A Male and Female Gametocyte Functional Viability Assay To Identify Biologically Relevant Malaria Transmission-Blocking Drugs A. Ruecker,a D. K. Mathias,b U. Straschil,a T. S. Churcher,c R. R. Dinglasan,b D. Leroy,d R. E. Sinden,a M. J. Delvesa Department of Life Sciences, Imperial College, London, United Kingdoma; W. Harry Feinstone Department of Molecular Microbiology and Immunology and the Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USAb; MRC Centre for Outbreak Analysis and Modelling, Department of Infectious Disease Epidemiology, School of Public Health, Imperial College, London, United Kingdomc; Medicines for Malaria Venture, Geneva, Switzerlandd

M

alaria is a disease of devastating human cost that has placed a tremendous economic burden on developing countries. Despite vigorous efforts by the global community and many successes, malaria has stubbornly resisted attempts at elimination from areas of high endemicity. Recent calls for malaria eradication have accepted that this problem will only be solved by combining and deploying different interventions that can act synergistically. One such class of interventions that is considered to be essential to the global eradication campaign includes therapeutics that prevent parasite transmission from the human host to the mosquito. These transmission-blocking therapies target the parasite at a critical population bottleneck when it is most vulnerable, and by preventing the onward spread of the disease, they limit the occurrence of new cases of infection (1). In Plasmodium falciparum, with every round of asexual intraerythrocytic infection, ⬃0.2 to 1% of parasites become committed to sexual development (2). After host erythrocyte reinvasion, these committed cells divert from the proliferative asexual pathway and differentiate into gametocytes—a process that takes ⬃10 to 12 days in P. falciparum (Fig. 1A). Gametocytes are solely responsible for onward parasite transmission to the mosquito. In P. falciparum, gametocyte development is divided into five stages (I to V) based upon morphological classification (3). Functionally, it is only the mature stage V gametocyte that is capable of transmission, with preceding stages invested in preparing the cell for its “one-shot” chance at infecting a mosquito. Upon gametocyte uptake in a mosquito blood meal, mature stage V gametocytes sense a change in environment due to a decrease in temperature and the presence of the gametocyte activating factor xanthurenic acid (XA) in the mosquito midgut and form gametes (4). Gametocytes are sexually dimorphic, with male and female gametocytes forming male and female gametes, respectively. Male gamete formation is a complex and rapid process completed within ⬃20 min involving gametocyte egress from the erythrocyte, increase in cell volume (rounding up), three rounds of DNA replication, assembly of axonemes, and the discharge of up to eight flagellate male gametes from the exflagellation center (5). Female gamete formation is

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visually less spectacular, involving egress from the erythrocyte and translation of mRNAs previously stored under translational repression that is controlled by the DDX class helicase DOZI (development of zygote inhibited) (6). The motile male gamete “swims” through the blood meal until contacting a female gamete, fertilization ensues, and this then results in the transformation of the newly formed zygote into an ookinete (7, 8). The motile ookinete then migrates to and through the midgut epithelial wall, where it colonizes the mosquito and develops into an oocyst (9). Over a period of 16 to 21 days, thousands of sporozoites may form within the oocyst, which eventually ruptures and releases the sporozoites into the mosquito hemolymph, where they travel to the salivary glands to await reinfection into a human host upon a subsequent mosquito bite. The current “gold standard” test of the ability of a compound to prevent transmission to the mosquito is the standard membrane feeding assay (SMFA) (Fig. 1E), which permits the feeding of gametocytes in the absence or presence of an intervention to mosquitoes in an experimentally controlled manner (10, 11). This assay has been universally adopted because it links data generated in the laboratory to that observed under field conditions (12). Generally for P. falciparum SMFAs (here, “PfSMFAs”), cultured mature stage V gametocytes are exposed to the compound of interest for 24 h and then either fed to mosquitoes without removing the compound so that it enters the mosquito blood meal and can

Received 18 June 2014 Returned for modification 18 July 2014 Accepted 18 September 2014 Published ahead of print 29 September 2014 Address correspondence to Michael J. Delves, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.03666-14. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.03666-14 The authors have paid a fee to allow immediate free access to this article.

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Malaria elimination will require interventions that prevent parasite transmission from the human host to the mosquito. Experimentally, this is usually determined by the expensive and laborious Plasmodium falciparum standard membrane feeding assay (PfSMFA), which has limited utility for high-throughput drug screening. In response, we developed the P. falciparum dual gamete formation assay (PfDGFA), which faithfully simulates the initial stages of the PfSMFA in vitro. It utilizes a dual readout that individually and simultaneously reports on the functional viability of male and female mature stage V gametocytes. To validate, we screen the Medicines for Malaria Venture (MMV) Malaria Box library with the PfDGFA. Unique to this assay, we find compounds that target male gametocytes only and also compounds with reversible and irreversible activity. Most importantly, we show that compound activity in the PfDGFA accurately predicts activity in PfSMFAs, which validates and supports its adoption into the transmission-stage screening pipeline.

Gametocyte Sex-Selective Transmission-Blocking Assay

A

Transmission Cell Biology

Human Mosquito

Gamete formation

Gametocyte development Fertilization

I

II

III

Zygote

IV

Ookinete

Oocyst

Stage V (infectious)

B

Immature gametocyte assays

“Early stage” Biology assayed “Late stage”

Cell biology only partially overlaps with PfSMFA

Drug exposure

C

PfDual gamete formation assay (PfDGFA)

Drug “washout”

D

PbOokinete Development assay (PbODA)

E

PfSMFA (gold-standard)

Simulates initial stages of PfSMFA

Drug “carryover” Drug “washout”

FIG 1 An overview of current transmission-stage drug assays highlighting the interrogated parasite cell biology (light blue lines) and parasite cell types receiving drug exposure (dark blue lines). (A) Overview of P. falciparum transmission to the mosquito. (B) “Early” and “late” gametocyte stage transmission-blocking drug screening assays interrogate immature stages of gametocyte development. (C) The P. falciparum dual gamete formation assay (PfDGFA) interrogates only functionally mature stage V gametocytes. (D) The P. berghei ookinete development assay (PbODA) interrogates early vector-stage parasite development. (E) The gold standard P. falciparum standard membrane feeding assay (PfSMFA) interrogates the broadest range of transmission cell biology from the mature stage V gametocyte through oocysts in the mosquito midgut.

also act during vector stage development (“carryover”), or the gametocytes are washed free of compound before feeding (“washout”). Transmission blocking is then assessed by counting mosquito midgut oocyst burden 7 to 10 days later. The cell biology of the washout format identifies compounds that act upon functionally mature stage V gametocytes to either kill them directly or irreversibly compromise their future onward development in the mosquito (Fig. 1E). These are currently considered the “ideal” properties for a transmission-blocking drug (13). By comparison to the washout format, the carryover format identifies those compounds that must be physically present in the mosquito blood meal to elicit a transmission-blocking response (Fig. 1E)—a property that is less than ideal but still considered “desirable” (13) as long as the compound’s stability permits efficacious concentrations for as long as infectious gametocytes are present in a patient (estimated to be 13.4 days after artemisinin combination therapy [ACT] [14]). The PfSMFA is a powerful assay; however, it is extremely expensive and technically challenging to perform, and with the use of mosquitoes will never be suitable for highthroughput screening. Currently reported high-throughput gametocyte transmission-blocking assays broadly focus on immature stages of gametocyte development and so crucially only share limited overlap in cell biology with the gold standard PfSMFA (Fig. 1B and E) (15–18). This may seed screening campaigns with many false-positive compounds that are active only against nontransmissible immature gametocytes. Such compounds will have limited utility in the clinic as at the point of seeking treatment, malaria patients frequently already have transmissible stage V gametocytes present in their blood (19, 20). Moreover, asymptom-

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atic submicroscopic (stage V) gametocyte carriers are thought to greatly contribute to the transmissible pool of parasites, thus further necessitating the development of drugs to target the stage V gametocyte population (21). Additionally, early stage gametocytes are already sensitive to many antimalarials currently in use in the field (22–24): consequently, screening programs to discover additional drugs to target them do not seem a priority for malaria eradication. To address the highlighted shortcomings of existing assays, we implement two assays that specifically match certain aspects of the cell biology interrogated by the PfSMFA and that represent the most relevance to transmission-stage drug discovery: (i) the novel P. falciparum dual gamete formation assay (PfDGFA), which identifies transmission-blocking compounds that affect the functional viability of the P. falciparum mature male and female stage V gametocytes (those directly responsible for transmission) and (ii) the previously reported Plasmodium berghei ookinete development assay (PbODA) (25), which identifies those compounds that affect early vector-stage development in P. berghei (Fig. 1C and D). By using this combination of assays, we have been able to study the entire 400-compound Malaria Box library (26), identifying those compounds with ideal gametocytocidal properties and those with desirable vector-stage activity. We then confirmed the predictive power of the assays with PfSMFAs of selected compounds. By accurately assaying the parasite cell biology responsible for transmission, we anticipate efficient identification and prioritization of transmission-blocking compounds that will have a greater probability of being efficacious under field conditions and thus make a significant contribution to malaria eradication.

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Drug “carryover”

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MATERIALS AND METHODS

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treatment; Thermo Scientific) which already contained 5 ␮l ookinete medium in each well (5 ␮M final concentration of xanthurenic acid). The cells were allowed to settle at room temperature. At ⬃8 min postinduction, a cellular monolayer was visible at the bottom of each well, and the plate was transferred to the automated stage of a Nikon Eclipse Ti inverted microscope. Using a custom algorithm written within the JOBS module of NIS Elements software (version 4.12.01) (Nikon), the plate was autofocused using a ⫻10 objective under phase contrast and a Neo sCMOS camera (Andor Technology) to maximize the captured field of view, and the z-coordinate at the center of each well was stored. At 20 min postinduction, exflagellation was captured in the center of each well using the prestored focused z-position. Each recording captured 20 frames/well at ⬃4 frames per second (fps), which resulted in a total recording time of 5 s/well. The wells were read in a meandering pattern from well B2 to well H11, which was repeated three times, in total taking ⬃11 min to complete. The plate was then transferred into a humidified incubator (80% relative humidity) at 26°C for 24 h to imitate mosquito midgut conditions and allow accumulation of Pfs25 on the surface of the female gamete. Female gamete formation readout. Upon induction of gamete formation, female gametocytes are released from their translational repression. One of the translationally repressed mRNAs encodes Pfs25, which following its translation is exported to the surface of the female gamete and is readily detectable by live immunostaining. Pfs25 surface expression is first detectable ⬃2 h after gamete formation; however, it is easier to observe by low-magnification fluorescence microscopy after 24 h. After a 24-h incubation (day 16), anti-Pfs25 4B7 monoclonal antibody (MAb) (from MR4) coupled to Cy3 (28) was added to the plate at a 1:3,000 dilution of a 2.0-mg/ml stock (0.67-␮g/ml final concentration). The plate was returned to the microscope, and activated female gametes were visualized at a ⫻20 objective using a Cy3 fluorescence filter. Another automated algorithm captured four high-resolution images at identical locations in each well of the plate. Notably this assay permits male and female gamete formation to be recorded and directly compared on exactly the same experimental well. PfDGFA—washout format. The PfDGFA was performed as described above. However, prior to triggering gamete formation, test compounds were washed from the gametocytes by removing 185 ␮l culture supernatant from each well and resuspending the settled cell pellet with fresh complete culture medium. The cells were allowed to settle over 2 h before repeating the process twice more over a total of 6 h. To prevent premature gamete formation due to the repeated plate manipulation, each washing step was executed swiftly, and then the plates were immediately returned to the 37°C incubator and hypoxia chamber. Gamete formation was then triggered, and the assay proceeded as described above. The PfDGFA was performed with four independent biological replicates containing one technical replicate for each compound and for each 12-point dose-response analysis (carryover and washout formats). Semiautomated data analysis for PfDGFA. P. falciparum male exflagellation events were identified and counted by a standardized algorithm within NIS Elements that was based upon a previously described algorithm (28). Briefly, male gamete formation is an event defined by the fast movement of the highly motile male gametes escaping the remnants of the gametocyte. In an erythrocyte monolayer, exflagellation can be readily identified by localized perturbation of the usually almost motionless surrounding erythrocytes. By comparing the 20 captured frames for each data point, it is possible by image subtraction to identify those areas of the field that are moving. These were then enhanced by applying smoothing and regional maxima filters to the composite image. Finally, exflagellation centers were identified by threshold determination based on pixel intensity, size, and circularity. The mean exflagellation count for each sample well (corresponding to the three recorded passes during data capture) was calculated and evaluated with reference to the mean of the six DMSO negative-control wells and six methylene blue (MB) positive-control wells. The percentage of inhibition was then calculated using the equation

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Open access Malaria Box library. The 400-compound open access Malaria Box library (26) was obtained as five 96-well plates from the Medicines for Malaria Venture (MMV) (Geneva, Switzerland) containing compounds stamped at 10 mM in dimethyl sulfoxide (DMSO). For verification of the hits obtained in the primary screen in this study, active compounds were repurchased as powders from ChemBridge, Molport, and Sigma, where available (see Data Set S1 in the supplemental material). Stock solutions were prepared from powders at 10 mM in DMSO. P. falciparum in vitro asexual parasite culture. Asexual P. falciparum NF54 strain cultures were routinely maintained between 0.3% and 3% parasitemia and 4% hematocrit O⫹ blood (NHS National Blood Service) supplemented with 0.16 mg/ml heparin in a 10-ml total culture volume under 3% O2–5% CO2–92% N2 gas. Complete culture medium (RPMI medium supplemented with 25 mM HEPES, 50 mg/liter hypoxanthine, 2 g/liter glucose, 2 g/liter sodium bicarbonate, and 10% A⫹ human serum) was replaced daily, and cultures were passaged every 48 to 72 h. Due to the progressive accumulation over time of mutations deleterious to gametocyte growth (27), low-passage-number cryovials were thawed every 6 weeks to maintain optimum numbers of functionally viable gametocytes in cultures. P. falciparum in vitro gametocyte culture. On day 0, an asexual culture with ⬃3% ring stages was used to seed gametocyte cultures at 1% rings and 4% hematocrit in a 10-ml total volume (25-cm2 flask; Nunc) under 3% O2–5% CO2–92% N2 gas. Spent complete culture medium was carefully replaced daily for 14 days, with all media, pipettes, and work surfaces heated to 37°C. Under these conditions, cultures follow a very steady and reproducible development, with asexual parasitemia rising to a peak and crashing at days 4 to 5, stage II gametocytes visible at day 7, stage III gametocytes visible at day 9, stage IV gametocytes visible at day 11, and stage V gametocytes visible at day 14. To manage culture workload, on day 7 of culture (stage II), two gametocyte culture flasks were routinely pooled into one flask with no demonstrable effect on final culture quality. On day 14, gametocyte cultures (stage V) were tested for maturity and functional viability by quantifying male gamete formation (exflagellation) as described previously (28). Briefly, 200 ␮l of the culture was withdrawn and quickly centrifuged, the supernatant was discarded, and the remaining pellet was resuspended in 5 ␮l ookinete medium (RPMI medium with 25 mM HEPES, 50 mg/liter hypoxanthine, 2 g/liter sodium bicarbonate, 100 ␮M xanthurenic acid, and 10% A⫹ human serum). The resuspended culture was added to a FastRead disposable hemocytometer slide (Immune Systems) and allowed to settle as a monolayer at room temperature for ⬃20 min (28). The culture was considered acceptable for the PfDGFA if ⬎50 exflagellation centers per field were observed under bright-field illumination and a ⫻10 objective. PfDGFA— carryover format. Complete culture medium (150 ␮l) was dispensed to individual inside wells of a sterile round-bottom 96-well plate (NuncMicroWell plate with Nunclon Delta surface treatment; Thermo Scientific). The outside wells were filled with equal amounts of sterile distilled water (dH2O) to prevent evaporation and act as a temperature buffer during plate handling. The Malaria Box compounds were then added to all inside wells at a final concentration of 1 ␮M or for a 12-point dose-response analysis (maximum assay concentrations of 10, 25, or 50 ␮M [compound dependent]), and plates were prewarmed at 37°C for 20 min. DMSO (0.25% final assay concentration) and methylene blue (MB [10 ␮M final assay concentration]) served as negative and positive controls, respectively. The day-14 gametocyte culture was resuspended in 7.5 ml complete culture medium, and 50 ␮l was dispensed into all inside wells. The plates were then transferred to a hypoxia chamber and incubated at 37°C for 24 h. Male gamete formation readout. On day 15, gamete formation was induced at room temperature (20°C) by rapidly discarding 100 ␮l supernatant from each well, resuspending gametocyte pellets in the remaining 100 ␮l complete culture medium, and transferring the mixture to a flatbottom 96-well plate (NuncMicroWell plate with Nunclon Delta surface

Gametocyte Sex-Selective Transmission-Blocking Assay

冋 冉 冊册 a⫺b c⫺b

100 ⫺ 100

冉 冊

Z⬘ ⫽ 1 ⫺ 3

d⫹f g⫺h

where d is the median absolute deviation of the positive controls, f is the median absolute deviation of the negative controls, g is the median of the positive controls, and h is the median of the negative controls. To incor-

December 2014 Volume 58 Number 12

SSMD ⫽

⌫[(4 ⫺ 1) ⁄ 2] ⌫[(4 ⫺ 2) ⁄ 2]

冑

2 d៮1 4 ⫺ 1 s1

where d៮1 is the mean difference between the measured compound value and median of plate negative-control values, s1 is the standard deviation of the individual differences between replicates, and ⌫ is the gamma function. Fifty percent inhibitory concentrations (IC50s) were calculated in GraphPad Prism version 6 from dose-response data using the following curve-fitting function: log(agonist) vs normalized response – variable slope. The similarity or difference between male and female dose-response curves was evaluated within the software by using the extra sumof-squares F test (P value cutoff of 0.05). Statistical analysis of PfSMFA oocyst intensity was carried out using generalized linear mixed models (GLMMs) with a negative binomial error structure (30). The impact of compound washout over both replicates was assessed for each intervention separately, with the treatment and washout statuses included as fixed effects and with oocyst intensity allowed to vary between experiments at random. The change in the efficacies of different interventions caused by washing out was determined by including all data in a single GLMM with intervention and treatment as fixed effects and the experiment number as a random effect. The impact of washing out was initially allowed to vary between interventions before the different interventions were grouped together until the most parsimonious model was identified (using the likelihood ratio test).

RESULTS

Development of the PfDGFA. We have recently shown that male gametocytes are generally more sensitive to antimalarial drugs than female gametocytes (28). This is highly significant as males may only represent ⬃20% of the total gametocyte population, and so assays without a sex-specific readout are not likely to identify male-targeted transmission-blocking compounds as they fall below the threshold of detection (31, 32). As both gametocyte sexes are required for transmission, a combined male and female gametocyte readout is essential to maximize the screening potential of a gametocyte assay. Our previously reported assay was performed in Eppendorf tubes with manual data capture taking male and female readouts independently and on separately prepared samples, which is not suitable for high-throughput screening. Here we have developed a 96-well format and utilized automated microscopy to greatly increase throughput and permit male and female readouts to be measured on exactly the same parasite population in a sample well (Fig. 2). To miniaturize the assay, round-bottom 96-well plates were used during gametocyte incubation with test compounds to better mirror the profile of an Eppendorf tube. The compact cell pellet formed at the bottom of the round well better facilitates the medium changes necessary for the washout format (described below) without removing cells. To ensure that the 96-well format faithfully reproduces data comparable to the existing assay, a panel of known and marketed antimalarials was screened (Fig. 3; see Table S1 in the supplemental material) and compared to reported data (28) (see Fig. S1 in the supplemental material). Previously, at 1 ␮M, the endoperoxides dihydroartemisinin, artemisinin, and artesunate had moderate activity (62.0 to 80.3% inhibition) against male gametocytes but no effect on females, even at 10 ␮M. In broad agreement, the 96-well assay demonstrated 62.6 to 68.6% inhibition against males

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where a is number of recorded exflagellation centers in sample well, b is the mean number of exflagellation centers recorded in the MB positivecontrol wells, and c is the mean number of exflagellation centers recorded in the DMSO negative-control wells. To identify female gametes, another algorithm was employed that applied a regional maximum filter to enhance and separate individual cells. The gametes then were identified by threshold determination based on pixel intensity, size, and circularity, and the counts were exported to MS Excel. For each well, four images were recorded and so were used to calculate the mean female gamete count for the well, which was then evaluated with reference to the control wells as described above. PbODA. All work involving laboratory animals was performed in accordance with the EU regulations EU Directive 86/609/EEC and within the regulations of the United Kingdom Animals (Scientific Procedures) Act 1986. The PbODA was set up identically to that described by Delves et al. (25) and differed only in data capture and analysis. Instead of quantifying ookinete development of the CTRPp.GFP reporter strain by measuring fluorescence intensity in a fluorescent plate reader, individual ookinetes were recorded by fluorescence microscopy at ⫻10 objective on a Nikon Ti inverted microscope by automated image capture. The same algorithm used to identify female gametes was then employed to identify ookinetes by using threshold determination with ookinete-specific pixel intensity, size, and circularity parameters. Percent inhibition was then calculated as described above. The PbODA was performed with four biological replicates. PfSMFAs. Test compounds in DMSO were added to separate wells of a gametocyte culture (P. falciparum NF54, 6-well plate, 2.5% gametocytemia) in duplicate at a final concentration of 1 ␮M and incubated for 24 h. For the carryover experiment, erythrocytes from one well per compound were resuspended in medium and pelleted at 900 ⫻ g in an Eppendorf 5810 centrifuge with a swinging-bucket rotor. Spent medium was then aspirated, and pellets were resuspended with equal volumes of prewarmed, heat-inactivated human serum to bring each erythrocyte/gametocyte pellet to 50% hematocrit. To dilute the gametocytemia to 0.1%, 40 ␮l of each suspension was transferred to new, prewarmed tubes and brought up to 1 ml with uninfected blood (50% hematocrit). Each test compound was added back to the diluted gametocytes at a final concentration of 1 ␮M, and then 250 ␮l was pipetted into two membrane feeders per compound. For each feeder, 50 to 60 mosquitoes (Anopheles gambiae Keele strain) in pint-sized cup cages were allowed to feed for 30 min, after which mosquitoes were chilled on ice for 10 min and unfed females were removed. Mosquitoes were then maintained on a solution of 10% sucrose at a constant temperature of 26°C and relative humidity of 80% until 8 days postfeeding, at which time midguts were dissected, stained (0.1% mercurochrome for 15 min), and examined for oocysts. For the washout experiment, compound-exposed cultures (one well per compound) were treated as described above with two changes. First, prior to pelleting erythrocytes/gametocytes, spent medium was aspirated from each well and washed with 5 ml of prewarmed complete culture medium. The cells were allowed to settle for 2 h at 37°C, and the wash was repeated twice for a total of three washes. Second, following dilution of compound-exposed cultures to 0.1% gametocytemia, no compounds were added back prior to feeding mosquitoes. Therefore, in the washout experiment, parasites were exposed to each compound in the gametocyte culture but not in the membrane feeder or the mosquito gut. Statistical analyses. The robust Z= factor of both primary screens was calculated for each 96-well plate using the formula

porate the four independent replicate data points generated for each compound in each screen and evaluate the robustness of the data, the strictly standardized mean difference (SSMD) was again utilized. The SSMD was calculated as described by Zhang et al. (29) using the formula

Ruecker et al.

Day 0 initiate Day 7 cultures pool 2x cultures at ~1% rings (daily medium change day 1-14)

Day 14 confirm Stage V gametocyte viability

Day 15 male read-out record exflagellation (movement)

Begin assay

carryover format

Day 16 female readout (fluorescence, shape, size)

drug exposure washout format 24 hr 37°C ~20 min RT

24 hr 26°C RT

FIG 2 Schematic overview of the P. falciparum dual gamete formation assay (PfDGFA) from culture induction to assay readout.

male female

100

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80

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40

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Am od i A r aqu te in m e et he Ar D r te H s Ar un A M t ef a lo H em te qu al is of ini i n M e an n e f (R tr lo i qu ace ne in m e ic ( Pa + ) m RS a P q ) Su rim uin lp aq e ha uin d e R iazi ib n At of e ov lav C aq in M yc uon e t lo hy g u e a l Az ene nil ith b r lu D om e ox y c Th ycy in P y i o s clin t rim r e e e pt Py tha on ro m i na ne r id in e

0

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Methylene Blue 100 male IC50 39.13 nM

50

female IC50 43.1 nM

0 10 0 -50

10 1

10 2

10 3

10 4

10 5

Concentration nM

FIG 3 Validation of the PfDGFA. (A) Twenty current antimalarials were evaluated in the 96-well-format PfDGFA at 1 ␮M in quadruplicate independent experiments. Compounds demonstrating ⬎50% inhibition were considered active. Assay data generally concur with those reported previously (28), with solid bars denoting a nonsignificant difference from previous data and hatched bars denoting a significant deviation (P ⫽ 0.05, unpaired Student’s t test). Full data comparison is shown in Fig. S1 in the supplemental material. Blue bars show the percentage of inhibition with male readout, and red bars show the percentage of inhibition with female readout. Error bars denote the standard error of the mean (SEM). (B) A dose-response curve of methylene blue showed low nanomolar equipotent activity against male and female gametocytes, and thus 10 ␮M was selected as a positive control for screening in the PfDGFA. Shown are results from quadruplicate independent experiments; error bars denote the SEM.

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Pf DGFA validation

at 1 ␮M, and additionally artemether showed weak activity with 51.6% inhibition. Pyrimethamine and cycloguanil were both confirmed to be male specific at 1 ␮M, with 73.2% and 76.9% inhibition, respectively. The activity of thiostrepton closely matched previous data, giving 74.2% and 26.2% inhibition of males and females, respectively, in the 96-well assay, compared to 92.5% and 22.9% previously observed. Pyronaridine, which was previously found to be weakly active with 97.9% inhibition at 10 ␮M against female gametocytes (but only 26.6% at 1 ␮M), showed greater activity in the 96-well assay, exhibiting 78.3% inhibition at 1 ␮M. Finally, by sampling male and female gametocytes from exactly the same well, it was possible to accurately determine that methylene blue has equipotent activity (male IC50, 39.1 nM; female IC50, 43.1 nM) (Fig. 3B). Consequently, methylene blue was selected as the positive control for both male and female readouts in subsequent library screening. Identification of Malaria Box compounds with transmission-blocking activity. The 400-compound open access Malaria Box library comprises a distillation of ⬃20,000 compounds showing activity against P. falciparum asexual parasites identified from larger screening campaigns designed to maximize chemical diversity and includes 200 “drug-like” and 200 “probe-like” compounds (26). This library was selected due to its “open” nature, which permits both a comparison of data to those from existing assays and serves as a benchmark by which to evaluate any future assays. The entire library was screened at 1 ␮M with four independent biological replicates in two distinct assays—the previously reported P. berghei ookinete development assay (PbODA) (25) and the 96-well format P. falciparum dual gamete formation assay (PfDGFA). The PfDGFA simultaneously assesses the functional viability of mature male and female stage V gametocytes as reported by their ability to form gametes—the initial event of “vector-stage” parasite development. For simplicity, the primary screen was performed with compounds present for 24 h with the gametocyte followed by continued exposure to the compound during gamete formation (carryover format) (Fig. 1C and 2). Therefore, active compounds either kill the gametocyte (have gametocytocidal activity), permanently damage the gametocytes in such a manner that they are alive but cannot complete gamete formation (sterilize them), or interfere directly with the process of gamete formation once triggered (are contraceptive) (Fig. 4). The presence of morphologically “normal” mature stage V gametocytes does not necessarily correlate with their ability to form gametes (33). In contrast, the functional viability of mature stage V gametocytes is very sensitive to culture manipulation and environmental factors, with a reduction of temperature by ⬎7°C enough to trigger gam-

Gametocyte Sex-Selective Transmission-Blocking Assay

transmission-blocking activity

“carryover”

“washout”

gamete formation +compound

gamete formation -compound

xx

xx

gametocytocidal (dead) sterilizing (alive, but onward viability compromised) contraceptive (interferes with the process of gamete formation)

phenotype post-induction of gamete formation

FIG 4 Conceptual phenotypes post-gamete formation for gametocyte-focused transmission-blocking drugs. Gametocyte-targeted transmissionblocking compounds can act to prevent gamete formation by three distinct mechanisms: (i) directly killing the gametocyte (gametocytocidal), (ii) compromising the gametocyte in such a manner that onward development is impossible to complete (sterilizing), and (iii) interacting directly with the process of gamete formation (contraceptive). By triggering gamete formation in the presence or absence of the test compound, it is possible to distinguish the irreversible gametocytocidal and sterilizing compounds from the reversible contraceptive compounds.

TABLE 1 Malaria Box hits identified by PfDGFA and PbODA Result with 1 ␮M compounda: PfDGFA Male gametocytes

Female gametocytes

PbODA

Compound ID no.

Subset

% inhibition

SEM

SMMD

% inhibition

SEM

SSMD

% inhibition

SEM

SSMD

MMV000448 MMV085203 MMV665827 MMV667491 MMV665980 MMV666691 MMV019918 MMV666026 MMV665977 MMV665878 MMV006389 MMV020439 MMV007116 MMV006457 MMV007127 MMV665830 MMV645672 MMV665972 MMV396749 MMV665941

Probe-like Probe-like Probe-like Probe-like Probe-like Probe-like Drug-like Probe-like Probe-like Drug-like Probe-like Drug-like Drug-like Probe-like Probe-like Probe-like Probe-like Probe-like Drug-like Probe-like

97.47 96.85 92.37 83.58 80.70 77.35 74.09 71.26 71.21 70.87 70.66 67.37 65.80 63.56 62.89 62.29 58.81 54.08 48.23 19.70

1.33 2.08 3.62 12.98 7.91 5.93 11.71 12.55 5.83 1.25 6.41 6.35 8.34 10.85 4.29 18.19 22.14 17.15 3.66 12.32

⫺2.21 ⫺2.13 ⫺2.24 ⫺0.85 ⫺0.97 ⫺2.54 ⫺0.52 ⫺0.61 ⫺3.32 ⫺2.13 ⫺0.83 ⫺2.38 ⫺2.05 ⫺1.48 ⫺0.80 ⫺0.69 ⫺0.65 ⫺0.52 ⫺0.63 ⫺0.63

24.60 ⫺15.86 ⫺15.24 27.26 73.75 ⫺7.34 38.13 ⫺5.89 ⫺14.99 36.64 11.01 ⫺15.45 9.93 2.21 8.27 56.10 ⫺16.41 10.70 24.02 44.59

7.82 25.95 16.94 10.76 12.33 12.62 12.60 7.11 6.52 7.76 11.19 11.63 9.76 13.53 6.96 12.13 15.92 17.59 17.48 18.58

⫺17.66 0.17 0.28 ⫺0.91 ⫺0.77 0.01 ⫺1.19 0.31 0.78 ⫺2.02 ⫺0.35 0.41 ⫺0.20 ⫺0.02 ⫺0.53 ⫺1.44 0.19 ⫺0.15 ⫺0.48 ⫺0.79

96.20 ⫺14.08 41.89 ⫺7.36 14.01 72.74 ⫺9.54 2.46 20.52 58.53 71.31 78.13 97.88 27.95 56.77 ⫺10.78 97.46 ⫺6.48 54.64 62.53

1.14 4.50 6.91 5.55 21.01 6.19 3.12 18.69 20.27 10.06 17.10 5.07 0.68 10.34 20.72 0.72 1.10 6.64 11.33 11.62

⫺2.04 1.03 ⫺1.83 0.01 ⫺0.13 ⫺1.72 0.29 0.10 ⫺0.39 ⫺1.31 ⫺0.91 ⫺2.12 ⫺2.08 ⫺1.09 ⫺0.68 1.00 ⫺1.50 0.07 ⫺2.16 ⫺1.32

a Shown is the mean percentage of inhibition compared to the DMSO control in the PfDGFA and PbODA. The screening data set for entire library can be found in Data Sets S1 and S2 in the supplemental material. SEM, standard error of the mean; SSMD, strictly standardized mean difference. Robustness of data: ⬍⫺5 or ⬎⫹5, extremely strong; ⬍⫺1.28 or ⬎⫹1.28, moderate; ⬍⫺0.5 or ⬎⫹0.5, weak; ⬍⫺0.25 or ⬎⫹0.25, extremely weak.

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reversible

irreversible

no effect

ete formation (34). All combined, these factors result in a highly variable baseline for gamete formation that shows relatively high inter- and intraplate variation. Consequently the PfDGFA screen displayed relatively low mean Z= factors of 0.43 ⫾ 0.31 (mean ⫾ standard deviation [SD]) for the male readout and 0.36 ⫾ 0.29 for the female assay. This was mitigated, however, by the use of the four independent replicates. In the PfDGFA screen, a compound was considered a “hit” if it gave ⬎50% inhibition at 1 ␮M. Additionally, as the screen contained independent replicate data, it was possible to calculate the strictly standardized mean difference (SSMD) of each compound tested in the screen (29, 35). The SSMD gives a statistical measure of the robustness of compound activity across all replicates by taking into consideration the interreplicate variation and plate-specific positive and negative controls. In total, 34 compounds (14 drug-like and 20 probe-like) met our hit criterion, with 12 having a “strong” SSMD in the male or female assay (SSMD between ⫺1.65 and ⫺5), 8 considered “moderate” hits (SSMD between ⫺1.64 and ⫺1), and 14 considered “weak” hits (SSMD between ⫺0.99 and ⫺0.5) (Table 1; see Data Set S1 in the supplemental material). In agreement with our previous finding that female gametocytes are less drug sensitive than male gametocytes, only two of these compounds (MMMV665980 and MMV665830) additionally inhibited female gamete formation ⬎50% in the primary screen. Indeed, no compounds were identified that possessed specific activity against female gametocytes. In parallel, the library was screened at 1 ␮M in the PbODA, which simulates in vitro the first 24 h of parasite development in the mosquito. In this assay, gametocyte-infected mouse blood is introduced to compounds simultaneously with the induction of

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TABLE 2 Malaria Box compounds analyzed for dose response Result froma: PfDGFA (IC50 [nM]) Female gamete formation

PfDGFA phenotype

Carryover

Washout

Carryover

Washout

PbODA (IC50 [nM])

MRC-5 fibroblasts (CC50 [nM])

Dual irreversible (gametocytocidal or sterilizing) MMV000448 MMV665830 MMV667491 MMV665941 MMV019918

103.3 139.2 182.7 977.3 25.54

247 73.19 81.64 419.2 65.07

1,074 188.8 473.1 445.5 159.40

4,019 223.6 173.6 292.9 512.20

292.2 6,505 7,731 991.4 7,819

2,864.97 4,148.62 3,494 353.55 4,028.10

Male irreversible (gametocytocidal or sterilizing) MMV007127 MMV085203 MMV665878 MMV666691 MMV666026 MMV396749

3,414 95.96 240.7 481.1 3,710 2,903

3,950 331.3 665.6 1,977.00 14,403 7,747.00

Inactive Inactive 383.9 Inactive Inactive 6,452.00

Inactive Inactive 13,136 Inactive Inactive Inactive

2,273 4,248 463.6 96.22 5,759 ND

⬎32,000 9,878.88 ⬎32,000 4,489.85 ⬎32,000 ⬎32,000

Male reversible (contraceptive) MMV665977 MMV020439 MMV006389 MMV645672 MMV665827 MMV007116 MMV006457

44.95 288.2 2,136 3,241 364.7 569.7 237

Inactive Inactive Inactive Inactive 16,546 20,742 1,363.00

Inactive Inactive Inactive Inactive Inactive Inactive Inactive

Inactive Inactive Inactive Inactive Inactive Inactive Inactive

2,174 487.1 1,065 12,845 2,192 287.1 207.5

⬎32,000 ⬎32,000 ⬎32,000 15,667.43 ⬎32,000 ⬎32,000 7,584.62

Dual reversible (contraceptive) MMV665980

383.9

8,068

614.1

Inactive

16,377

⬎32,000

a

Nineteen active compounds from the primary screen were characterized further by dose-response analysis in the PfDGFA carryover and washout formats and the PbODA. For comparison, corresponding CC50s are also displayed from previously published cytotoxicity data for the Malaria Box compounds against human fetal lung fibroblasts (MRC-5 cells) available in the ChEMBL database (https://www.ebi.ac.uk/chembl/malaria/assay/inspect/CHEMBL2095143). By comparing the carryover and washout IC50s for male and female gametocytes, compounds were classified to exhibit reversible or irreversible activity and to target both gametocyte sexes or only males. ND, not determined.

gamete formation. Therefore, this assay will identify vector stagetargeted compounds that act between the induction of gamete formation and the development of the mature ookinete (Fig. 1D). The screen of the Malaria Box in the PbODA was very robust, with a Z= factor of 0.79 ⫾ 0.11 (mean ⫾ SD). Eighteen compounds (7 drug-like and 11 probe-like) were found to give ⬎50% inhibition, with 8 having an SSMD considered strong, 9 with an SSMD considered moderate, and 1 with an SSMD considered weak (Table 1; see Data Set S2 in the supplemental material). Of these hits, 8 were common with the PfDGFA (see Fig. S2 in the supplemental material). Twenty of the compounds determined to be active in either screen were available from commercial vendors (see Data Set S1 in the supplemental material) and were purchased fresh to confirm the validity of the screens by dose-response testing and IC50 determination. Fourteen of these displayed submicromolar IC50s in the PfDGFA, with 7 also having submicromolar activity in the PbODA (Table 2 and Fig. 5A; see Fig. S3 and S4 in the supplemental material). Interestingly, compounds tested for dose response in the PfDGFA carryover format displayed one of three characteristics: (i) those with equipotent activity against male and female gametocytes, such as MMV665830; (ii) those that were active against both sexes but more potent against males, such as MMV000448; (iii) and those such as MMV085203 that were active

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against males but show no activity against females at the highest concentration tested (which was between 10 and 50 ␮M, depending on the activity of the molecule in the original primary screen). Only MMV665941, which fell just below our hit criteria in the PfDGFA but was active in the PbODA, showed a slight (2-fold) bias in potency toward females (390.9 nM for males and 178.2 nM for females); however, this bias was not statistically significant (P ⫽ 0.05, F test). Of the retested compounds, only MMV665972, which showed 54.1% inhibition against males and a weak SSMD of ⫺0.52 in the primary screen was found to be a false positive after dose-response testing. To further characterize these molecules, dose-response determination was repeated in the washout format, in which gametocytes are exposed to the compounds for 24 h and then undergo three medium changes over 6 h to wash the compound from the cells (Fig. 2). Gamete formation is then induced in the absence of the compound; therefore, this method identifies only those compounds that irreversibly act on stage V gametocytes and not those that must be present during gamete formation to have inhibitory effects (Fig. 4). When comparing data between both assay formats, an even greater range of phenotypes emerged (Table 2): in the washout format, five compounds, such as MMV665941, retained activity (less than 5-fold difference) against male and female gametocytes similar to that displayed in the carryover format (Table 2). We infer that these com-

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Male gamete formation

Gametocyte Sex-Selective Transmission-Blocking Assay

A

dual irreversible

MMV007116

100

IC50 c-o:569.7 nM

50

50

0 10 1

10 2

10 3

10 4

10 5

0 10 0

-50

10 1

10 2

10 3

10 4

10 5

10 0

-50

100

IC50 c-o: inactive IC50 w-o: inactive

100

IC50 c-o:473.1 nM

10 1

10 2

10 3

10 4

10 5 -50

10 1

10 2

10 3

10 5

10 3

10 4

10 5

IC50 c-o: inactive

10 5

10 0 -50

10 1

10 2

Concentration nM

female carryover

MMV007116 96.80

48.53

92.24

48.00

Carry-over MMV667491 99.61

59.32

94.29

64.14

MMV085203 99.78

76.58

90.47

42.97

MMV007116 74.64

3.35

62.76

17.65

Wash-out MMV667491 96.91

30.47

95.16

38.46

MMV085203 98.81

61.61

98.20

64.29

Replicate 1

10 4

Concentration nM

male washout

O in ocy hi st bi tio inte n (% nsit ) y R pr edu ev ct al ion en i ce n (% ) O o in cy hi st bi tio inte n (% nsit ) y Re pr du ev ct al ion en i ce n (% )

B

10 4

0

10 0

Concentration nM

male carryover

10 3

50

0

10 0

10 2

IC50 w-o: inactive

50

0

10 1

-50

IC50 w-o:173.6 nM

50

-50

50

0 10 0

IC50 c-o:95.96 nM IC50 w-o:331.3 nM

IC50 w-o:81.6 nM

IC50 w-o:20742 nM

100

MMV085203 100

IC50 c-o:182.7 nM

Replicate 2

female washout

C 120 Oocyst intensity (%of DMSO control)

100

male irreversible

MMV667491

carryover

100

washout

80 60 40 20 0 D

M

SO M

M

0 V0

16 71 M

M

6 V6

74

91 M

M

8 V0

52

03

FIG 5 Three exemplar compounds showing different activities in the PfDGFA were selected to validate the assay against the current gold standard assay—the P. falciparum standard membrane feeding assay (PfSMFA). (A) MMV007116, MMV667491, and MMV085203 displayed male-specific reversible, dual irreversible, and male-specific irreversible activities, respectively, in the PfDGFA carryover (c-o) and washout (w-o) formats. Blue continuous lines and solid circles represent male readout with the carryover format, blue dotted lines and open squares represent male readout with the washout format, red continuous lines and solid circles represent female readout with the carryover format, and red dotted lines and open squares represent female readout with the washout format. Shown are results from quadruplicate independent experiments; error bars denote the SEM. (B) The same compounds were tested in the PfSMFA using similar carryover and washout formats at 1 ␮M in two independent replicates (n ⫽ 13 to 91 mosquitoes). (C) All three compounds strongly inhibited transmission to the mosquito in the carryover format. As predicted by the PfDGFA, if washed out before feeding, there was a significant reduction in the inhibition of MMV007116 (P ⫽ 4.945 ⫻ 10⫺11).

pounds irreversibly kill or sterilize mature stage V male and female gametocytes and thus exemplify the “ideal” functional properties of a transmission-blocking compound. Seven compounds that showed male-specific activity when present during gamete formation substantially lost activity when washed out. For example, MMV020439 showed a ⬎34-fold increase in IC50 after washout. We infer that these compounds are male-specific “contraceptives” that directly inhibit the process of male gamete formation rather than killing or sterilizing the gametocyte. MMV665980 showed activity against both sexes in the carryover format, but inhibition was reversed for both sexes in the washout format (IC50 of 383.9 nM for male carryover and 614.1 nM for female carryover versus IC50 of 8,068 nM for male and inactive female washout). These data suggest that MMV665980 reversibly inhibits a gamete formation induction pathway common to both male and female game-

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tocytes. Interestingly, MMV085203 which was found to be male specific in the carryover format, retained activity but with slightly reduced potency in the washout format (IC50 of 95.96 nM versus 331.3 nM, respectively). MMV007127 also showed this irreversible male gametocyte-specific activity (IC50 of 3,414 nM for carryover versus 3,950 nM for washout) but with less potency. Finally, highlighting the diversity of compound activity against gametocytes, MMV665878 gave similar potencies against male and female gametocytes in the carryover format (IC50s of 240.7 nM for males and 383.9 nM for females), but when washed out, it only retained potent activity against males (IC50s of 665.6 nM for males and 13,136 nM for females). We infer that this compound irreversibly inhibits the functional viability of male gametocytes, but its effects are reversible for females. The same compounds were also tested in dose response in the

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Inhibition of gamete formation (%)

Inhibition of gamete formation (%)

male reversible

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duced activity but not a total loss of activity in the washout PfSMFAs from 96.8% inhibition in the carryover to 74.6% inhibition in the washout for replicate 1 and 92.2% to 62.8% inhibition in the carryover and washout formats, respectively, for replicate 2. To determine whether this reduction was a real effect or was due to experimental variation, a generalized linear mixed model (GLMM) (30) was fitted to the whole data set. This showed that washing out MMV007116 results in a statistically significant reduction in efficacy compared to carrying it over into the mosquito (P ⫽ 4.945 ⫻ 10⫺11) (Fig. 5C). This validates the prediction of reversibility made for MMV007116 in the PfDGFA; however, total loss of efficacy was predicted. This discrepancy is likely due to the extended cell biology interrogated by the PfSMFA compared to the PfDGFA. It is possible that MMV007116 may cause some irreversible “silent” damage to the gametocyte that manifests only after gamete formation and hence is not identified in the PfDGFA. This is supported by its IC50 of 287.1 nM in the PbODA and is suggestive that a combination of both assays, which is easily achieved, could best predict data obtained in the PfSMFA. DISCUSSION

We present an experimentally validated novel in vitro transmission-blocking 96-well screening assay that faithfully mirrors the initial stages of P. falciparum transmission from the mature stage V gametocyte to formation of functional gametes. Critically, the assay greatly extends the transmission cell biology under investigation over existing gametocyte assays by distinguishing the more drug-sensitive male gametocytes in the assay readout and is not limited to identification of gametocytocidal compounds only but also identifies those that sterilize and have contraceptive effects. The assay permits for the first time the study of compound action exclusively on functionally viable mature stage V gametocytes with a readout that is not confounded by earlier nontransmissible immature stages, which could provide false-positive hits that are not active against this critical target population. The Malaria Box screen has resulted in two surprising discoveries that have profound consequences for the future optimal discovery and development of urgently required malaria transmission-blocking drugs. The first is that the inclusion of a sex-specific male readout more than triples the number of transmission-blocking compounds identified (from 6 confirmed female-active compounds to 19 confirmed male-active compounds in this study). Second, male-specific and contraceptive (reversible) compounds have substantially better cytotoxicity profiles than those that irreversibly target both sexes. An irreversible gametocyte-focused transmission-blocking drug has the “ideal” characteristic (26) in that, on being delivered to the afflicted individual, it acts upon the gametocyte and then permanently prevents parasite transmission to the mosquito. However, if this class of compounds is generally biased toward a cytotoxic profile, significant downstream medicinal chemistry will be required to develop viable lead compounds that eventually result in efficacious transmission-blocking drugs. Indeed, the main impediment to the development of transmission-blocking drugs is that drugs with delayed benefit to the patient (i.e., benefit gained through reducing the number of new cases of malaria in the surrounding population) must be extremely safe to justify their use (36). Compounds with poor selectivity for parasites may not be the optimum starting point for this type of therapeutic. A transmission-blocking contraceptive compound adminis-

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PbODA (Table 2; see Fig. S4 in the supplemental material). The seven compounds with submicromolar IC50s in the PbODA also had submicromolar activity in the PfDGFA. Compared to activity in the PfDGFA, compounds active in the PbODA were found in the various PfDGFA categories: male-specific reversible; malespecific irreversible; and dual irreversible. Given the overlap in cell biology between these two assays (Fig. 1C and D), it is unsurprising that they correlate at least in part. Some compounds, such as MMV665977 and MMV665827, showed activity but considerably less potency in the PbODA. Gamete formation is very rapid, and in the PbODA, parasites commence compound exposure simultaneously with gamete formation. Therefore, it is likely that reduced compound potency in the PbODA is a result of suboptimal exposure time in this assay before gamete formation proceeds, which could be improved by preincubating parasites with compound before inducing gamete formation. However, maintaining robust P. berghei gametocyte functional viability ex vivo has not yet proven possible (25). Alternatively, the difference in potency may be accounted for by species differences between P. berghei and P. falciparum. Those compounds active in the PfDGFA but not the PbODA are likely either targeted to the mature stage V gametocyte, which is not interrogated by the PbODA, or show P. falciparum-specific activity. The Malaria Box compounds all have activity against P. falciparum asexual parasites. Therefore, compounds active in all assays likely target conserved cell biology fundamental to multiple life stages or are generally cytotoxic. To investigate this further, assay data were compared to previously published cytotoxicity data for the Malaria Box compounds (i.e., 50% cytotoxic concentration [CC50]) against human fetal lung fibroblasts (MRC-5 cells) available in the ChEMBL database (https://www.ebi.ac.uk /chembl/malaria/assay/inspect/CHEMBL2095143) (Table 2). Interestingly, dual irreversible-acting compounds generally had much poorer selectivity for Plasmodium gametocytes versus human-derived cells than male-specific reversible, male-specific, or dual reversible compounds, which, with the exception of MMV645672, MMV085203, MMV666691, and MMV006457, all reportedly did not reach the CC50 by the highest concentration tested (32 ␮M). Validation of screening assay with current gold standard PfSMFAs. Having identified compounds active in the PfDGFA and PbODA, to prove potential utility, it was necessary to validate the predictive power of these novel assays against the current gold standard PfSMFA. Three compounds were selected for evaluation as they showed a range of different properties in the PfDGFA: MMV667491, showing dual irreversible activity; MMV085203, showing male-specific irreversible activity; and MMV007116, showing male-specific reversible activity plus submicromolar activity in the PbODA (Fig. 5A). All three compounds were tested at 1 ␮M in the two different formats (carryover and washout) of the PfSMFA (Fig. 1E) to mirror the two formats of the PfDGFA. Each set of feeds was repeated in two independent replicates (Fig. 5B; see Table S2 in the supplemental material). True to the activity predicted from the PfDGFA carryover format, all three compounds had strong inhibition in the PfSMFA carryover format, exhibiting between 96.8 and 99.8% inhibition of oocyst intensity in the first replicate and between 90.5 and 94.3% inhibition in the second. Similarly, the two predicted irreversible compounds MMV667491 and MMV085203 also demonstrated 96.9 and 98.8% inhibition (replicate 1) and 95.2 and 98.2% inhibition (replicate 2), respectively, in the PfSMFA washout format. MMV007116, which was predicted to be reversible, showed re-

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these assays will only identify compounds active against females as molecules specifically targeting the lesser male population will fall below the limits of assay detection. This is reflected in the fact that the Mitotracker assay only identified 50% of the compounds that we have characterized to be male specific or reversible, and the alamarBlue assay as performed by Sun et al. (38) only identified 14%. (With the alamarBlue assay as performed by Bowman et al. [37], there were none in common.) It is surprising that they identify any reversible compounds. Most likely, some compounds that are reversible over the 24-h incubation in the PfDGFA may become irreversible over the prolonged 96-h incubations of the other assays. We suggest that the limitations of published latestage gametocyte assays to identifying compounds that have increased likelihood of a cytotoxic profile or that require a prolonged incubation time to kill gametocytes should be considered when evaluating future screens with these assays. We contend that in this initial phase of transmission-blocking drug discovery, high-throughput screening of large compound libraries should be as inclusive as possible within the bounds of cell biology relevant to transmission and should embrace all compound action phenotypes described here. Currently the PfDGFA operates routinely a 96-well format; however recently we have successfully adapted the methodology to a 384-well format with a Z= factor of ⬎0.6, which will be utilized in future screening of large libraries of compounds. ACKNOWLEDGMENTS This work was supported by Medicines for Malaria Venture grant MMV 08/2800. M.J.D. and R.E.S. were supported by the Bill and Melinda Gates Foundation (OPP1043501). D.K.M. and R.R.D. are supported in part by the Bloomberg Family Foundation through the Johns Hopkins Malaria Research Institute and National Institutes of Allergy and Infectious Diseases of the National Institutes of Health (grant R01AI082587-01) and by the NIH National Center for Research Resources (grant UL1 RR 025005). D.L. is a paid employee of the Medicines for Malaria Venture. The following reagent was obtained through MR4 as part of the BEI Resources Repository, NIAID, NIH: Plasmodium falciparum anti-Pfs25 MAb 4B7, MRA-28, deposited by D. C. Kaslow. We also thank Hilary Hurd and Paul Eggleston for the Anopheles gambiae Keele strain.

REFERENCES 1. Sinden RE. 2010. A biologist’s perspective on malaria vaccine development. Hum. Vaccin. 6:3–11. http://dx.doi.org/10.4161/hv.6.1.9604. 2. Sinden RE. 1983. Sexual development of malarial parasites. Adv. Parasitol. 22:153–216. http://dx.doi.org/10.1016/S0065-308X(08)60462-5. 3. Alano P. 2007. Plasmodium falciparum gametocytes: still many secrets of a hidden life. Mol. Microbiol. 66:291–302. http://dx.doi.org/10.1111/j .1365-2958.2007.05904.x. 4. Billker O, Lindo V, Panico M, Etienne AE, Paxton T, Dell A, Rogers M, Sinden RE, Morris HR. 1998. Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito. Nature 392: 289 –292. http://dx.doi.org/10.1038/32667. 5. Toyé PJ, Sinden RE, Canning EU. 1977. The action of metabolic inhibitors on microgametogenesis in Plasmodium yoelii nigeriensis. Z. Parasitenkd. 53:133–141. http://dx.doi.org/10.1007/BF00380457. 6. Mair GR, Braks JA, Garver LS, Wiegant JC, Hall N, Dirks RW, Khan SM, Dimopoulos G, Janse CJ, Waters AP. 2006. Regulation of sexual development of Plasmodium by translational repression. Science 313: 667– 669. http://dx.doi.org/10.1126/science.1125129. 7. van Dijk MR, Janse CJ, Thompson J, Waters AP, Braks JA, Dodemont HJ, Stunnenberg HG, van Gemert GJ, Sauerwein RW, Eling W. 2001. A central role for P48/45 in malaria parasite male gamete fertility. Cell 104: 153–164. http://dx.doi.org/10.1016/S0092-8674(01)00199-4. 8. Janse CJ, Mons B, Rouwenhorst RJ, Van der Klooster PF, Overdulve JP, Van der Kaay HJ. 1985. In vitro formation of ookinetes and functional

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tered to a patient needs to maintain an efficacious concentration in the bloodstream either through a long serum half-life or by multiple dosing to be bioavailable in each and every mosquito blood meal where infectious gametocytes are present. However, the excellent selectivity of these molecules for Plasmodium over human cells suggests perhaps that hits generated with this profile may be converted into lead molecules more efficiently, assuming their serum half-lives are adequate or can be improved and that prolonged exposure in the human host does not cause greater cytotoxicity. Male-specific irreversible compounds may provide the best of both worlds in that they permanently disable male gametocyte functional viability, thus fulfilling the ideal target candidate profile, and by virtue of only targeting one gametocyte sex, they are more likely to target parasite-specific cell biology and less likely to display a general cellular toxicity. The PfDGFA is currently the only in vitro assay that has the capability to identify such compounds. To date, three previous reports have described screens of the Malaria Box in late-stage gametocyte assays (15, 37, 38); however, all of these studies only partially match the cell biology interrogated by the PfDGFA and the gold standard PfSMFA (Fig. 1). Bowman et al. (37) and Sun et al. (38) both screened the Malaria Box library against immature stage III to V gametocytes with a cell viability (mitochondrial respiration) readout indicated by alamarBlue (16). Despite screening at a high concentration of 5 ␮M with a prolonged compound incubation of 96 h, Bowman and coworkers (37) only identified 18 confirmed hits. Similarly Sun and coworkers (38) performing the same assay identified 17 compounds with submicromolar IC50s; however, there is very little concordance between the screens both of hits identified and of IC50s of compounds common to both studies (see Fig. S5 in the supplemental material). In contrast, Duffy and Avery (15) determined that approximately one-quarter of the Malaria Box compounds (106 compounds) were active against stage IV to V gametocytes when screened at 0.5 and 5 ␮M for a total of 96 h using a microscopic readout relying on the distinctive “elongated” shape of stage IV to V gametocytes and Mitotracker Red to determine parasite viability. None of these screens has yet reported confirmation of hits in PfSMFAs. Our PfDGFA screen was performed at 1 ␮M and with only 24 h of incubation with gametocytes (plus 20 min of incubation with gametes for the male readout and an extra 24 h with gametes for the female assay). Our reasoning for these conditions was to identify only those compounds that have potent and rapid transmission-blocking activities. All of the compounds identified as dual irreversible in the PfDGFA correlate with hits identified in both the Mitotracker and alamarBlue assay as described by Sun et al. (38), whereas only 2 out of 5 compounds are in common with the alamarBlue assay as described by Bowman et al. (37). This level of agreement is entirely to be expected as this is where the cell biology of all the assays best overlap. Alternatively, this concordance may be also accounted for by the unfavorable general cytotoxicity of these compounds. Supporting this assertion, four out of five of the dual irreversible compounds (MMV665941, MMV665830, MMV019918, and MMV667491), but none we identify from any other activity class we describe, were also recently identified as having activity against the unrelated trematode parasite Schistosoma mansoni (39). Late-stage gametocyte assays will only identify gametocyte-killing compounds and hence only those with irreversible effects (and not sterilizing or contraceptive). Additionally, by lacking a sex-specific readout,

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