 1% BSA (****, P < 0.0001; **, P < 0.005; *, P < 0.05 [by a Kruskal-Wallis test followed by Dunn’s test]). Red bars indicate the means. Calculated IC50s were: MMV030734, 34 nM; MMV688854, 157 nM; MMV687800, 95 nM; MMV687807, 154 nM; and MMV688703, 9.7 nM.
FIG 3
FIG 3 P. falciparum sporozoite viability after treatment with each of the five active pathogen box compounds. P. falciparum sporozoites were incubated with a fixable live/dead stain and the indicated pathogen box compound for 30 min at 20°C and then moved to 37°C for 1 h in the continued presence of the stain and compound. Following this, sporozoites were fixed, stained with antibodies specific for CSP, and quantified by immunofluorescence microscopy. (A) Representative images of sporozoites stained for CSP (red) and with live/dead stain (green). Bars, 50 μm. (B) Quantification of sporozoite viability after treatment with compounds. Total sporozoites (CSP) (red) and those that were stained with the live/dead stain (green) were counted to determine percent viability (n ≥ 100 sporozoites under each condition). Bars representing control samples are shown in black. Shown are the means ± standard deviations (SD) from two independent experiments.
TABLE 1
TABLE 1 Summary of activity and possible targets of the five inhibitory compounds
ParameterDescription
MMV030734MMV688854MMV687800MMV687807MMV688703
Target organism in the pathogen box or reference compoundMalariaCryptosporidiumReference compound clofazimineMycobacterium tuberculosisToxoplasma
 
Potential targetPfCDPK1TgCDPK1 (PfCDPK4)  cGMP-dependent protein kinase
Results from this study     
P. falciparum sporozoite motilitySignificant inhibition at 0.0625 μMSignificant inhibition at 0.25 μMSignificant inhibition at 0.25 μMSignificant inhibition at 0.25 μMSignificant inhibition at 0.0156 μM
P. berghei sporozoite motility72% inhibition at 1 μM45% inhibition at 1 μM38% inhibition at 1 μM49% inhibition at 1 μM71% inhibition at 1 μM
P. falciparum oocyst formationSignificant inhibition at 1 μM, with 0.1% gametocytemiaSignificant inhibition at 1 μM, with 0.1% gametocytemiaSignificant inhibition at 1 μM, with 0.03% gametocytemiaNo effect on oocyst developmentSignificant inhibition at 1 μM, with 0.03% gametocytemia
P. falciparum asexual-stage growthEC50, 15 ± 1 nM; schizont egress defect was observedInactiveNo dataNo dataNo data
MMV data sheet     
P. berghei liver-stage development, IC50  determination by a luciferase assay100% inhibition at 10 μM; IC50, 0.32 μMNo dataNo dataNo dataNo data
P. falciparum asexual-stage growth3D7, IC50 = 0.41 μMNo dataNo dataNo dataNo data
DD2, IC50 = 0.2 μM
W2, IC50 = 1.1 μM
P. falciparum (NF54) inhibition of late- stage (IV–V) gametocyte development9% inhibition at 10 μMNo dataNo dataNo dataNo data
 CytotoxicityaHepG2, CC20 = 5.8 μM and CC50 > 10 μMHepG2, CC20 = 22.6 μMHepG2, CC20 = 6.6 μMHepG2, CC50 = 0.7 μMHepG2, CC20 = 2.7 μM
HL60, CC50 > 50 μM
Results from S. Duffy et al.b     
P. falciparum asexual-stage growth3D7, IC50 < 5 μM3D7, IC50 > 20 μM84% inhibition at 20 μM (3D7)3D7, IC50 = 1.82 μM3D7, IC50 = 3.16 μM
Inactive (NF54)
P. falciparum inhibition of late-stage  (IV–V) gametocyte developmentIC50 = 5–10 μMIC50 = 5–10 μMInactiveIC50 = 5.07 μMIC50 > 20 μM
a
CC20 and CC50, cytotoxic concentrations of the compounds that cause the death of 20% and 50% of viable cells, respectively.
b
See reference 21.

Testing the pathogen box inhibitory compounds against Plasmodium berghei sporozoite motility.

A previous study from Douglas et al. screened the 400 compounds in the MMV malaria box for their impact on the sporozoite motility of the rodent malaria parasite Plasmodium berghei (16). These compounds are different from those in the MMV pathogen box. They found that three of the malaria box compounds, MMV665953, MMV665852, and MMV007224, inhibited motility by >75%; however, due to toxic effects on hepatocytes, these compounds were not pursued further. To determine if there was any cross-species activity of our hits, we tested the active pathogen box compounds on P. berghei sporozoites. Interestingly, we found that these sporozoites make tighter circles than P. falciparum sporozoites, complicating the quantification of the area occupied by the trails using high-content imaging (Fig. S1). To further confirm the difference in the circle diameters of gliding P. berghei and P. falciparum sporozoites, we imaged sporozoite motility in live gliding assays (on antibody-coated slides) and measured the diameters of their circular trajectories. As shown in Fig. S1C and D, P. falciparum sporozoites move in larger circles than P. berghei. Thus, we optimized the screening method for P. berghei by quantifying stained trails using fluorescence microscopy and measuring the total fluorescence intensity using ImageJ. We verified this assay with mAb 3D11, an inhibitory antibody specific for the P. berghei CSP repeats, and cytochalasin D (Fig. 4A and B). As shown, treatment with mAb 3D11 and cytochalasin D inhibited sporozoite motility in a dose-dependent manner. We then assessed the 5 inhibitory pathogen box compounds, and all of them showed significant inhibitory effects on P. berghei sporozoite motility (Fig. 4C and D). MMV030734 and MMV688703 showed >70% inhibition in the presence of 1 μM compound, while MMV688854, MMV687807, and MMV687800 were less potent. These findings are similar to the inhibitory activity of these compounds against P. falciparum sporozoite motility at 1 μM, with MMV030734 and MMV688703 being the most potent and MMV687800 demonstrating the least inhibitory activity (Fig. 2B). Thus, the rodent model can be used for screening compounds targeting motility, although ultimately, compounds need to be screened against human malaria parasites.
FIG 4
FIG 4 Testing the inhibitory pathogen box compounds against Plasmodium berghei sporozoite motility. (A and B) Validation of the P. berghei sporozoite motility assay. Sporozoites were preincubated with the indicated concentrations of mAb 3D11 or cytochalasin D for 30 min and then allowed to glide for 1 h in the continued presence of the antibody or cytochalasin D. Sporozoites and trails were stained for CSP, and the total fluorescence intensities of sporozoites and trails were quantified by using ImageJ. (A) Representative images of CSP-stained sporozoites and trails. Bars, 20 μm. (B) Quantification of the total fluorescence intensity of CSP-stained sporozoites and trails for each treatment group. Twenty-five images were acquired per well, with each dot representing the fluorescence intensity from one image. Data are pooled from 2 independent experiments, and all conditions were compared to each other (****, P < 0.0001; *, P < 0.05 [by a Kruskal-Wallis test followed by Dunn’s test]). Red bars indicate the means. (C and D) Testing of inhibitory pathogen box compounds against P. berghei sporozoites. Sporozoites were preincubated with each of the five inhibitory pathogen box compounds at 1 μM for 30 min, added to plates, and allowed to glide for 1 h in the continued presence of the compound. Sporozoites and trails were stained for CSP, and the fluorescence intensities of sporozoites and trails were quantified by using ImageJ. (C) Representative images of CSP-stained sporozoites and trails incubated with the indicated pathogen box compounds. Bars, 20 μm. (D) Quantification of the fluorescence intensities of CSP-stained sporozoites and trails in the presence of the indicated compounds. Twenty-five images per well were acquired, with each dot representing the fluorescence intensity of one image. Data were pooled from 3 independent experiments, and each inhibitor was compared to 1% BSA (****, P < 0.0001 [by a Kruskal-Wallis test followed by Dunn’s test]). Red bars indicate the means.

Testing the pathogen box inhibitory compounds in transmission-blocking assays.

We next determined whether any of our active compounds had inhibitory activity against the transmission of Plasmodium parasites to mosquitoes. To do this, we added each of the 5 compounds at 1 μM to the P. falciparum gametocyte blood meal fed to mosquitoes. Nine days later, mosquito midguts were dissected, and oocysts were counted (Fig. 5A and B). We performed this assay using two different gametocyte concentrations, 0.1% and 0.03% of the total erythrocytes. As shown in Fig. 5C, when fed on blood containing low gametocyte counts, there was a significant reduction in the number of oocysts in mosquitoes fed with four of the compounds, MMV030734, MMV688854, MMV687800, and MMV688703. In contrast, when mosquitoes were fed on blood with higher gametocyte counts, only MMV030734 and MMV68854 had inhibitory activity against transmission. One compound, MMV687807, had no effect on transmission, even when blood contained low numbers of gametocytes (Fig. 5C and D).
FIG 5
FIG 5 Testing the inhibitory pathogen box compounds for mosquito-transmission-blocking activity. (A) Schematic representation of Plasmodium transmission to mosquitoes. (B) Schematic representation of the assay setup. The mature gametocyte culture was mixed with each compound to a final concentration of 1 μM and fed to A. stephensi mosquitoes. Nine days later, mosquito midguts were dissected, and oocysts were counted. (C and D) A. stephensi mosquitoes were fed a blood meal at either 0.03% gametocytemia (C) or 0.1% gametocytemia (D) in the presence of the indicated compound or the DMSO control. On day 8 after the blood meal, individual mosquitoes were dissected, and oocysts were counted for 15 to 20 mosquitos per group. Each group was compared to the DMSO control (****, P < 0.0001; ***, P < 0.0005; **, P < 0.005; *, P < 0.05 [by a Kruskal-Wallis test followed by Dunn’s test]). Red bars indicate the means.

Asexual blood-stage parasites treated with MMV030734 exhibit egress defects.

Although these compounds had been previously tested against asexual blood-stage parasites of P. falciparum in a high-throughput assay (21), we wanted to confirm these results for the two compounds (MMV030734 and MMV688854) that had strong inhibitory activity against parasites at both transmission stages (Fig. 6A). Synchronized P. falciparum ring-stage parasites were grown in the presence of 1 μM MMV030734 or MMV688854 or 0.1% dimethyl sulfoxide (DMSO) for 60 h. At the end of the experiment, Giemsa-stained blood smears were made, and ring-stage parasites were counted. MMV688854 had no impact on parasite growth; however, no ring-stage parasites were observed in the culture treated with MMV030734 (Fig. 6B). Interestingly, the Giemsa-stained slides from the MMV030734-treated culture showed that parasite growth was halted at the schizont stage, suggesting an egress defect (Fig. 6C). To confirm this, we counted ring-, trophozoite-, and schizont-stage parasites in infected cells. As shown in Fig. 6D, all parasites were at the schizont stage in the culture treated with MMV030734, while the majority of the population in the culture treated with MMV688854 and the DMSO control was ring-stage parasites. To further characterize MMV030734, we determined its half-maximal effective concentration (EC50) and found that its inhibitory activity against blood-stage parasite growth was in the nanomolar range (Fig. 6E).
FIG 6
FIG 6 Pathogen box compound MMV030734 reduces asexual-stage parasite growth and inhibits egress. (A) Schematic representation of the Plasmodium asexual blood-stage cycle. (B) Quantification of ring-stage parasitemia. Synchronized P. falciparum ring-stage parasites were incubated with compound MMV030734 or MMV688854 at 1 μM or 0.1% DMSO for 60 h, at which point Giemsa-stained blood smears were made and ring-stage parasites were counted. The means ± standard deviations (SD) from two technical replicates are shown (*, P < 0.01 [by analysis of variance {ANOVA} for multiple comparisons]). (C) Representative images of Giemsa-stained asexual-stage parasites at 0 h and 60 h in the indicated treatment groups. Bars, 10 μm. (D) Giemsa-stained blood smears taken at 60 h were scored based on the numbers of ring-, trophozoite-, and schizont-stage parasites. The percentage of each life cycle stage is shown for 100 infected erythrocytes from each treatment group. (E) Dose-response plot for parasites grown in the presence of MMV030734 or chloroquine for 72 h. Data were pooled from two biological replicates, each with four technical replicates. The mean parasitemias ± SD normalized to the DMSO control are shown. The half-maximal effective concentration (EC50) is presented as the mean ± SD.

DISCUSSION

In this study, we have established a quantitative, moderate-throughput assay of P. falciparum sporozoite motility and used it to screen 400 drug-like compounds targeting neglected diseases. Five compounds had a significant inhibitory effect on P. falciparum sporozoite motility, MMV688854, MMV687800, MMV687807, MMV688703, and MMV030734, with MMV688703 and MMV030734 having IC50 values in the low-nanomolar range (Fig. 2). These 5 compounds were further assessed in P. falciparum transmission-blocking assays, and 2 of them, MMV030734 and MMV688854, had strong inhibitory activity against transmission to the mosquito, while 2 others, MMV687800 and MMV688703, had moderate activity in this assay (Fig. 5). Three of the compounds that had dual-transmission-blocking activity, MMV030734, MMV687807, and MMV688703, had previously been shown to inhibit the growth of P. falciparum asexual-stage parasites (21), with our study extending those findings, with the demonstration that MMV030734 impacts egress from infected erythrocytes (Fig. 6). These inhibition data are summarized in Fig. 7.
FIG 7
FIG 7 Summary of the five motility-inhibitory compounds across the Plasmodium life cycle. Pathogen box compounds were screened for their impact on P. falciparum sporozoite motility, and 5 compounds (MMV030734, MMV688854, MMV687800, MMV687807, and MMV688703) showed significant inhibition in this assay. Of these compounds, MMV030734 and MMV68854 inhibited the transmission of P. falciparum gametocytes to mosquitoes at high gametocytemia, and MMV687800 and MMV688703 inhibited transmission only at low gametocytemia. MMV030734, MMV687807, and MMV688703 had a significant inhibitory effect on asexual blood-stage growth. Thus, two compounds, MMV030734 and MMV688703, inhibited all three P. falciparum life cycle stages. *, the blood-stage-inhibitory effect of MMV688703 and MMV687807 was confirmed by S. Duffy et al. (21).
Testing of the 5 inhibitory compounds in motility assays with the rodent malaria parasite P. berghei demonstrated that all 5 compounds had inhibitory activity, with MMV030734 and MMV688703 having the greatest inhibitory activity against both species. These data highlight the conservation of the gliding motility machinery across the genus and the usefulness of the rodent model in testing compounds targeting motility. Nonetheless, in the course of our studies, we also discovered distinct differences between the species that impacted the assay design: P. falciparum sporozoites are less adherent than P. berghei and thus require anti-CSP antibody to attach to 2-dimensional surfaces. Additionally, the trajectories of P. falciparum in this assay were less constrained, with their trails having larger circles and occasional linear portions (see Fig. S1 in the supplemental material). Perhaps, the stronger adhesion of P. berghei sporozoites increases the likelihood of remaining in a tight circle after motility is initiated. However, in this assay, it could be due to or enhanced by the affinity of the capture antibody since mAb 3D11 is a higher-affinity antibody than mAb 2A10. Importantly, intravital imaging of P. berghei and P. falciparum sporozoites in the skin revealed no striking differences in displacement and speed (10), suggesting that these differences may become apparent only in 2-dimensional assays.
Interestingly, three of the compounds, MMV688703, MV030734, and MMV688854, are known to target protein kinases (Table 1). MMV688703, a substituted pyrrole, is also known as compound 1, a cGMP-dependent protein kinase (PKG) inhibitor in Toxoplasma gondii and Eimeria tenella (22, 23); MMV030734, a trisubstituted imidazole, binds to P. falciparum calcium-dependent protein kinase 1 (PfCDPK1) (24) and inhibits blood-stage parasites; and MMV688854, a pyrazolopyrimidine bumped kinase inhibitor derivative, is a known inhibitor of Toxoplasma gondii CDPK1 (TgCDPK1) (25, 26). The other compounds that had inhibitory effects on sporozoite motility were MMV687800 and MMV687807, both of which have antimycobacterial activity. MMV687800 is clofazimine, which is used together with dapsone to treat leprosy. Although its precise mechanism of action is unclear, clofazimine interacts with bacterial membrane phospholipids and interferes with K+ uptake and ATP production (27). MMV687807 is a salicylanilide derivative with significant cytotoxicity (28). There are also 26 reference compounds in the pathogen box, and of these, doxycycline (MMV000011), primaquine (MMV000023), amphotericin B (MMV689000), and bedaquiline (MMV689758) had significant inhibitory activity against asexual blood stages (21); however, they had no impact on sporozoite motility (Fig. S2B and C).
Among the five compounds that inhibited sporozoite motility, MMV688703, or compound 1, had the highest potency, with activity in the low-nanomolar range. This inhibitor also had activity in blocking parasite transmission to mosquitoes. The compound’s target molecule, PKG, is likely the sole mediator of cGMP signaling in Plasmodium parasites, with phosphoproteomics studies showing hundreds of downstream substrates (29; reviewed in reference 30). Thus, it is not surprising that PKG regulates many cellular processes in Plasmodium, including egress from red blood cells, gamete formation, and motility (3134). The critical role of PKG in cellular pathways occurring across the life cycle in both mosquito and mammalian hosts can be explained by the recent demonstration that PKG controls cytosolic Ca2+ levels, which in turn regulate a variety of stage-specific effector pathways (35). Its role in motility has been demonstrated in P. berghei, where it has been shown to inhibit the regulated secretion of micronemes, a calcium-dependent process necessary for gliding motility, and the phosphorylation of central components of the actin-myosin motor (34, 36). Our current findings demonstrate that PKG signaling is critical for motility in P. falciparum. Additionally, the modest but significant transmission-blocking activity of MMV688703 extends previous findings with this compound on gametogenesis and ookinete motility to show that these defects impact mosquito transmission. Taken together, these data, covering a wide range of phenotypic assays in both human and rodent malaria parasites, make a compelling case that targeting PKG could have potent multistage activity.
Interestingly, the two compounds that had strong transmission-blocking activity in addition to their inhibitory activity against sporozoite motility were MMV688854 and MMV030734, which target kinases of the CDPK family (2426). CDPKs are serine/threonine protein kinases that are mediators of calcium signaling in Plasmodium and other apicomplexans (37, 38), containing calcium-binding EF hand domains that, when bound to Ca2+, lead to conformational changes that enable them to rapidly respond to Ca2+ fluxes. Apicomplexan parasites have multiple CDPK family members, with P. falciparum having 7 CDPKs and the rodent malaria parasites having orthologs of all but one of these. Toxoplasma CDPK1, the target of MMV688854, has been shown to regulate microneme secretion, and the inhibition of TgCDPK1 results in the blockade of parasite motility, host cell invasion, and egress from host cells (39). The Plasmodium ortholog of TgCDPK1 is CDPK4. Studies with P. berghei demonstrated a role for CDPK4 in sporozoite gliding motility and hepatocyte invasion (36, 40), and in both P. berghei and P. falciparum, CDPK4 controls microgametocyte activation and exflagellation (4143). MMV030734 has been shown to target PfCDPK1, which is expressed throughout the Plasmodium life cycle and phosphorylates motor complex proteins such as myosin A tail domain-interacting protein (MTIP) and glideosome-associated protein 45 (GAP45) (44), consistent with the recent demonstration in P. berghei that it plays a critical role in sporozoite motility and invasion (40). Furthermore, the deletion of PfCDPK1 results in the slower growth of asexual blood stages and the formation of gametocytes that are not infectious to mosquitoes (45). Recently, the trisubstituted imidazole MMV030084, which is an analog of MMV030734, has been identified as a multistage-targeting compound, inhibiting P. berghei liver-stage development, P. falciparum asexual blood-stage development, and male gamete activity (46). Using a chemoproteomics approach, those researchers found that MMV030084 targeted both PfCDPK1 and PKG; however, conditional knockdown and molecular modeling studies pointed to PKG as being the primary target (46).
Our findings that three of the dual-transmission-blocking compounds target either PKG or the CDPK family of kinases highlight the central role of calcium signaling as Plasmodium parasites move between their mammalian and mosquito hosts and support the idea that PKG, the master regulator of parasite Ca2+ levels, and the CDPKs, the effectors of calcium signaling (30, 36, 47), are excellent targets for multistage-inhibitory drugs. Our new moderate-throughput screening strategy for sporozoite motility facilitates compound testing on P. falciparum preerythrocytic stages and future work on identifying transmission-blocking compounds and preerythrocyte-stage vaccine candidates.

MATERIALS AND METHODS

Ethics statement.

All animal work was conducted according to the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (48). The protocol was approved by the Johns Hopkins University Animal Care and Use Committee (protocol number M017H325), which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care.

Mosquito infection with P. falciparum NF54.

Mosquito infection with P. falciparum NF54 was performed as previously described (49). Asexual cultures were maintained in vitro in O+ erythrocytes at 4% hematocrit in RPMI 1640 (Corning) supplemented with 74 μM hypoxanthine (Sigma), 0.21% (wt/vol) sodium bicarbonate (Sigma), and 10% (vol/vol) heat-inactivated human serum. Cultures were maintained at 37°C in a candle jar made from glass desiccators. Gametocyte cultures were initiated at 0.5% parasitemia and 4% hematocrit. Medium was changed daily for up to 15 to 18 days without the addition of fresh blood to promote gametocytogenesis. Adult Anopheles stephensi mosquitoes (3 to 7 days after emergence) were allowed to feed through a glass membrane feeder for up to 30 min on gametocyte cultures at 40% hematocrit containing fresh O+ human serum and O+ erythrocytes. Infected mosquitoes were maintained for up to 19 days at 25°C with 80% humidity and were provided with a 10% (wt/vol) sucrose solution.

Mosquito infection with P. berghei WT-ANKA.

Mosquito infection with P. berghei wild-type ANKA (WT-ANKA) was performed as previously described (50). Swiss Webster mice (Taconic) were infected with P. berghei ANKA wild-type parasites, and once abundant gametocyte-stage parasites were observed, A. stephensi mosquitoes (3 to 7 days after emergence) were allowed to feed on infected mice. Infected mosquitoes were maintained for up to 25 days at 18°C with 80% humidity and were provided with a 10% (wt/vol) sucrose solution.

Pathogen box compounds.

Pathogen box compounds were obtained from the Medicines for Malaria Venture (MMV) and consisted of 400 compounds at a concentration of 10 mM, dissolved in dimethyl sulfoxide (DMSO; Sigma). Compounds were diluted to 1 mM in DMSO, aliquoted into 96-well storage microplates (catalog number CLS3363; Sigma), and stored at −80°C.

P. falciparum moderate-throughput sporozoite motility assay.

Freshly dissected P. falciparum salivary gland sporozoites were aliquoted such that 15,000 sporozoites in 60 μL of 2% (wt/vol) bovine serum albumin (BSA) in Hanks’ balanced salt solution (HBSS) at pH 7.4 were mixed with 60 μL of each pathogen box compound at 2 μM and added to a well of a U-bottom 96-well plate (catalog number 353077; Corning). The final concentration of each pathogen box compound was 1 μM in 1% (wt/vol) BSA in HBSS. Controls were incubated with either HBSS alone, 0.1 to 10 μg/mL of mAb 2A10, or 0.1 to 10 μM cytochalasin D. Plates were incubated for 30 min at 20°C, and the sporozoite-compound mixture was transferred to a 96-well glass-bottom plate (catalog number 655892; Greiner) coated with 5 μg/mL of mAb 2A10 in phosphate-buffered saline (PBS). The plate was centrifuged for 3 min at 300 × g and incubated for 1 h at 37°C. Wells were fixed in 4% paraformaldehyde (PFA) in PBS, blocked with 1% (wt/vol) BSA in PBS (pH 7.4), and stained with biotinylated mAb 2A10 in 1% (wt/vol) BSA in PBS (pH 7.4) for 1 h at room temperature, followed by detection with Alexa Fluor 488 streptavidin (catalog number S11223; Invitrogen) diluted at 1:500 in PBS for 1 h at room temperature. Samples were preserved in a glycerol-PBS solution (ratio, 9:1) at 4°C, and the plate was imaged within 1 week. Imaging was performed on 25 positions per well (5 by 5, 500 μm apart) by using the ImageXpress Micro XLS wide-field high-content analysis system (Molecular Devices) with a 40× Plan Fluor objective.

Quantification of the area occupied by trails using Cell Profiler software.

Image analysis was automated with the open-source Cell Profiler software (version 3.0.0) (51). All images were run through a pipeline designed to threshold the images and quantify the area occupied by trails. The image intensity was rescaled from 0 to 0.007 to 0 to 1, and the rescaled image was used to set the threshold, which was set automatically by using the minimum cross-entropy setup in the Cell Profiler pipeline. Following this, object pixel diameter sizes of between 5 and 1,000 were counted and exported to an Excel file.

Testing pathogen box compounds against P. berghei sporozoite motility.

Coverslips were placed into 24-well plates, coated with 5 μg/mL of mAb 3D11 in PBS for 1 h at room temperature, and then washed. A total of 50,000 sporozoites in HBSS with 2% (wt/vol) BSA (pH 7.4) were mixed with pathogen box compounds, cytochalasin D, or mAb 3D11 in a low-retention 1.5-mL tube (catalog number MCT-175-L-C; Axygen) and preincubated for 30 min at 20°C. Each sporozoite-compound mixture was then added to a mAb 3D11-coated well, centrifuged onto the coverslip for 3 min at 300 × g, and incubated for 1 h at 37°C. Wells were fixed in 4% (vol/vol) paraformaldehyde in PBS, blocked in 1% (wt/vol) BSA in PBS (pH 7.4), and stained with biotinylated mAb 3D11 diluted at 1:500 in 1% (wt/vol) BSA in PBS (pH 7.4) for 1 h at room temperature, followed by detection with Alexa Fluor 488 streptavidin (Invitrogen) diluted at 1:500 in PBS for 1 h at room temperature. Samples were mounted in gold antifade mountant (catalog number P36935; Invitrogen) and imaged by fluorescence microscopy (Nikon E600) using a 40× objective. Twenty-five positions per slide were acquired using identical exposure settings, and acquired images were batch processed using Fiji (https://fiji.sc/) to measure the total fluorescence intensity.

Sporozoite viability assessment.

Freshly dissected sporozoites were aliquoted such that 15,000 sporozoites were preincubated with pathogen box compounds at a 1 μM concentration and a 1:1,000 dilution of live/dead fixable green stain (catalog number L23101; Invitrogen) in HBSS with 1% (wt/vol) BSA (pH 7.4) at 20°C for 30 min, transferred to a 48-well plate containing a coverslip, centrifuged at 300 × g for 3 min, and further incubated at 37°C for 1 h to replicate the treatment of sporozoites in the gliding assay. After incubation, sporozoites were fixed with 4% (vol/vol) PFA, blocked with 1% (wt/vol) BSA in PBS (pH 7.4), and stained with 1 μg/mL of mAb 2A10 in 1% (wt/vol) BSA in PBS (pH 7.4), followed by detection with Alexa Fluor 594 goat anti-mouse secondary antibody (catalog number A11032; Invitrogen). Samples were mounted in gold antifade mountant (Invitrogen) and observed by fluorescence microscopy. For each condition, 100 sporozoites were identified by CSP staining (red), and dead sporozoites were counted by live/dead staining (green) to quantify viability.

Live sporozoite gliding assay.

Lab-Tek 8-well glass slides (Nunc, catalog number 177402; Thermo Scientific) were coated with 5 μg/mL of mAb 3D11 or mAb 2A10 in PBS for 1 h at room temperature and then washed. Freshly isolated sporozoites in 50 μL were mixed with an equal volume of 2% (wt/vol) BSA in HBSS (pH 7.4) and incubated at 37°C for 5 min to activate the sporozoites. The Lab-Tek chambers were removed, leaving the gasket intact; the sporozoites (~75 μL) were placed into a Lab-Tek well; and the top was covered by a cover glass (22 by 50 mm, catalog number 48393-195; VWR). The slide was spun at 200 × g for 2 min, and moving sporozoites were imaged for 2 min at 1 frame/s using an upright Nikon E600 microscope with a 10× objective. The movies were analyzed using Fiji (http://fiji.sc/): maximum projections of sporozoite trajectories were generated, and following this, circle diameters were manually measured.

Testing pathogen box compounds using the standard membrane feeding assay (SMFA).

The pathogen box compounds (MMV030734, MMV688854, MMV687800, MMV687807, and MMV688703) or DMSO in HBSS was mixed with gametocyte cultures, such that the final concentration of the compound was 1 μM and that of DMSO was 0.1% (vol/vol), and fed to adult female A. stephensi mosquitoes (3 to 7 days after emergence). Cultures with final gametocytemias of 0.3% and 0.01% were used for feeding. A. stephensi mosquitoes were allowed to feed for up to 30 min. Infected mosquitoes were maintained at 25°C with 80% humidity and were provided with a 10% (wt/vol) sucrose solution. Oocyst development was quantified on day 9 after blood feeding by staining mosquito midguts with 0.1% (wt/vol) mercurochrome (catalog number M7011; Sigma) in PBS and counting by bright-field microscopy with a 10× objective.

Testing pathogen box compounds against Plasmodium falciparum asexual stages.

The P. falciparum transgenic NF54attB line (52), which has a half-maximal effective concentration (EC50) of chloroquine similar to that of wild-type NF54 (53, 54), was cultured in O+ erythrocytes at 2% hematocrit and maintained in 25-cm2 gassed flasks (94% N2, 3% O2, and 3% CO2) at 37°C. The cultures were kept in RPMI 1640 medium with l-glutamine (catalog number R8999; U.S. Biological) supplemented with 20 mM HEPES, 0.2% (wt/vol) sodium bicarbonate, 12.5 μg/mL hypoxanthine, 5 g/L Albumax II (catalog number S7563; Life Technologies), and 25 μg/mL gentamicin. Cultures were synchronized using 5% (wt/vol) sorbitol as previously outlined (55), and synchronized ring parasites were seeded at 1% parasitemia and 1% hematocrit and treated with 1 μM the indicated pathogen box compounds, 0.1% (vol/vol) DMSO, or 1 μM chloroquine for 60 h. After incubation at 37°C for 60 h, parasite growth was quantified using Giemsa-stained blood smears. To determine the EC50 of MMV030734, sorbitol-synchronized ring parasites were seeded at 3% parasitemia and 2% hematocrit in a 96-well flat-bottom plate and grown in the presence of the inhibitor at 37°C for 72 h. Concentration series of MMV030734 (17 pM to 3 μM) and chloroquine (487 pM to 250 nM) were tested. After 72 h, parasite growth was quantified using SYBR green I (Invitrogen) to stain DNA and an Attune NxT flow cytometer as described previously (56, 57). Parasitemias from the MMV030734 and chloroquine samples were normalized to that of the 0.1% (vol/vol) DMSO control. Data from two independent biological replicates, each with four technical replicates, were fit to a four-parameter sigmoidal dose-response curve using Prism (version 8.4; GraphPad).

Statistical analysis.

All statistical analyses were performed with GraphPad Prism (version 7 or 8.4).

ACKNOWLEDGMENTS

We thank the team of the parasitology and insectary core facilities at the Johns Hopkins Malaria Research Institute, the Johns Hopkins School of Medicine Microscopy Facility (MicFac), and Hoku West-Foyle for invaluable assistance.
This work was supported by the National Institutes of Health (R01 AI132359 to P.S. and R01 AI065853 to S.T.P.), Johns Hopkins Malaria Research Institute postdoctoral fellowships (S.K. and R.E.), Bloomberg Family Philanthropies, and NIH grants that funded the MicFac high-content imager (R01 GM28007-S1 and R01 GM66817-S1).

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Published In

cover image Antimicrobial Agents and Chemotherapy
Antimicrobial Agents and Chemotherapy
Volume 66Number 920 September 2022
eLocator: e00418-22
PubMed: 35943271

History

Received: 29 March 2022
Returned for modification: 29 April 2022
Accepted: 12 July 2022
Published online: 9 August 2022

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Keywords

  1. Plasmodium falciparum
  2. antimalarial agents
  3. calcium signaling
  4. drug screening
  5. motility
  6. sporozoite
  7. transmission blocking

Contributors

Authors

W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA
Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA
Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA
Sukanat Kanchanabhogin
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA
Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA
Natasha Vartak
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA
Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA
Abhai K. Tripathi
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA
Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA
Sean T. Prigge
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA
Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA
Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA

Notes

The authors declare no conflict of interest.

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  • There is a 3- to 4-day delay in article usage, so article usage will not appear immediately after publication.
  • Citation counts come from the Crossref Cited by service.

Citations

If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. For an editable text file, please select Medlars format which will download as a .txt file. Simply select your manager software from the list below and click Download.

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