Novel Endochin-Like Quinolones Exhibit Potent In Vitro Activity against Plasmodium knowlesi but Do Not Synergize with Proguanil

Quinolones, such as the antimalarial atovaquone, are inhibitors of the malarial mitochondrial cytochrome bc₁ complex, a target critical to the survival of both liver- and blood-stage parasites, making these drugs useful as both prophylaxis and treatment. Recently, several derivatives of endochin have been optimized to produce novel quinolones that are active in vitro and in animal models. While these quinolones exhibit potent ex vivo activity against Plasmodium falciparum and Plasmodium vivax, their activity against the zoonotic agent Plasmodium knowlesi is unknown.

ABSTRACT

Quinolones, such as the antimalarial atovaquone, are inhibitors of the malarial mitochondrial cytochrome bc₁ complex, a target critical to the survival of both liver- and blood-stage parasites, making these drugs useful as both prophylaxis and treatment. Recently, several derivatives of endochin have been optimized to produce novel quinolones that are active in vitro and in animal models. While these quinolones exhibit potent ex vivo activity against Plasmodium falciparum and Plasmodium vivax, their activity against the zoonotic agent Plasmodium knowlesi is unknown. We screened several of these novel endochin-like quinolones (ELQs) for their activity against P. knowlesi in vitro and compared this with their activity against P. falciparum tested under identical conditions. We demonstrated that ELQs are potent against P. knowlesi (50% effective concentration, <117 nM) and equally effective against P. falciparum. We then screened selected quinolones and partner drugs using a longer exposure (2.5 life cycles) and found that proguanil is 10-fold less potent against P. knowlesi than P. falciparum, while the quinolones demonstrate similar potency. Finally, we used isobologram analysis to compare combinations of the ELQs with either proguanil or atovaquone. We show that all quinolone combinations with proguanil are synergistic against P. falciparum. However, against P. knowlesi, no evidence of synergy between proguanil and the quinolones was found. Importantly, the combination of the novel quinolone ELQ-300 with atovaquone was synergistic against both species. Our data identify potentially important species differences in proguanil susceptibility and in the interaction of proguanil with quinolones and support the ongoing development of novel quinolones as potent antimalarials that target multiple species.

INTRODUCTION

Malaria continues to place a significant burden on humanity, with around 228 million infections estimated in 2018, an increase from the 217 million infections estimated for 2014 (1). The World Health Organization currently recommends artemisinin-based combination therapies (ACT) as the first-line treatment for uncomplicated malaria. These are composed of a potent but short-lived artemisinin derivative combined with a long-acting partner drug (2). By using drugs in combination with different targets, the intention is to delay the emergence of resistance to the individual components. However, recent evidence of resistance to both artemisinin (3, 4) and current partner drugs (5, 6) has emerged in the Greater Mekong Subregion. There is, therefore, an urgent need to develop new drugs and novel combination regimens before reduction in ACT efficacy occurs more widely.

Quinolones have been investigated as potential antimalarial agents since World War II (7). However, the only successful candidate from this class to emerge from these studies as an antimalarial has been atovaquone. Atovaquone targets the mitochondrial cytochrome bc₁ complex (8, 9) and is highly potent against Plasmodium species. Unfortunately, recrudescence after atovaquone monotherapy occurs rapidly. Atovaquone is therefore used in combination with a synergistic partner drug, proguanil (10), but even this combination is vulnerable to mutations in pfcytb, especially in areas of cycloguanil resistance (11, 12). Although the target of proguanil is not currently known, proguanil has recently been shown to increase in potency against Plasmodium falciparum after a longer in vitro exposure (13). Considering that the mechanism of resistance to the components of current ACT differs from the mechanism of resistance to the mitochondrion-targeting atovaquone-proguanil combination, this combination has been investigated as an alternative treatment of multidrug-resistant malaria infections (14), though it should be noted that the atovaquone-proguanil combinations tested performed poorly (only 90 to 92% effective at 42 days posttreatment) in northwestern Cambodia and are unlikely to be useful as treatment. Furthermore, drugs targeting the mitochondria kill both liver- and blood-stage malaria parasites and so can be used for both prophylaxis and treatment.

New quinolones based on endochin, a compound shown to be active at clearing avian malaria (15), have recently been synthesized and tested against malaria parasites (16–18). These endochin-like quinolones (ELQ) are equally effective in ex vivo blood-stage screens against P. falciparum and Plasmodium vivax clinical field isolates and are also effective against exoerythocytic forms of rodent and monkey (Plasmodium cynomolgi) malaria (16). However, the activity of ELQs against the zoonotic Plasmodium knowlesi, an increasingly important cause of human malaria in Southeast Asia, is unknown. Importantly, recent articles have identified differences in in vitro (19, 20) and ex vivo (21) susceptibility to established and experimental antimalarial agents between P. knowlesi and P. falciparum. In particular, in vitro studies demonstrated that P. knowlesi is up to 8-fold less susceptible than P. falciparum to inhibitors of dihydroorotate dehydrogenase (e.g., DMS265) (20), 6-fold less susceptible to ATP4 inhibitors (e.g., cipargamin and SJ733) (19), around 3-fold less susceptible to cladosporin and pentamidine, and 66-fold less susceptible to the oxaborole AN13762 (19). Conversely, P. knowlesi was shown to be 10-fold more susceptible to dihydrofolate reductase inhibitors (e.g., pyrimethamine and cycloguanil) (20), around 4.5-fold more susceptible to ganaplacide (KAF156), and over 3-fold more susceptible to halofantrine (19). In spite of the reduced susceptibility of P. knowlesi compared with P. falciparum, many antimalarials remain potent against P. knowlesi in vitro (e.g., 6 nM for cipargamin), and any clinical significance of these reported species differences is yet to be established.

Here, we tested the in vitro activity of endochin and an ELQ series against P. knowlesi and compared this to their activity against a quinolone-sensitive reference P. falciparum line (3D7) under identical experimental conditions, exposed for a single asexual erythrocytic parasite life cycle (i.e., 27 h for our P. knowlesi A1-H.1 clone [22] and 48 h for the P. falciparum 3D7 clone). We then assessed the impact of longer exposures to proguanil and selected ELQs on the susceptibility of our P. knowlesi and P. falciparum lines. Finally, we used isobologram analysis in vitro to test for evidence of synergy between proguanil or atovaquone and ELQ compounds against both species.

RESULTS AND DISCUSSION

Endochin and six endochin-like quinolones (ELQ) were screened under identical in vitro conditions across one complete asexual erythrocytic life cycle against both the P. knowlesi A1-H.1 and the P. falciparum 3D7 lines (Table 1). All but one (ELQ-271) of the ELQ compounds were potent against the P. knowlesi line, with 50% effective concentrations (EC₅₀) under 100 nM. The potencies of endochin and the ELQ compounds against P. knowlesi and P. falciparum were similar, with a <2-fold difference observed between species. With the exception of ELQ-300, all the quinolones screened were more active against P. falciparum (Table 1), though for endochin and ELQ-331, the differences were not significant (P = 0.6233 and P = 0.5014, respectively) (Table 1). ELQ-400 and ELQ-480 were both active at under 10 nM, making them more potent than chloroquine but not as active as dihydroartemisinin (Table 1).

TABLE 1.

Comparison of the in vitro susceptibility of Plasmodium knowlesi (clone A1-H.1) and Plasmodium falciparum (clone 3D7) exposed to novel endochin-like quinolones for one complete life cycle

Compound	EC50 (nM)a		Fold differenceb	Pc
	P. knowlesi A1-H.1 (27-h exposure)	P. falciparum 3D7 (48-h exposure)
Endochin	18.9 ± 1.2	18.1 ± 0.5	1.04	0.6233
ELQ-271	117 ± 12	64.5 ± 3.1	1.81	0.0081
ELQ-300	15.4 ± 0.9	23.1 ± 1.2	0.67	0.0215
ELQ-316	47.1 ± 2.6	33.5 ± 2.3	1.41	0.0097
ELQ-331	49.0 ± 6.2	45.4 ± 1.6	1.08	0.5014
ELQ-400	6.80 ± 0.26	4.95 ± 0.26	1.37	0.0030
ELQ-480	7.06 ± 0.32	5.81 ± 0.26	1.22	0.0433
Chloroquine	33.1 ± 2.0	17.7 ± 1.3	1.87	<0.0001
Dihydroartemisinin	1.52 ± 0.07	3.64 ± 0.42	0.42	0.0112

^aMeans ± standard errors of the means from at least 4 experiments, each performed in duplicate.

^bCalculated as P. knowlesi EC₅₀/P. falciparum EC₅₀.

^cCalculated by comparing EC₅₀ values for P. knowlesi versus P. falciparum using Student’s two-tailed paired t test.

P. falciparum exhibits significantly enhanced susceptibility to proguanil when incubated for more than one life cycle (13). Therefore, in preparation for in vitro combination analysis (isobolograms), we screened ELQ-300 and ELQ-400 as well as proguanil and atovaquone using a longer incubation time (2.5 life cycles). We had previously found no activity for proguanil at 10 μM (the highest concentration we tested) after a single-life-cycle exposure against either P. knowlesi or P. falciparum (data not shown). However, with a longer exposure (2.5 cycles), we observed an EC₅₀ value of proguanil of 2,461 ± 236 nM for P. knowlesi, over 10-fold higher than the EC₅₀ value that we observed for P. falciparum 3D7 (228 ± 29 nM) (Table 2). We expect natural variability within our EC₅₀ values, because our assays were run using asynchronous parasite populations and because the parasites have different life cycle lengths, meaning that drugs are exposed longer to P. falciparum per life cycle than to P. knowlesi. Hence, we consider only >3-fold changes in EC₅₀ between species as potentially important species differences (19).

TABLE 2.

Comparison of the in vitro susceptibility of Plasmodium knowlesi (clone A1-H.1) and Plasmodium falciparum (clone 3D7) exposed to proguanil and selected quinolones for 2.5 life cycles

Compound	EC50 (nM)a		Fold differenceb	Pc
	P. knowlesi A1-H.1 (68-h exposure)	P. falciparum 3D7 (120-h exposure)
Proguanil	2,461 ± 236	228 ± 29	10.79	0.0007
Atovaquone	0.71 ± 0.02	0.74 ± 0.09	0.99	0.1211
ELQ-300	5.31 ± 0.3	15.29 ± 1.2	0.35	0.0011
ELQ-400	1.32 ± 0.2	2.66 ± 0.3	0.50	0.0072

^aMeans ± standard errors of the means from at least 3 experiments, each performed in duplicate.

^bCalculated as P. knowlesi EC₅₀/P. falciparum EC₅₀.

^cCalculated by comparing EC₅₀ values for P. knowlesi versus P. falciparum using Student’s two-tailed unpaired t test.

Atovaquone, ELQ-300, and ELQ-400 were all more potent after the longer exposure. Atovaquone potency increased around 3-fold from 2.5 nM (20) to 0.7 nM (Table 2) and was not significantly different between species. ELQ-300 and ELQ-400 were also more potent after longer in vitro exposures (Tables 1 and 2). Both compounds were now more active against P. knowlesi than P. falciparum (P < 0.0072), though the difference between species was small (<3-fold).

Based on these data, combination studies were then designed to explore the in vitro interactions between the compounds. These experiments were also conducted over multiple life cycles to take into account the increased potency of proguanil after longer exposures (13). As shown previously (10, 13, 23), atovaquone is synergistic in combination with proguanil against P. falciparum (Fig. 1A; Table 3). The investigational quinolones ELQ-300 and ELQ-400 were also synergistic when combined with proguanil against our P. falciparum line (Fig. 1B and C), confirming previous observations for ELQ-300 (16). Surprisingly, neither atovaquone nor the ELQ compounds demonstrated a synergistic interaction in combination with proguanil when tested against P. knowlesi. Instead, all interactions were additive or indifferent (Fig. 1D to F; Table 3). Without knowing the target of proguanil or understanding its mechanism of action, it is not possible to speculate on the reason for this species difference. Clearly, the 10-fold-lower proguanil activity against P. knowlesi (not observed with quinolone activity) coupled with the lack of synergism with quinolones suggests a species-related difference in the inhibitory activity of this biguanide.

FIG 1.

Comparison of the in vitro interaction of proguanil with selected quinolones against P. falciparum (clone 3D7) (A to C) and P. knowlesi (clone A1-H.1) (D to F). Fractional inhibitory concentration (FIC) data are averaged from at least three independent experiments, each run in triplicate. Error bars show standard errors of the means. FIC values of <1.0 are considered to indicate synergy, while FIC values of 1 are considered to indicate an additive or indifferent effect.

TABLE 3.

Mean FICs for the drug combinations tested in this study (Fig. 1 and 2)

Combination	Mean FICa
	P. knowlesi A1-H.1	P. falciparum 3D7
Proguanil-atovaquone	0.986 (0.949–1.024) ADD	0.545 (0.503–0.586) SYN
Proguanil–ELQ-300	1.077 (0.970–1.184) ADD	0.660 (0.619–0.700) SYN
Proguanil–ELQ-400	0.995 (0.858–1.132) ADD	0.631 (0.571–0.690) SYN
Atovaquone–ELQ-300	0.867 (0.814–0.920) M–SYN	0.816 (0.785–0.848) M–SYN
Atovaquone–ELQ-400	0.980 (0.961–0.998) ADD	1.016 (0.980–1.052) ADD

^aCalculated from all FICs within each experiment and for all experiments performed. Values in parentheses are 95% confidence intervals. SYN, synergistic interaction; M-SYN, moderately synergistic interaction; ADD, additive or indifferent interaction.

An alternative drug combination strategy for quinolones is suggested by recent data indicating that quinolones can inhibit the cytochrome bc₁ complex at either the quinol oxidase (Q_o) or quinone reductase (Q_i) site (24). Atovaquone and ELQ-400 are Q_o site inhibitors (8, 24), while ELQ-300 was shown to target the Q_i site (24). Isobolograms combining a Q_o site inhibitor (atovaquone) with a Q_i site inhibitor (ELQ-300) previously demonstrated a moderately synergistic interaction against P. falciparum strain D6 in vitro (25). We confirm this moderately synergistic interaction between atovaquone and ELQ-300 against our P. falciparum 3D7 line (Fig. 2A) and show also a moderately synergistic interaction with this combination against our P. knowlesi line (Fig. 2C; Table 3). Combinations of atovaquone with ELQ-400, both inhibitors of the Q_o site, were additive or indifferent against P. falciparum (Fig. 2B) and P. knowlesi (Fig. 2D; Table 3). Therefore, a more appropriate combination partner for the Q_o site inhibitor atovaquone might be a Q_i site inhibitor (such as ELQ-300) which (i) is considerably more potent than proguanil and (ii) demonstrates moderate synergism in combination with atovaquone against both P. falciparum and P. knowlesi in vitro, unlike combinations with proguanil.

FIG 2.

Comparison of the in vitro interaction of atovaquone with two endochin-like quinolones against P. falciparum (clone 3D7) (A and B) and P. knowlesi (clone A1-H.1) (C and D). Fractional inhibitory concentration (FIC) data are averaged from three independent experiments, each run in triplicate. Error bars show standard errors of the means. FIC values of <1.0 are considered to indicate synergy, while FIC values of 1 are considered to indicate an additive or indifferent effect.

In vivo, proguanil is metabolized to cycloguanil by the liver cytochrome P450 (CYP2C19) (26, 27). Cycloguanil is an inhibitor of the enzyme dihydrofolate reductase (DHFR), a component of the folate pathway in malaria parasites. Thus, the drug combination atovaquone-proguanil actually serves as a triple drug therapy consisting of atovaquone (cytochrome bc₁ inhibitor), proguanil (target unknown), and its metabolite cycloguanil (DHFR inhibitor). Cycloguanil, like atovaquone, has been shown to be highly potent against P. knowlesi in vitro (20). Therefore, even though antagonistic interactions between atovaquone and cycloguanil have been described in vitro (13), the low-nanomolar potency of both cycloguanil and atovaquone (20) should still support this combination for P. knowlesi infections, despite the reduced activity of proguanil and its lack of synergy reported here.

In light of the above-mentioned data, the recent strategy proposed to block the cyclization of proguanil, thereby reducing its metabolism to cycloguanil, ought to be approached with caution (13). In the absence of cycloguanil, and with the reduced activity of proguanil, atovaquone may be exposed as a monotherapy against P. knowlesi infections. It will therefore be critical to screen the cyclization-blocked tert-butyl proguanil (13) for its activity against P. knowlesi and to test it in combination studies with quinolones in this species.

To our knowledge, this is the first study to demonstrate differences in drug interactions between two human malaria parasite species. We reported previously that P. knowlesi exhibited reduced in vitro susceptibility to compounds in human trials for malaria (e.g., DSM 265 and cipargamin) (19, 20), similar to our observations here for proguanil. Considering that all new malaria treatments will likely comprise combinations of drugs, it will be critical to ensure that new combinations involving compounds to which P. knowlesi exhibits reduced susceptibility interact similarly across species.

Resistance to 10 nM atovaquone (5× EC₅₀) is induced readily in vitro after exposure to only 10⁵ parasites of the P. falciparum clone W2 or exposure to 10⁶ parasites of the 3D7 or FCR3 clone (28). Furthermore, exposure of 10⁸ parasites of the P. falciparum Dd2 clone to 10 nM atovaquone (10× EC₅₀) also selected resistant parasites, but no resistant parasites emerged after treatment with 150 nM ELQ-300 (also 10× EC₅₀) at the same inoculum (16). This suggests that the new endochin-like quinolones demonstrate a lower propensity to induce resistance in that parasite clone (16). Similar tests should be performed on the P. knowlesi A1-H.1 line and other newly adapted P. knowlesi lines to explore the propensity of this species to develop resistance to the various quinolones.

In conclusion, novel endochin-like quinolones exhibit strong antimalarial activity (EC₅₀ values of <117 nM) against P. knowlesi in vitro and are equipotent against P. falciparum. We demonstrate for the first time that quinolone combinations with proguanil lack synergy against P. knowlesi in vitro, suggesting distinct mechanisms of action in the malaria parasites. In contrast, combinations of inhibitors targeting the cytochrome bc₁ complex at the Q_o site (e.g., atovaquone) with those targeting the Q_i site (e.g., ELQ-300) show moderate synergism against both species.

MATERIALS AND METHODS

Drugs and experimental compounds.

Proguanil hydrochloride (product no. G7048) was purchased from Sigma-Aldrich UK. Atovaquone was obtained from the Medicines for Malaria Venture. Endochin-like quinolones were synthesized as described below.

Chemical synthesis.

The chemical synthesis of endochin was performed as originally described by Salzer and others in 1948 (29), while methods for ELQ-271 and ELQ-300 were described by Nilsen et al. in 2014 (17). Methods for preparing ELQ-316 were described by Doggett et al. in 2012 (30). Preparation of ELQ-331 was described previously by Frueh et al. (31). Chemical synthesis of ELQ-400 was carried out by the methods of Stickles and coworkers (32). The synthesis and characterization of ELQ-480 are described below.

5-Fluoro-7-methoxy-2-methyl-3-(4-(4-(trifluoromethoxy)phenoxy)phenyl)quinolin-4(1H)-one (ELQ-480) was synthesized according to the methods described by Nilsen et al. in 2014 (17). Purity of ELQ-480 was assessed as >95% by proton nuclear magnetic resonance (NMR). ¹H-NMR spectra were obtained using a Bruker AMX-400 NMR spectrometer operating at 400.14 MHz in dimethyl sulfoxide (DMSO) D₆. The NMR raw data were analyzed using iNMR Spectrum Analyst software. Proton chemical shifts (δ) were reported in parts per million relative to the residual proton at 2.54 ppm in deuterated DMSO D₆. J coupling constants values are in hertz. Coupling constants for ¹⁹F NMR operating at 376 MHz were also obtained for compounds containing fluorine elements for additional validation of structure. The NMR spectrum of ELQ-480 is as follows: ¹H-NMR (400 MHz; DMSO D₆): δ 11.55 (s, 1H), 7.42 (d, J = 8.1 Hz, 2H), 7.25 (d, J = 8.3 Hz, 2H), 7.15 (d, J = 8.7 Hz, 2H), 7.06 (d, J = 8.2 Hz, 2H), 6.76 (s, 1H), 6.63 (d, J = 13.3 Hz, 1H), 3.85 (s, 3H), 2.18 (s, 3H).

Parasite culture.

P. knowlesi parasites (clone A1-H.1) and P. falciparum parasites (clone 3D7) were grown in RPMI 1640 supplemented with 25 mM HEPES, 25 mM Na₂HCO₃, 10 mM d-glucose, 2 mM l-glutamine, 50 mg/liter hypoxanthine, 25 mg/liter gentamicin sulfate, 5 g/liter Albumax II, and 10% (vol/vol) donor horse serum (P30-0702; Pan Biotech). All parasites were grown in human A⁺ red blood cells (National Health Blood and Transplant, United Kingdom). Parasites were incubated in sealed flasks at 37°C under a culture gas mixture of 96% N₂, 3% CO₂, and 1% O₂.

Growth inhibition assays and isobologram testing.

Drug susceptibility was assessed precisely as described previously, with parasites being exposed to the drugs for one complete life cycle (27 h for P. knowlesi and 48 h for P. falciparum) or 2.5 life cycles (68 h for P. knowlesi and 120 h for P. falciparum) (19). Drug combination studies were performed as described previously (19, 23), with the exception that parasites were exposed to drugs for 2.5 cycles instead of 1 life cycle and the starting parasitemia was reduced to 0.5%, while the hematocrit was maintained at 1%. The fractional inhibitory concentrations (FICs) were calculated as described previously (33). The SYBR green I method was used to determine parasite viability (19, 34).