Antiapicoplast and Gametocytocidal Screening To Identify the Mechanisms of Action of Compounds within the Malaria Box

Abstract

Malaria remains a significant infectious disease that causes millions of clinical cases and >800,000 deaths per year. The Malaria Box is a collection of 400 commercially available chemical entities that have antimalarial activity. The collection contains 200 drug-like compounds, based on their oral absorption and the presence of known toxicophores, and 200 probe-like compounds, which are intended to represent a broad structural diversity. These compounds have confirmed activities against the asexual intraerythrocytic stages of Plasmodium falciparum and low cytotoxicities, but their mechanisms of action and their activities in other stages of the parasite's life cycle remain to be determined. The apicoplast is considered to be a promising source of malaria-specific targets, and its main function during intraerythrocytic stages is to provide the isoprenoid precursor isopentenyl diphosphate, which can be used for phenotype-based screens to identify compounds targeting this organelle. We screened 400 compounds from the Malaria Box using apicoplast-targeting phenotypic assays to identify their potential mechanisms of action. We identified one compound that specifically targeted the apicoplast. Further analyses indicated that the molecular target of this compound may differ from those of the current antiapicoplast drugs, such as fosmidomycin. Moreover, in our efforts to elucidate the mechanisms of action of compounds from the Malaria Box, we evaluated their activities against other stages of the life cycle of the parasite. Gametocytes are the transmission stage of the malaria parasite and are recognized as a priority target in efforts to eradicate malaria. We identified 12 compounds that were active against gametocytes with 50% inhibitory concentration values of <1 μM.

INTRODUCTION

More than 40% of the world's population is at risk of contracting malaria, and the continued emergence of drug resistance is a constant threat. As a result, the identification and characterization of new leads for the development of antimalarial drugs with different mechanisms of action are of the highest priority. Historically, antimalarial drug discovery and development have focused on the asexual intraerythrocytic stages of the life cycle of the parasite since these stages are responsible for the pathology. Gametocytes are the sexual stage of the malaria parasite and are essential for the transmission of the parasite to the mosquito. Most current antimalarials have little or no effect on gametocytes; thus, treated patients can still transmit malaria. The development of new drugs that have a broader spectrum of activity, including activity against gametocytes, is recognized as a highly favorable quality in the effort to eradicate malaria (1).

In order to catalyze the development of new antimalarials, the Medicines for Malaria Venture (MMV) and Scynexis, Inc., assembled the Malaria Box (2), an open-access library composed of 400 compounds originally identified by phenotypic screening of >4,000,000 compounds from the research libraries of Saint Jude Children's Research Hospital, Novartis, and GlaxoSmithKline (3). The in vitro antimalarial activities of the Malaria Box compounds span a range of 50% inhibitory concentration (IC₅₀) values from 30 nM to 4 μM (2). The 400 compounds in the Malaria Box were selected based on several factors, including low toxicity, oral bioavailability, and chemical diversity; however, the primary selection was based on their commercial availability to ensure accessibility for follow-up experiments (2). Substantial information about these compounds is available at the ChEMBL-NTD archive (see https://www.ebi.ac.uk/chemblntd); this knowledge base is expected to grow iteratively as more research is performed. The Malaria Box compounds were selected based on global phenotypic screening against asexual intraerythrocytic stages of Plasmodium falciparum; however, their mechanisms of action and their efficacies against gametocyte stages remain unknown.

Malaria parasites contain a vestigial plastid called the apicoplast, which performs vital functions such as biosynthesizing isoprenoid precursors, fatty acids, and heme (4, 5). Plasmodium parasites utilize the methylerythritol phosphate (MEP) pathway to synthesize isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which is essential for parasite growth (6, 7). This pathway is absent in humans, who rely on the mevalonate pathway instead. Recently, it was suggested that the MEP pathway and the biosynthesis of the isoprenoid precursors IPP and DMAPP represent the sole essential function of this organelle during asexual intraerythrocytic development of the parasites (8). The strongest support for this stems from the observation that loss of the apicoplast can be chemically complemented by supplementing the growth medium with IPP. Therefore, the inhibitory effect of drugs that directly target the biosynthesis of isoprenoid precursors or indirectly disrupt their biosynthesis by interfering with processes essential for apicoplast biogenesis, such as apicoplast DNA replication, transcription, and protein translation, may be reverted by IPP supplementation (8). As a result, the reversal of growth inhibition by IPP supplementation can be used as a phenotypic screening diagnostic to identify compounds within the Malaria Box that target the apicoplast, thus identifying their mechanisms of action and narrowing their potential molecular targets (8).

Several antibiotics, such as doxycycline (DOX), azithromycin, solithromycin, chloramphenicol, and clindamycin, kill malaria parasites by interfering with apicoplast genome replication, transcription, protein translation, posttranslation modification, or protein turnover (9–14). Parasites treated with these antibiotics initially present normal morphology and continue to grow and release daughter cells (merozoites) that are capable of invading new host cells. However, these progeny will not complete the following intraerythrocytic cycle since the apicoplast will fail to replicate and segregate; this phenomenon is known as the delayed-death phenotype (15). In contrast to those antibiotics, fosmidomycin (FOS) is an inhibitor that directly targets the metabolism of the apicoplast (7, 16–18). FOS shows a rapid onset of action, with IC₅₀s between 300 and 1,200 nM for P. falciparum in vitro culture, depending on the strain (17, 18). FOS is a specific inhibitor of the second enzyme in the MEP pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), which is essential for the survival of P. falciparum (6, 17, 18). Therefore, a reversal of growth inhibition can aid in the characterization of the mechanisms of action to, ultimately, identify the molecular targets among the Malaria Box compounds.

In the present work, we aimed to identify the mechanisms of action of the compounds of the Malaria Box. By using cell-based phenotypic assays, we identified one drug-like compound that targets the apicoplast. Further analyses revealed that the mechanism of action of this compound is more similar to that of FOS than to that of DOX. In addition, we probed the Malaria Box for compounds that have activity against late-stage gametocytes, which is the stage that is most insensitive to the current antimalarials (19, 20). We identified 12 gametocytocidal compounds with IC₅₀s of <1 μM.

MATERIALS AND METHODS

Parasites.

The following P. falciparum strains were obtained through the MR4 Malaria Reagent Repository (ATCC, Manassas, VA) as part of the BEI Resources Repository (NIAID, NIH): D10-ACP_L(leader)-GFP, MRA-568, deposited by A. F. Cowman; NF54, MRA-1000, deposited by M. Dowler, Walter Reed Army Institute of Research; and Dd2, MRA-150, deposited by D. Walliker. NF54 is sensitive to chloroquine. Dd2 is a pyrimethamine-, chloroquine-, and mefloquine-resistant strain. D10-ACP_L-GFP has been transfected with a plasmid containing the leader sequence of the acyl carrier protein (ACP), which is targeted to the apicoplast and fused to green fluorescent protein (GFP), allowing observation of apicoplast development during the intraerythrocytic cycle of the parasite. The D10-ACP_L-GFP plasmid is maintained by 0.1 μM pyrimethamine treatment (21). P. falciparum resistant to FOS was a generous gift from David Fidock (22) and was maintained by 1 μM FOS treatment.

Malaria Box.

The Malaria Box compound collection was provided by Scynexis, Inc. (Research Triangle Park, NC), for the MMV and supplied at 10 mM in dimethyl sulfoxide (DMSO). Additional amounts of MMV008138 were purchased from Sigma-Aldrich, and the results were reproduced using this commercial source.

P. falciparum cultures and drug sensitivities at asexual stages.

Parasites were maintained in O-positive human erythrocytes (4% hematocrit) in RPMI 1640 medium supplemented with 5 g/liter Albumax I (Gibco Life Technologies), 2 g/liter glucose (Sigma-Aldrich), 2.3 g/liter sodium bicarbonate (Sigma-Aldrich), 370 μM hypoxanthine (Sigma-Aldrich), 25 mM HEPES, and 20 mg/liter gentamicin (Gibco Life Technologies). The parasites were kept at 37°C under reduced-oxygen conditions (5.06% CO₂, 4.99% O₂, and 89.95% N₂).

The effect of each compound identified as gametocytocidal was further evaluated against asexual parasites from P. falciparum strains NF54 and Dd2 using the SYBR green assay as described previously (23, 24). Ten-point dilutions were used to test the dose responses at concentrations ranging from 5 μM to 0.002 μM. The IC₅₀ calculation was performed with GraFit 5 (Erithacus Software Ltd.) using nonlinear regression curve fitting and represents the average of two independent experiments and the standard deviation (SD). We determined the IC₅₀s for MMV008138, FOS, and DOX at 72 h and 96 h by a SYBR green assay to assess for the delayed-death phenotype. MMV008138 was also tested against FOS-resistant (FOS^r) parasites by the SYBR green assay.

Reversal of growth inhibition by isopentenyl diphosphate supplementation.

Recently, it was demonstrated that parasites can survive in vitro without the apicoplast when IPP is supplied at 200 μM. A reversal of growth inhibition by IPP supplementation was proposed as a method for potentially identifying compounds that target apicoplast function (8). We assessed whether compounds from the Malaria Box were targeting the apicoplast by measuring their growth recoveries in the presence of inhibitor and IPP as described previously (8). Briefly, Dd2 strain ring stages were cultured under the following conditions: control (no drug), 5 μM Malaria Box compounds, and 5 μM Malaria Box compounds with 200 μM IPP, which is the necessary concentration to achieve 100% growth recovery (8). FOS was used as a control drug for growth inhibition and the reversal of growth inhibition by IPP. All conditions were set in 96-well plates (200 μl/well with 1% hematocrit and 1% parasitemia), incubated for 72 h, and analyzed by a SYBR green assay as described previously (23, 24). The percentage of growth was normalized to that of untreated control parasites. Since several compounds within the Malaria Box did not inhibit growth completely at 5 μM, we considered reversal of growth inhibition only when a compound alone exhibited >95% growth inhibition and >60% growth recovery in the presence of inhibitor and IPP. Uninfected erythrocytes were used for background determinations. Two independent experiments were performed.

To establish dose dependency for the reversal of growth inhibition, ring-stage parasite cultures (200 μl/well, 1% hematocrit, and 1% parasitemia) were grown for 72 h in the presence of increasing concentrations of the inhibitor and in the presence or absence of 200 μM IPP. The growth of the parasite was assessed by a SYBR green assay as described previously (23, 24).

Activity of MMV008138 against Toxoplasma gondii.

The T. gondii RH-HX-KO-YFP2-DHFR(m2m3) strain tachyzoites were maintained in human fibroblasts, and parasite growth was measured by a fluorescence assay as described previously (25). Drug assays were performed by 2-fold serial dilutions of MMV008138 dissolved in 100% DMSO using concentrations ranging from 5 μM to 0.039 μM. DMSO was added to a final concentration of 0.05% for all doses. Six replicates were used for each dose and control.

Gametocytocidal activity assay.

Malaria parasites do not replicate their genome during gametocytogenesis; however, they are metabolically active, especially their mitochondria (26). Therefore, gametocyte viability was measured using alamarBlue, a fluorescent oxidoreduction indicator, as a general measure of metabolic activity. In this assay, the fluorometric readings are proportional to the numbers of living gametocytes and correspond to their metabolic activities. Damaged and nonviable gametocytes have lower metabolic activities and, therefore, generate proportionally lower signals (dose dependent) than healthy gametocytes. We investigated whether the compounds from the Malaria Box were active in killing late-stage (stage V) gametocytes using the alamarBlue viability assay described previously by Tanaka and Williamson (27). Gametocytes were developed from the P. falciparum NF54 strain (a chloroquine-sensitive strain). Asexual stages were synchronized by sorbitol treatment 2 days before gametocyte cultures were started. P. falciparum subcultures for the generation of gametocytes were set at 1% parasitemia and 4% hematocrit, and the culture medium was not changed until day four after the subculture. Parasite development was evaluated by Giemsa-stained thin blood smears on days 4, 8, 12, and 13 after the initial subculture. Starting on day nine of gametocyte culture, the parasites were treated for 4 consecutive days with 5% sorbitol for 10 min at 37°C to remove the asexual parasites. Sorbitol treatment effectively removed >99% of the asexual parasites. Gametocyte recovery and concentration were achieved on day 13 using a NycoPrep 1.077 cushion, and the gametocytes were counted using a Neubauer chamber. About 30,000 to 50,000 gametocytes per well (100 μl final volume) were added to black flat-bottom half-area 96-well plates containing the drug candidates, 60 nM epoxomicin (28) (positive control), or DMSO (negative control). The plate was incubated in a humidified chamber at 37°C and under low-oxygen conditions (5.06% CO₂, 4.99% O₂, and 89.95% N₂) for 72 h. alamarBlue was added on day 16 postinduction at 10% of the well volume, and the plates were returned to the chamber for an additional 24 h. Fluorescence was determined at 585 nm after excitation at 540 nm. The initial screen was performed at 5 μM (see Data Set S1 in the supplemental material). Compounds exhibiting >85% inhibition of metabolic activity compared to that of control were selected for further dose-dependent analyses using 10 μM as the highest concentration (see Fig. S3 in the supplemental material). The IC₅₀ calculation was determined from dose-response curves calculated from normalized percent activity values and log₁₀-transformed concentrations using GraFit 5 software. Only compounds that showed a sigmoidal response were selected to report their gametocytocidal activity (see Table 2; see also Fig. S3 in the supplemental material). The final figures were prepared using GraphPad Prism 6 software (see Fig. S3).

TABLE 2.

In vitro antimalarial activity (IC₅₀) against gametocytes (NF54 strain) and parasites at asexual stages (NF54 and Dd2 strains)

MMV no.	Set	IC50 (nM) (mean ± SD) against:
		Gametocytesa in strain NF54	Parasites in asexual stagesb in strain:
			NF54	Dd2
MMV000248	Drug-like	310 ± 50	50 ± 6	90 ± 10
MMV019918	Drug-like	320 ± 60	280 ± 80	620 ± 280
MMV006172	Probe-like	420 ± 30	40 ± 3	60 ± 3
MMV019555	Probe-like	470 ± 60	20 ± 1	70 ± 5
MMV666125	Probe-like	480 ± 120	120 ± 20	50 ± 2
MMV019266	Drug-like	520 ± 30	310 ± 50	380 ± 20
MMV665943	Probe-like	570 ± 80	190 ± 30	190 ± 20
MMV665794	Probe-like	680 ± 50	90 ± 10	90 ± 5
MMV666693	Drug-like	730 ± 150	40 ± 6	20 ± 2
MMV011438	Probe-like	820 ± 160	50 ± 5	130 ± 20
MMV007384	Probe-like	870 ± 180	100 ± 7	190 ± 20
MMV396794	Drug-like	870 ± 200	180 ± 20	60 ± 6
MMV000448	Probe-like	1000 ± 280	50 ± 5	350 ± 40
MMV007591	Probe-like	1150 ± 150	920 ± 170	840 ± 400
MMV666079	Probe-like	1460 ± 180	210 ± 30	260 ± 20
MMV666687	Probe-like	1660 ± 210	270 ± 50	190 ± 8
MMV007224	Probe-like	1980 ± 180	320 ± 20	270 ± 20
MMV666686	Probe-like	2060 ± 260	450 ± 90	360 ± 30
Epoxomicin	Positive control	3.8 ± 0.2	NDc	ND

^aGametocytocidal activity was measured using alamarBlue, a fluorescent oxidoreduction indicator of gametocyte metabolic activity. The IC₅₀ values were obtained from dose-response curves calculated from normalized percent activity values and log₁₀-transformed concentrations (see Fig. 3S in the supplemental material). Epoxomicin and DMSO were used as positive and negative controls, respectively.

^bIn vitro antimalarial activity against parasites in asexual stages was evaluated using the SYBR green assay.

^cND, not determined.

Fluorescence microscopy.

A Zeiss Axio Imager M1 microscope with a ×100/1.4-numerical-aperture oil immersion objective and an AxioCam MRm digital camera was used to image live parasites as described previously with minor modifications (8). Briefly, control (untreated), parasites treated with IPP alone, drug-treated parasites, and drug-treated parasites supplemented with 200 μM IPP D10-ACP_L-GFP were incubated with 100 nM MitoTracker Red CM-H2XRos (Life Technologies) for 15 min at 37°C. Parasites were washed once with complete medium, and 0.25 μg/ml Hoechst 33342 (Life Technologies) was added to each condition immediately before imaging. The final figures were prepared using Adobe Photoshop CS2.

RESULTS

Identification of compounds that act on the apicoplast.

We used reversal of growth inhibition by IPP supplementation to interrogate the Malaria Box to identify compounds that have a mechanism of action that targets the apicoplast (8). From the 400 compounds tested, only the drug-like compound MMV008138 met our criteria of >95% growth inhibition at 5 μM for the compound alone and >60% growth recovery in the presence of inhibitor and 200 μM IPP, as detailed in Materials and Methods. MMV008138 showed at least 85% recovery of growth inhibition in the presence of IPP (Fig. 1A) when retested at 2.5 μM, indicating that this compound interferes with the function of the apicoplast as its mechanism of action. As expected, growth inhibition by FOS was 100% reversed by IPP supplementation. The complete data set listing growth inhibition at 5 μM for all the inhibitors tested, as well as the respective growth recovery in the presence of IPP, is available online (see Data Set S1 in the supplemental material).

FIG 1.

(A) MMV008138 at 2.5 μM showed at least 85% recovery of growth inhibition in the presence of 200 μM IPP after 72 h of incubation starting at ring stage. FOS was used as the positive control, as its inhibition is reversed by IPP supplementation. (B) Dose-dependent activity and growth recovery of MMV008138 in the presence of 200 μM IPP after 72 h of incubation. All experiments were performed using the P. falciparum Dd2 strain.

We determined that MMV008138 has an IC₅₀ of 350 nM for the Dd2 strain (Table 1). We observed >85% growth recovery at concentrations up to 2.5 μM MMV008138 and 200 μM IPP, but only ∼70% growth recovery was observed at 5 μM MMV008138 and 200 μM IPP in the Dd2 strain (Fig. 1B), which may indicate off-target effects at high concentrations. To avoid potential off-target effects, all of the following studies were performed at concentrations of ≤2.5 μM. We also noted that the kinetics of MMV008138 inhibition resembled that of FOS activity (Table 1, compare the 72- and 96-h IC₅₀s) (29–31). For comparison, we used DOX, which blocks protein translation in the apicoplast and shows a pronounced delayed-death phenotype (Table 1). Next, we decided to test MMV008138 against a FOS-resistant (FOS^r) strain to investigate DXR as the potential molecular target. The resistance in the FOS^r is due to an increase in the copy number of the Pfdxr gene, which encodes the enzyme targeted by FOS, allowing the parasite to overcome FOS-mediated inhibition of isoprenoid biosynthesis (22). Intriguingly, whereas FOS showed a 4-fold increase in the IC₅₀ for the FOS^r, relative to its parental strain Dd2, MMV008138 exhibited no increase in its IC₅₀ against FOS^r (Table 1, compare Dd2 and FOS^r IC₅₀s at 72 h).

TABLE 1.

In vitro activity (IC₅₀) of DOX, FOS, and MMV008138 against asexual stages in different P. falciparum strains^a

Compound	IC50 (nM) against:
	Dd2		NF54		D10-ACPL-GFP at 72 h	FOSr parasites at 72 h
	72 h	96 h	72 h	96 h
DOX	>4,500	900 ± 180	2,370 ± 410	380 ± 60	NDb	ND
FOS	880 ± 70	430 ± 50	980 ± 60	ND	1,610 ± 160	3,330 ± 150
MMV008138	350 ± 50	180 ± 30	300 ± 10	ND	370 ± 30	250 ± 10

^aData shown are mean ± SD.

^bND, not determined.

T. gondii is an apicomplexan parasite that, like P. falciparum, harbors an apicoplast that is essential for survival of the parasite. T. gondii causes toxoplasmosis in humans and can be life-threatening to immunocompromised individuals. Currently, antifolates form the basis of chemotherapy against both primary and secondary (recurrent) infection by T. gondii. Antifolate therapy is effective in limiting the growth of the acute tachyzoite stage but does not cure chronic infection. New drugs with different modes of action are, therefore, highly desirable for this pathogen as well. We tested MMV008138 against T. gondii, but no effect on the growth of the parasite was observed (see Fig. S1 in the supplemental material). This lack of activity may be due to the lack of transport of MMV008138, which is similar to previous observations using FOS. While T. gondii relies on the MEP pathway for isoprenoid precursor biosynthesis, the parasite is insensitive to high concentrations of FOS due to the lack of drug uptake at the level of the parasite plasma membrane (7).

Effect of MMV008138 on apicoplast development.

To investigate the effect of MMV008138 on apicoplast development, we used transgenic P. falciparum cell line D10-ACP_L-GFP to visualize the apicoplast (21). In this line, green fluorescent protein (GFP) is targeted to the lumen of the organelle, and its developmental changes along the intraerythrocytic cycle are readily observed. Microscopic analysis showed that the apicoplast elongation and division process was arrested in MMV008138-treated parasites. A single stunted apicoplast was present at a point where controls showed a well-developed tubular apicoplast or an organelle that already had given rise to multiple daughters. This pattern was indistinguishable from that of parasites treated with FOS (Fig. 2; see also Fig. S5 in the supplemental material). We also costained cells with MitoTracker, which accumulates in the mitochondria of living cells and is dependent on the mitochondrial membrane potential. We noted diminished labeling of the mitochondrion in both FOS- and MMV008138-treated parasites. A diminished membrane potential is consistent with the role of the apicoplast isoprenoid pathway in ubiquinone biosynthesis. It has been demonstrated that FOS inhibits ubiquinone biosynthesis (17, 32). Ubiquinones participate in the electron transport chain, and their biosynthesis relies on the MEP pathway to provide precursors for the isoprene side chain that anchors them into the inner membrane of the mitochondria. Importantly, the addition of 200 μM IPP rescued both apicoplast development and mitochondrial membrane potential in FOS- and MMV008138-treated parasites (Fig. 2), and this recovery was also observed at 1 μM and 350 nM MMV008138 (see Fig. S2 in the supplemental material). As mentioned above, the effect of DOX treatment on apicoplast development was not observed until the following intraerythrocytic cycle, explaining the slow action of this drug (11). As expected, 40 h of DOX treatment did not affect apicoplast development at this early time point (see Fig. S5B in the supplemental material), but the inhibition of apicoplast elongation was observed after 120 h of treatment (Fig. 2; see also Fig. S5C). In contrast, IPP supplementation restored the mitochondrial membrane potential but not apicoplast morphology in DOX-treated parasites. The fact that MMV008138 was fully rescued by IPP suggests a metabolic function for its target, potentially in IPP biosynthesis.

FIG 2.

Wide-field epifluorescence microscopy for the nucleus (Hoechst), apicoplast (ACP-GFP), and mitochondria (MitoTracker), comparing schizonts in untreated control parasites, FOS-treated parasites (10 μM), FOS-treated parasites (10 μM) in the presence of IPP (200 μM), MMV008138-treated parasites (2.5 μM), and MMV008138-treated parasites (2.5 μM) in the presence of IPP (200 μM) after 40 h of treatment. Parasites were treated with DOX (2 μM) in the presence of IPP (200 μM) for 120 h, since inhibition of apicoplast elongation was not observed at 40 h. Additional microscopic fields from each condition can be found in the supplemental material (see Fig. S5A, B, and C in the supplemental material).

Gametocytocidal activity among the Malaria Box compounds.

In our efforts to elucidate the mechanisms of action of compounds within the Malaria Box, we also evaluated their activities against other stages of the life cycle of the parasite. We decided to probe the Malaria Box collection for gametocytocidal activity, since new transmission-blocking antimalarials are needed and the gametocytocidal potentials of the Malaria Box compounds have not been evaluated to date. We identified a set of 22 compounds that showed >85% inhibition of gametocyte viability at 5 μM as assayed by alamarBlue. The complete data for all compounds are available online (see Data Set S1 in the supplemental material). From the set of 22 active compounds, 18 compounds acted in a clear dose-dependent manner, and 12 of them showed IC₅₀s of <1 μM (Table 2; see also Fig. S3). At a 5 μM concentration, MMV008138 did not have activity against late-stage gametocytes (see Data Set S1). The set of 18 compounds was tested in asexual intraerythrocytic stages against drug-resistant and drug-sensitive parasites. Three compounds, MMV000248, MMV006172, and MMV019555, showed midnanomolar IC₅₀s in late-stage gametocytes and low-nanomolar IC₅₀s in asexual stages (Table 2). An IC₅₀ of 3.8 nM was obtained for epoxomicin, a compound for which gametocytocidal activity had been reported previously (28) (Table 2; see also Fig. S3).

DISCUSSION

MMV008138 inhibits apicoplast elongation.

The apicoplast is indispensable for parasite survival and is unique to the parasite. The apicoplast is already a well-established and clinically used target for antibiotics in malaria and toxoplasmosis therapy (4). The apicoplast harbors hundreds of proteins, and there are likely numerous additional targets to be identified and exploited (4, 5, 33, 34). Recently, it was demonstrated that parasites can survive in vitro without the apicoplast when IPP is supplied at 200 μM. A reversal of growth inhibition by IPP supplementation was proposed as a method for potentially identifying compounds targeting apicoplast function (8). Here, we used the IPP supplementation assay to interrogate Malaria Box compounds that target the apicoplast in order to establish their mechanisms of action, and we identified MMV008138 as a new antiapicoplast lead (Table 1). Interestingly, we noted that the mechanism of action of MMV008138 is more like that of FOS than that of DOX. In general, drugs like DOX, which target apicoplast genome replication, transcription, protein translation, posttranslation modification, or protein turnover, exhibit a delayed-death phenotype (9–15). The kinetics of MMV008138 provided circumstantial support for the hypothesis that this drug acts more directly on the metabolism of the organelle than through interference with its biogenesis.

More directly, we showed that MMV008138 inhibited apicoplast elongation and disturbed the mitochondrial membrane potential and that these phenotypes were reversed by the presence of IPP (Fig. 2). FOS also showed inhibition of apicoplast elongation, as reported previously (7), and we have reported here that this phenotype can be reversed by IPP supplementation. A potential mechanism by which FOS prevents apicoplast elongation could be through inhibition of the isoprenoid modification of the tRNA on the adenine bases, which is essential for binding to the mRNA-ribosome complex and ensures translational integrity (33, 35). Four tRNAs in Plasmodium are predicted to be isoprenylated in the apicoplast (33). However, no studies have been reported so far that directly demonstrate tRNA isoprenylation in Plasmodium. Furthermore, FOS also disrupted the mitochondrial membrane potential, which was expected, as FOS inhibits ubiquinone biosynthesis (17, 32). The rescue of apicoplast elongation by IPP supplementation was not observed in parasites treated with DOX (Fig. 2), similar to chloramphenicol (8), which inhibits protein synthesis in the apicoplast by targeting the ribosomes. This suggests that DOX and MMV008138 act on different targets and that MMV008138 does not interfere with protein synthesis.

Altogether, our results are consistent with the hypothesis that MMV008138 may have its molecular target within the MEP pathway; therefore, we also tested this compound against FOS^r parasites. As mentioned above, the resistance in this parasite strain is due to an increase in the copy number of the Pfdxr gene (22). The FOS^r strain exhibited no change in its IC₅₀ for MMV008138 compared with that in its parental strain Dd2, suggesting that DXR is most likely not the molecular target (Table 1). Since the apicoplast is indispensable for apicomplexan parasites and is an attractive drug target, we tested MMV008138 against T. gondii, another important human pathogen, but it was inactive, similar to FOS, perhaps due to poor transport (7). Our efforts to identify the potential molecular target(s) for MMV008138 in P. falciparum may also help to establish the basis of its lack of activity in T. gondii.

It has been shown that antibiotics targeting the apicoplast, including FOS, do not affect late-stage gametocytes (20), and we observed that MMV008138 also did not affect late-stage gametocytes. This lack of activity was expected, since the apicoplast does not grow during gametocytogenesis, but others have noted that the mitochondrion undergoes remarkable morphological development during gametocytogenesis (36), which requires an active MEP pathway to supply IPP and DMAPP for ubiquinone biosynthesis. The low activities of FOS and MMV008138 against late-stage gametocytes might then be attributed to either (i) poor transport or (ii) prior biosynthesis and storage of the needed isoprenoids.

Due to its short half-life in plasma, FOS is not suitable for monotherapy; however, combination therapy studies in humans revealed success in maintaining total parasite clearance when FOS was used with artesunate or clindamycin (37–43). Thus far, our results indicate that MMV008138 affects apicoplast function and that its molecular target may differ from current antiapicoplast drugs, like FOS. Whether a compound like MMV008138 could comprise a new monotherapy or a useful adjunct to other antimalarials will depend upon many factors, including pharmacokinetic parameters.

Structure-activity relationship of MMV008138.

The most notable structural features of MMV008138 are its pipecolic acid and tetrahydro-β-carboline moieties. A review of the 400 compounds in the Malaria Box revealed that only MMV008138 possessed both of these functionalities. One other tetrahydro-β-carboline (MMV019690) and two related structures (MMV396719 and MMV396715) (Fig. 3) were identified among the 400 compounds, with only MMV019690 possessing the 1-aryl substituent present in MMV008138. Since none of the three other related structures possessed antiapicoplast activity, we conclude that the appropriate 1-aryl group and 3-carboxylic acid (i.e., pipecolic acid) moiety contribute to antiapicoplast activity. Interestingly, the clinical candidate, NITD609, has a scaffold structure that is very similar to that of MMV008138 and is currently in human clinical trials as a monotherapy for malaria (44–47). In addition to possessing the 1-aryl-tetrahydro-β-carboline, this potential antimalarial features a transposed pipecolic acid moiety in the form of a spiroamide and a methyl group in place of the carboxy group of MMV008138 (Fig. 3). It should be noted that MMV008138 is present in the Malaria Box as an unspecified mixture of four stereoisomers that differ in their configurations at C-1 and C-3. After we confirmed that MMV008138 was specifically targeting the apicoplast, MMV008138 was purchased from Sigma-Aldrich (again, an unspecified mixture of stereoisomers), and the repeat assay results matched those of the MMV sample. Of these possible stereoisomers, the (1R,3R)-stereoisomer of MMV008138 coincidentally provides a structure that closely mimics that of NITD609 (see Fig. S4 in the supplemental material). NITD609 targets PfATPase4, a plasma membrane Na⁺ efflux pump (44), and it is active against gametocyte stages (45). Is the (1R,3R)-stereoisomer of MMV008138 present in the Malaria Box, and if so, does it have activity similar to that of NITD609? At this point, we cannot address this question since, as mentioned, the low gametocytocidal activity of MMV008138 may be due to poor transport in late-stage gametocytes.

FIG 3.

Structures of MMV008138, three other tetrahydro-β-carbolines or close analogs in the Malaria Box, and clinical candidate NITD609.

It is also too early to know what off-target activities MMV008138 might possess. Tetrahydro β-carbolines comprise a large group of natural and synthetic indole alkaloids. These compounds are of great interest due to their diverse biological activities, and they possess a broad spectrum of pharmacological properties, including sedative, anxiolytic, hypnotic, anticonvulsant, antitumor, antiviral, and antiparasitic as well as antimicrobial activities (48). Additionally, their molecular targets span from the inhibition of topoisomerase, monoamine oxidase, and cyclic nucleotide phosphodiesterases to interaction with benzodiazepine receptors and 5-hydroxy serotonin receptors. However, to the best of our knowledge, MMV008138 has no other biological or enzymatic activities reported beyond its antimalarial activity in asexual intraerythrocytic stages (29–31).

New potential leads for transmission-blocking development.

Currently, primaquine is the only drug that is active against late-stage gametocytes, and only a small set of new transmission-blocking drugs are in clinical trials (49), supporting the urgent need for new leads against late-stage gametocytes. Therefore, we decided to interrogate the Malaria Box against late-stage gametocytes not only as a complementary assay to study their mechanisms of action but also to identify new gametocytocidal leads. Twelve compounds with IC₅₀s of <1 μM against late-stage gametocytes were identified. Three compounds, MMV000248, MMV006172, and MMV019555, showed midnanomolar IC₅₀s in late-stage gametocytes and low-nanomolar IC₅₀s in asexual stages (Table 2).

Among these three compounds, two basic pharmacophores were identified, the benzimidazole imine scaffold and the 9-aminoacridine/9-amino-tetrahydroacridine scaffold. MMV000248 (Fig. 4) is a promising drug-like compound that showed the best overall potency in late-stage gametocytes and asexual stages (Table 2). It possesses the bicyclic guanidine or benzimidazole imine scaffold, which is also present in four other compounds in the Malaria Box (Fig. 4). MMV396723 and MMV665902 contain pendant tertiary amine moieties that are similar to that of MMV000248. Three of the four compounds (MMV665902, MMV000445, and MMV000444) also resemble MMV000248 in bearing a secondary alcohol embedded in a 2- to 4-atom linker connecting the endocyclic nitrogen of the imidazole imine to an aromatic ring. The lack of confirmed gametocytocidal activity of these four compounds, combined with 10- to 20-fold increases in their previously reported asexual stage IC₅₀s (2), is quite striking. Overall, these data indicate strict structural requirements for both gametocytocidal and asexual-stage activity within this scaffold (Fig. 4).

FIG 4.

MMV000248 and related benzimidazole imine structures in the Malaria Box. The IC₅₀s for MMV396723 (∼1.25 μM), MMV665902 (∼1 μM), MMV000445 (∼1.135 μM), and MMV000444 (∼1.25 μM) in asexual intraerythrocytic stages were previously reported and can be found in the ChEMBL database (see https://www.ebi.ac.uk/chemblntd).

MV006172 and MMV019555 possess the 9-aminoacridine and 9-amino-tetrahydroacridine scaffolds, respectively, known to be antimalarial pharmacophores (50). MV006172 was shown to have moderate activity against the liver stage of Plasmodium yoelii (30), and this compound was also found to bind to the recombinant human α-2C adrenergic receptor (51). MMV019555 is a dimeric molecule that was originally explored as an acetylcholinesterase inhibitor (52). A search of the Malaria Box revealed three other 9-aminoacridines (MMV000304, MMV000448, and MMV006513) and no additional 9-amino-tetrahydroacridines (Fig. 5). Interestingly, MMV006172 bears a close resemblance to the three other 9-aminoacridines and quinacrine, which is a known potent antimalarial that is active against intraerythrocytic asexual stages of the chloroquine-resistant P. falciparum Dd2 strain (53) and inactive against P. falciparum gametocytes (54, 55). The asexual-stage potency of MMV006172, MMV019555, and MMV000448 (Table 2) may stem from their structural resemblance to quinacrine (IC₅₀, 5 to 10 nM [53]). Like quinacrine, both of these compounds feature a 5- to 7-atom tether to a basic amine moiety. The three other 9-aminoacridines in Fig. 4 either feature a shorter tether or lack a pendant basic amine. However, since quinacrine is inactive against P. falciparum gametocytes, this structural motif is clearly not sufficient for gametocytocidal activity.

FIG 5.

MMV006172, MMV019555, three related 9-aminoacridines in the Malaria Box, and quinacrine.

In conclusion, we identified one promising drug-like compound, MMV000248, showing activity against both asexual and sexual intraerythrocytic stages at low and midnanomolar values, respectively, and is a potential multistage lead that could be developed to cure malaria and stop its transmission.