Assessment In Vitro of the Antimalarial and Transmission-Blocking Activities of Cipargamin and Ganaplacide in Artemisinin-Resistant Plasmodium falciparum

ABSTRACT

Artemisinin resistance in Plasmodium falciparum has emerged and spread widely in the Greater Mekong Subregion, threatening current first-line artemisinin combination treatments. New antimalarial drugs are needed urgently. Cipargamin (KAE609) and ganaplacide (KAF156) are promising novel antimalarial compounds in advanced stages of development. Both compounds have potent asexual blood stage activities, inhibit P. falciparum gametocytogenesis, and reduce oocyst development in anopheline mosquitoes. In this study, we compared the asexual and sexual stage activities of cipargamin, ganaplacide, and artesunate in artemisinin-resistant P. falciparum isolates (n = 6; K13 mutations C580Y, G449A, and R539T) from Thailand and Cambodia. Asexual blood stage antimalarial activity was evaluated in a SYBR-green I-based 72-h in vitro assay, and the effects on male and female mature stage V gametocytes were assessed in the P. falciparum dual gamete formation assay. Ganaplacide had higher activities than cipargamin and artesunate, with mean (standard deviation [SD]) 50% inhibitory concentrations (IC₅₀s) against asexual stages of 5.6 (1.2) nM and 6.9 (3.8) nM for male gametocytes and 47.5 (54.7) nM for female gametocytes. Cipargamin had a similar potency against male and female gametocytes, with mean (SD) IC₅₀s of 115.6 (66.9) nM for male gametocytes, 104.9 (84.3) nM for female gametocytes, and 2.4 (0.7) nM for asexual stages. Both cipargamin and ganaplacide showed significant transmission-blocking activities against artemisinin-resistant P. falciparum in vitro.

INTRODUCTION

Plasmodium falciparum malaria remains an important cause of disease and death in countries where malaria is endemic, with an estimated 241 million infections causing 627,000 deaths worldwide in 2020 (1). Artemisinin-based combination therapies (ACTs), which combine a rapidly eliminated artemisinin derivative and a second long-acting antimalarial drug with a different mechanism of action, are currently the first-line treatments for uncomplicated falciparum malaria in all areas of endemicity. Artemisinin resistance in P. falciparum, first reported in western Cambodia 14 years ago (2, 3), has subsequently emerged and spread widely throughout the Greater Mekong Subregion (GMS) (4–6), and it has recently emerged in several other areas, notably Rwanda (7, 8) and Uganda (9–11). Artemisinin resistance in vivo is characterized by slow parasite clearance, in a low-transmission setting, measured as a parasite clearance half-life of more than 5 h (4). Reduced sensitivity of early-stage gametocytes toward artemisinins in vitro (12) and in vivo (4) has also been described. Artemisinin and partner drug resistance has led to a high rates of treatment failure with several ACTs in the eastern part of the GMS. New antimalarial drugs are needed urgently for malaria treatment and elimination.

Cipargamin (KAE609) and ganaplacide (KAF156) represent novel classes of antimalarial drugs that are currently in extended phase 2 and phase 3 studies (13). The compounds were discovered and identified in phenotypic screens against asexual blood stages. Cipargamin is a synthetic antimalarial belonging to the spiroindolone class. The mechanism of action is inhibition of P. falciparum P-type ATPase 4 (PfATP4) (14, 15), which is an Na⁺ efflux transporter on the plasma membrane of malaria parasites. Inhibition causes osmotic disruption, swelling, augmented splenic clearance, and death of blood stage parasites (16–20). Cipargamin has transmission-blocking activity in a standard membrane feeding assay—completely blocking parasite transmission at 500 nM (21). Ganaplacide is an imidazolopiperazine with potency in the nanomolar range against multiple stages of the P. falciparum life cycle (liver stage and asexual and sexual blood stage inhibitory activities) (22). Activity against P. falciparum and Plasmodium vivax has been confirmed in preclinical (22, 23) and clinical studies (24), and transmission-blocking activity has been shown both in vitro and in vivo (22). Ganaplacide also has causal prophylactic efficacy in the controlled human malaria infection (CHMI) model (25). The mechanism of action remains to be elucidated. The related imidazolopiperazine compound (GNF179) inhibits protein trafficking, blocks the establishment of new permeation pathways, and causes endoplasmic reticulum expansion (26).

Gametocytocidal activity is important to reduce malaria transmission from the treated infection. Currently, primaquine is the only widely approved drug with substantial gametocytocidal activity against mature stage V gametocytes of P. falciparum. A single low primaquine dose of 0.25 mg base/kg is highly effective and safe even in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency (27). The activities of cipargamin and ganaplacide on the asexual and sexual blood stages in artemisinin-resistant parasites have not been reported so far. In this study, we evaluated the antimalarial activities and gametocytocidal effects of cipargamin and ganaplacide against the asexual and sexual blood stages of artemisinin-resistant P. falciparum isolates from Thailand and Cambodia.

RESULTS

Artemisinin-resistant gametocyte-producing P. falciparum isolates (n = 6; K13 mutations R539T [n = 2], C580Y [n = 3], and G449A [n = 1]) and a reference artemisinin-sensitive P. falciparum laboratory strain (NF54) (Table 1) were cultured and tested with serial dilutions of test compounds. Antimalarial activity against the asexual blood stage was evaluated by a standard SYBR green I-based 72-h in vitro assay (28, 29). Cipargamin, ganaplacide, and artesunate were all potent, with 50% inhibitory concentration (IC₅₀) estimates in the low-nanomolar range (Table 2). The asexual IC₅₀s were significantly lower than activity against male gametocytes (P < 0.001) for cipargamin and artesunate and female gametocytes (P < 0.001) for all compounds. Gametocyte production was then induced in the same parasites over 3 to 4 passages. The gametocytes were matured subsequently under controlled conditions for 12 to 14 days. The viability of the mature male gametocytes was considered verified if the number of exflagellating cells was ≥0.06% of total red blood cells (RBCs) in all parasite lines (Table 1). The mature stage V gametocytes were exposed to a range of concentrations of cipargamin, ganaplacide, and artesunate for 24 h. After this, the drug effects on male and female mature stage V gametocyte functional activity were assessed by the P. falciparum dual gamete formation assay (PfDGFA), which assesses exflagellation in male gametocytes and Pfs25 expression in female gametocytes (30–32).

TABLE 1.

P. falciparum origin, K13 mutations, parasite clearance half-life in vivo, and percentages of gametocytemia and exflagellation

Parasite	Origin, yr	K13 profile	Parasite clearance half-life (h)a	Mean (SD) % of:
				Gametocytemia	Exflagellation
NF54	Reference strain	Wild type	-	2.1 (0.6)	0.10 (0.04)
ANL9G	Cambodia, 2010	R539T	5.84	1.8 (0.3)	0.06 (0.00)
APS2G	Thailand, 2011	R539T	6.09	2.7 (1.0)	0.07 (0.02)
APS9G	Thailand, 2015	C580Y	7.90	3.6 (1.9)	0.13 (0.03)
APL5G	Cambodia, 2014	C580Y	7.70	1.3 (0.4)	0.11 (0.04)
APL9G	Cambodia, 2015	C580Y	6.67	3.0 (0.9)	0.14 (0.02)
ARN2G	Thailand, 2011	G449A	6.30	3.0 (0.7)	0.09 (0.03)

^aFor details, see references 4 and 44.

TABLE 2.

Effects of cipargamin, ganaplacide, and artesunate against asexual blood stages in artemisinin-resistant P. falciparum isolates

Parasite (K13 profile)	Mean (SD) IC50 (nM) of asexual blood stages witha:
	Cipargamin	Ganaplacide	Artesunate
NF54 (wild type)	2.9 (0.2)	7.7 (0.3)	0.9 (0.2)
Artemisinin-resistant isolates (n = 6)b	2.4 (0.7)	5.6 (1.2)	1.4 (0.7)
ANL9G(R539T)	1.5 (0.1)	5.9 (0.3)	2.2 (0.8)
APS2G(R539T)	1.9 (0.3)	5.3 (1.0)	1.2 (0.3)
APS9G(C580Y)	2.9 (0.3)	4.3 (1.2)	1.9 (0.5)
APL5G (C580Y)	2.3 (0.5)	7.2 (0.3)	1.3 (0.2)
APL9G(C580Y)	2.7 (0.5)	5.3 (1.2)	0.8 (0.2)
ARN2G(G449A)	3.2 (0.2)	6.1 (0.9)	0.9 (0.3)

^aIC₅₀ values were calculated from at least three independent replicates.

^bData obtained with different parasite isolates were pooled.

Cipargamin, ganaplacide, and artesunate all showed gametocytocidal activity against the cultured P. falciparum isolates. They inhibited male and female mature stage V gametocyte functional activity of P. falciparum in the nanomolar drug concentration range. For cipargamin, the mean (standard deviation [SD]) IC₅₀s for inhibition of functional activity in parasite isolates were 115.6 (66.9) nM for male gametocytes and 104.9 (84.3) nM for female gametocytes (P = 0.258) (Table 3 and Fig. 1; see Supplemental Fig. S1 at https://drive.google.com/file/d/1sbj9P0QYdyX1OdtSFreKAMLc1axIk9PO/view?usp=sharing). The respective ratios for sexual to asexual stage inhibitory activities of artemisinin-resistant parasites were mean values of 48.6 for male gametocytes and 44.1 for female gametocytes (Table 3). Cipargamin exposure at the highest studied dose (1 μM) resulted in mean (SD) inhibition of 76.0% (10.1%) and 80.9% (12.1%) for male and female gamete formation, respectively (see Table S1 and Fig. S1). Of the three drugs evaluated, ganaplacide had the most potent activity against mature gametocytes, with mean (SD) IC₅₀s of 6.9 (3.8) nM and 47.5 (54.7) nM for male and female gametocytes, respectively (Table 3; see Fig. S1). The IC₅₀ ratios of sexual to asexual stage inhibitory activities for ganaplacide in artemisinin-resistant parasites were 1.2 for male gametocytes and 8.5 for female gametocytes (Table 3). Thus, male gametocytes were significantly more sensitive to ganaplacide than female gametocytes (P < 0.001) (Fig. 1) with inhibitory activity similar to that against asexual stages. The maximal studied concentration of ganaplacide of 250 nM resulted in 99.8% (0.8%) inhibition of male gamete formation and 60.6% (18.5%) inhibition of female gamete formation (see Table S1 and Fig. S1). Artesunate had less potency against both mature stage V male and female gametocytes. The mean (SD) IC₅₀s against male and female gametocytes were 317.7 (197.7) nM and 493.0 (240.2) nM, respectively. The IC₅₀ ratios of sexual to asexual stage activities for artesunate were 224.2 for male gametocytes and 347.9 for female gametocytes in artemisinin-resistant parasite isolates (Table 3). At the maximal studied dose (1 μM), artesunate gave 63.8% (14.1%) inhibition of male gamete formation and 58.1% (9.4%) inhibition of female gamete formation (see Table S1 and Fig. S1).

TABLE 3.

Effects of cipargamin, ganaplacide, and artesunate against mature stage V male and female gametocytes from artemisinin-resistant P. falciparum isolates

Parasite (K13 profile)	Cipargamina				Ganaplacidea				Artesunatea
	Male gametocytes		Female gametocytes		Male gametocytes		Female gametocytes		Male gametocytes		Female gametocytes
	IC50 (nM)	IC50 ratio	IC50 (nM)	IC50 ratio	IC50 (nM)	IC50 ratio	IC50 (nM)	IC50 ratio	IC50 (nM)	IC50 ratio	IC50 (nM)	IC50 ratio
NF54 (wild type)	168.9 (61.8)	58.0	375.9 (267.6)	129.2	5.1 (3.0)	0.7	NAc	-	86.7 (26.3)	100.3	NAc	-
Artemisinin-resistant isolates (n = 6)b	115.6 (66.9)	48.6	104.9 (84.3)	44.1	6.9 (3.8)	1.2	47.5 (54.7)	8.5	317.7 (197.7)	224.2	493.0 (240.2)	347.9
ANL9G(R539T)	50.6 (24.5)	34.6	57.8 (6.7)	39.5	4.4 (1.0)	0.8	45.3 (25.0)	7.7	602.6 (84.4)	272.2	813.9 (103.1)	367.6
APS2G(R539T)	104.2 (36.6)	55.3	136.1 (44.6)	72.2	5.3 (2.5)	1.0	99.0 (91.1)	18.6	283.3 (247.8)	233.4	NAd	-
APS9G(C580Y)	125.1 (76.1)	43.3	37.7 (15.8)	13.0	7.6 (1.8)	1.8	29.0 (20.4)	6.7	137.6 (33.7)	71.9	249.9 (116.3)	130.6
APL5G (C580Y)	114.6 (45.0)	50.8	259.1 (50.3)	114.8	5.2 (2.2)	0.7	NAc	-	196.3 (107.9)	152.4	506.0 (196.2)	392.9
APL9G(C580Y)	96.6 (45.6)	35.2	67.7 (17.4)	24.7	12.4 (4.8)	2.3	57.6 (2.7)	10.8	377.4 (84.2)	451.2	427.0 (218.8)	510.5
ARN2G(G449A)	184.0 (102.9)	57.2	74.8 (50.6)	23.3	8.2 (4.6)	1.3	6.4 (2.9)	1.1	470.5 (212.8)	543.6	370.1 (154.5)	427.6

^aIC₅₀ values are presented as mean (SD) and were calculated from at least three independent replicates. The IC₅₀ ratio was calculated from the ratio of the IC₅₀ of male or female gametocytes to the IC₅₀ of asexual blood stages.

^bData obtained with different parasite isolates were pooled.

^cNA, not available because data from these parasite isolates could not be plotted in a dose-response curve, so the IC₅₀ could not be calculated because there was less than 50% inhibition of female gamete formation.

^dNA, not available because there were no inhibition data against female gamete formation.

FIG 1.

Drug sensitivity of cipargamin, ganaplacide, and artesunate against asexual blood stages (triangles), male gametocytes (circles), and female gametocytes (squares) in artemisinin-resistant P. falciparum isolates. Except for ganaplacide against male gametocytes, asexual blood stages were more susceptible to all compounds than sexual stages. Data obtained with different parasite isolates were pooled. The mean and error bar show the standard deviation. Asterisks represent P < 0.05 (*) and P < 0.001 (***), respectively.

Morphological assessment by light microscopy of a thin blood film in the control group after incubation for 24 h showed mature and healthy gametocytes, characterized by elongated sexual stage parasites inside the red blood cell, with no pyknotic forms. Gametocyte morphology in the drug-treated groups remained unchanged at the highest drug concentrations (cipargamin and artesunate at 1 μM and ganaplacide at 250 nM), except after cipargamin exposure, which resulted in a slightly more rounded shape of the gametocytes (Fig. 2).

FIG 2.

Morphology of gametocytes after drug exposure (24 h). No drug was added in the control group. Gametocytes in the control group developed into mature stage V forms, and morphology was normal and healthy. Cipargamin was incubated with mature stage V gametocytes at 1 μM. Gametocyte morphology was normal, but the shape was slightly rounder than in the control group. Ganaplacide and artesunate were incubated with mature stage V gametocytes at 250 nM and 1 μM, respectively. The morphologies of gametocytes with these two drugs were normal (i.e., no different from the control group), and there were no pyknotic cells.

DISCUSSION

New antimalarial drugs are needed. The emergence and spread of artemisinin resistance in the GMS over the last 15 years and the consequent development of ACT partner drug resistance emphasize the urgency of the situation. The ideal antimalarial drug should target multiple stages of the parasite life cycle to cure the disease, block transmission, and provide some protection against early reinfection. In malaria elimination strategies, transmission-blocking activity is an important component.

The results of this study confirmed the potent asexual stage activities of cipargamin and ganaplacide against artemisinin-resistant P. falciparum isolates with potencies in the low-nanomolar range (IC₅₀, <10 nM). GNF179, which is an imidazolopiperazine ganaplacide analogue, has also been shown to have potent activity against artemisinin-resistant P. falciparum in vitro (33). Both cipargamin and ganaplacide have shown potent antimalarial activity in clinical trials (20, 24, 34). The ring stage survival (35, 36) and trophozoite maturation inhibition assays (37) were most commonly used to assess artemisinin resistance in P. falciparum. The IC₅₀ values of artesunate from a standard SYBR green I-based 72-h in vitro assay [mean (SD), 1.4 (0.7) nM] do not discriminate artemisinin-sensitive and -resistant parasites.

The gametocytocidal activity of the artemisinin derivatives is thought to have contributed to their beneficial effects in reducing malaria incidence in low-transmission settings. This property is reduced in artemisinin-resistant infections, and this enhances the spread of resistance (12). Thus, it is important to study the gametocytocidal activity against artemisinin-resistant parasites of the new antimalarial drugs in clinical development. The PfDGFA (30–32) is an assay that assesses male and female mature stage V gametocytes after induction with a specific ookinete medium. In this study, cipargamin and ganaplacide had significant inhibitory effects against both male and female gametocytes. As is often the case, male gametocytes were more susceptible to the antimalarials than female gametocytes (28). In our study, the same was observed for ganaplacide and artesunate, but interestingly, not for cipargamin. Artesunate retained some activity against artemisinin-resistant parasite isolates, with 63.8% inhibition of male gametocyte exflagellation and 58.1% inhibition of female gametocytes at 1 μM. These results are similar to previously reported responses in male gametocytes with 3D7 (66.95% inhibition) (32) and NF54 (62.57% inhibition) at 1 μM (31). In this study, there was also evidence of some inhibition of female gametocytes of artemisinin-resistant P. falciparum isolates and NF54, with 58.1% and 43.7% inhibition at 1 μM, respectively (see Table S1). This contrasts with no effects of artesunate in 3D7 and NF54 female gametocytes at 1 μM and 10 μM in previous reports (31, 32).

Cipargamin has been reported to inhibit both early- and late-stage development of P. falciparum NF54 gametocytes at a range of 5 to 500 nM (21). In this study, the mean IC₅₀ for male gametocytes was 115.6 nM (equivalent to 46.2 ng/mL), and that for female gametocytes was 104.9 nM (equivalent to 41.9 ng/mL). These concentrations are well within the range of drug plasma concentrations observed in clinical studies. Pharmacokinetic studies of oral cipargamin (30 mg per day for 3 days) in phase 2 studies reported mean (SD) peak plasma concentrations (C_max) of 911 (349) ng/mL and 1,340 (374) ng/mL at day 1 and day 3, respectively (20). In the induced blood stage malaria (IBSM) model of P. falciparum, the mean (SD) C_max of cipargamin after a 10-mg single dose was 101 (18.3) ng/mL (34). In the phase 2 clinical study, gametocyte clearance was observed in vivax malaria patients by 8 h but could not be assessed in P. falciparum infections as none of the falciparum malaria patients were gametocytemic (20). The osmotic disruption that reduced infected red blood cell deformability was the likely cause of the accelerated splenic clearance of ring stage P. vivax and P. falciparum (38). In our study, the gametocyte morphology after cipargamin treatment showed slight swelling of the gametocytes, which could accelerate their clearance in vivo. In the IBSM study, P. falciparum 3D7 gametocytes were detected and showed peak gametocytemia approximately 17 days after 10 mg of cipargamin administration in all subjects, but they did not transmit to mosquitoes in the direct membrane feeding assay (34).

Previous studies reported that ganaplacide inhibited early-stage P. falciparum gametocyte development. No oocysts were observed in the standard membrane feeding assay when the gametocyte culture was treated on days 8 to 12 with ganaplacide at 5 and 50 nM (22). There was a >90% reduction in oocyst numbers when mature stage V gametocytes were treated at 500 nM for 15 min (22). In this study, the potency of ganaplacide on male gametocytes in artemisinin-resistant P. falciparum isolates (mean IC₅₀, 6.9 nM; equivalent to 4.2 ng/mL) was approximately similar to the asexual stage activity (mean IC₅₀, 5.6 nM; equivalent to 3.4 ng/mL). Female gametocytes (mean IC₅₀, 47.5 nM; equivalent to 28.9 ng/mL) were less susceptible than male gametocytes. Pharmacokinetic studies showed that the mean (SD) ganaplacide C_max values were 1,850 (401) ng/mL after a single dose of 800 mg ganaplacide and 2,270 (496) ng/mL after 800 mg ganaplacide plus 1,280 mg piperaquine in healthy volunteers (39), 1,800 (404) ng/mL after an 800-mg single oral administration, and 795 (182) ng/mL and 1,440 (299) ng/mL at days 1 and 3, respectively, after a 400-mg oral administration for 3 days in malaria patients (24). This suggests that potent transmission-blocking activity would be observed in vivo. In the phase 2 clinical trial in falciparum and vivax malaria, gametocytemia was observed in two falciparum malaria patients from baseline up to 54 h and 72 h after drug administration and also in two vivax malaria patients from baseline to 16 h after drug administration. (24). It is important to note that gametocyte sterilization may precede clearance. Following 8-aminoquinoline treatment, gametocyte clearance is substantially slower than inhibition of gamete formation and transmissibility (40). Overall, these laboratory data support a potent transmission-blocking effect of ganaplacide in vitro in artemisinin-resistant P. falciparum. Mutations in the P. falciparum cyclic amine resistance locus (pfcarl) (41, 42), caused resistance to imidazolopiperazine compounds (up to 458-fold reduction in susceptibility), and this extended to sexual stages (290-fold reduction in susceptibility) (42). Other mutations, including in an acetyl coenzyme A (acetyl-CoA) transporter gene (pfact) and a UDP-galactose transporter gene (pfugt), were also observed in drug resistance selection in vitro (43). Until these drugs are deployed (in combinations), the risks of resistance emergence remain unclear. In conclusion, cipargamin and, particularly, ganaplacide showed high potency against the asexual stage and inhibition activities against both male and female gametocytes of artemisinin-resistant P. falciparum isolates. Further information is needed from mosquito feeding assessments and clinical studies.

MATERIALS AND METHODS

Compounds and parasite culture.

Cipargamin (KAE609 hemihydrate; batch no. 1010017450) and ganaplacide (KAF156 diphosphate; batch no. 1010010507) were kindly supplied by Novartis Institute for Tropical Diseases (NITD). Cipargamin hemihydrate and ganaplacide diphosphate stock solutions were prepared at 100 mM in dimethyl sulfoxide (DMSO)–50% ethanol (1:1 [vol/vol]). Artesunate [Artesunate for Injection; registration no. 1C 3/35 (N)] was purchased from Guilin No.2 Pharmaceutical Factory. It was dissolved in 5% NaHCO₃ to make 60 mg/mL (equivalent to 156 mM) as a stock solution. All stock solutions were kept at −80°C, used within 6 months, and thawed only once before being used in the assay.

Artemisinin-resistant gametocyte-producing P. falciparum isolates (n = 6) were obtained from the Thailand-Cambodia border in 2010–2015 as a part of clinical studies (TRAC I and II). All parasite isolates were mycoplasma free. Parasite isolates underwent continuous asexual stage culture at 5% parasitemia and 5% hematocrit at 37°C in a 5% CO₂ incubator. The culture medium, which consisted of RPMI 1640 (Sigma catalog no. R6504) supplemented with 50 mg/L hypoxanthine (Sigma catalog no. H9377), 3 mg/L thiamine (Sigma catalog no. T1270), 6 mg/L l-ascorbic acid (Sigma catalog no. A5960), 30 mg/L CaCl₂ (Sigma catalog no. C4901), 26 mg/L KH₂PO₄ (Merck catalog no. A681173), 16 mg/L MgSO₄ (Sigma catalog no. M8150), 1 g/L d-glucose (Sigma catalog no. G7021), 5.96 g/L HEPES (Sigma catalog no. H3375), 2 g/L NaHCO₃ (Sigma catalog no. S5761), and 10% human serum, was replaced daily. To induce gametocyte production, gametocyte cultures were initiated at 4 to 6% parasitemia (mainly ring stage) and 5% hematocrit in a 20-mL final volume. Culture medium was changed daily for 14 days and maintained at 37°C under a gas mixture of 5% CO₂, 5% O₂, and 90% N₂. Parasites were examined by Field’s-stained thin blood films under light microscopy daily. The number of gametocytes per 1,000 red blood cells (RBCs) was counted, and the stage of gametocyte development was determined by morphological assessment. Mature stage V gametocytes appeared 10 to 14 days after induction. The stage and sex of gametocytes were distinguished by morphology, color, and pigment granules.

SYBR green I-based 72-h in vitro susceptibility test.

Parasite culture at predominantly ring stage development was prepared and incubated with 2-fold serial dilutions of cipagarmin (0.2 to 100 nM), ganaplacide (0.2 to 100 nM), and artesunate (0.02 to 10 nM) in technical duplicates. The final conditions of parasite suspension were 1% parasitemia and 1% hematocrit. Plates were incubated at 37°C for 72 h. After incubation, a mixture of 2× SYBR-green I lysis buffer (0.1% [wt/vol] saponin [Sigma catalog no. 47036]), 1% (vol/vol) Triton X-100 (Bio-Rad catalog no. 161-0407), 5 mM EDTA (Sigma catalog no. E7889), and 20 mM Tris-HCl (Sigma catalog no. T5941) was added to each well. Plates were incubated in the dark for 30 min before fluorescence signal was measured on a microplate reader (Synergy H1, BioTek) using a 485-nm excitation filter and a 520-nm emission filter. Assays were performed for at least three independent replicates. The percentage of growth and IC₅₀ were calculated using GraphPad version 8.

In vitro male and female gamete formation assay.

Gametocyte cultures which had more than 80% purity of mature stage V gametocytes with an exflagellating cell proportion of ≥0.06% of total red blood cells were considered suitable for setting up the assay. Exflagellation was assessed in mature stage V male gametocytes by incubating parasite suspensions with precooled ookinete medium (RPMI 1640 supplemented with 50 mg/L hypoxanthine, 5.96 g/L HEPES, 2 g/L NaHCO₃, 100 μM xanthurenic acid [Sigma catalog no. D120804], and 10% human serum) in 1:1 ratio. The suspension was added into a hemocytometer and incubated at room temperature for 15 min, then exflagellating cells and RBCs were quantitated under light microscopy at ×40 magnification. The numbers of exflagellating cells were counted and calculated as a percentage of total RBCs (30).

In vitro drug susceptibility tests of cipargamin, ganaplacide, and artesunate on P. falciparum gametocytes were done in serial 2-fold drug dilutions to obtain final concentrations ranging from 0.24 to 250 nM ganaplacide and 0.98 to 1,000 nM cipargamin and artesunate. Gametocytes were added into drug susceptibility test plates to make a final 2% hematocrit and incubated for 24 h at 37°C in a 5% CO₂ incubator. The dose-response assay was performed for at least three independent experiments.

The P. falciparum dual gamete formation assay was performed to assess male and female gamete formation after drug treatment. Male microgametes were identified by exflagellation, and female macrogametes were identified by Pfs25 antibody staining (30–32). Briefly, gametocytes were induced to form gametes by adding 85 μL ookinete medium into new flat-bottom 96-well plates (Thermo catalog no. 167008) and adjusted in a 15-μL parasite suspension to make a final 0.3% hematocrit. The plate was incubated for 10 min at room temperature and was transferred to an inverted fluorescence microscope (Nikon Eclipse Ti, model Ti-DH; 10× lens objective) for readout of exflagellation at 20 min after induction. The exflagellation was quantified by using an automated algorithm in NIS Elements software version 4.50, which captured 20 frames/well. Then, anti-Pfs25 antibody labeled with Cy3 fluorescence dye (anti-Pfs25 antibody–phosphate-buffered saline [PBS] in a 1:500 ratio) was added to the same plate for detection of activated female gametes. After incubation overnight in the dark at room temperature, the plate was read under an inverted fluorescence microscope (20× lens objective) by using another automated algorithm, which captured 4 high-resolution images of each well. The numbers of exflagellations and activated female gametes were identified and counted using the NIS algorithm. The percentage of inhibition was calculated by comparing the inhibitory activity of tested compound with that of control wells. In addition, a thin blood smear was made at 24 h of incubation before the PfDGFA was performed.

Statistical analysis.

The dose-response relationships and IC₅₀ values were analyzed by fitting a curve using a nonlinear regression model from quantitative data normalized to control values in GraphPad Prism version 8. IC₅₀ data were compared within and between groups by the nonparametric Kruskal-Wallis or Mann-Whitney U tests. All statistical analyses and graphical figures were performed using GraphPad Prism version 8.