Antagonistic antimalarial properties of a methoxyamino chalcone derivative and 3-hydroxypyridinones in combination with dihydroartemisinin against Plasmodium falciparum

Introduction

Artemisinin (ART)-based combination therapy is recommended by the World Health Organization for the first-line treatment of malaria in uncomplicated Plasmodium falciparum infections. ART and its derivatives are biotransformed by the liver to the active metabolite, dihydroartemisinin (DHA) (Fig. 1), which can be activated by iron, resulting in endoperoxide radicals that damage proteins via the formation of covalent adducts. The accumulation of damaged, polyubiquitinated proteins rapidly induces lethal endoplasmic reticulum stress (Bridgford et al., 2018). ART-resistant P. falciparum parasites emerged in Western Cambodia in the 2000s showing a slow clearance phenotype in malaria patients and an increased ring survival rate (Fairhurst & Dondorp, 2016; Tilley et al., 2016). Mutations in the kelch domain of the kelch13 (k13) gene have been established as genetic markers for ART-resistant malaria parasites (Ariey et al., 2014; Straimer et al., 2015); however, ART resistance is modulated by other genetic factors in some resistant parasites that do not contain k13 mutations (Balikagala et al., 2017; Ikeda et al., 2018). A genome-wide association study identified variants in the ferredoxin (fd), apicoplast ribosomal protein S10 (arps10), multidrug resistance protein 2 (mdr2), and chloroquine resistance transporter (crt) genes as additional factors contributing to ART resistance, among which a missense fd mutation (fd-D193Y) was the most frequent variant among resistant parasites (Miotto et al., 2015).

Ferredoxin (Fd) is an iron-sulfur (Fe-S) electron carrier protein that functions with ferredoxin NADP⁺ reductase (FNR) in apicoplast metabolism, particularly isoprenoid biosynthesis, fatty acid desaturation, and heme oxygenation. However, only the isoprenoid biosynthesis pathway is essential during intraerythrocytic developmental stages (Gisselberg et al., 2013; Rohrich et al., 2005). The Fd-D97Y mutant protein (mutated residue corresponding to residue 193 in the full-length pre-processed Fd protein) has an increased binding affinity for FNR. This mutation reduces FNR function; hence, loss-of-function mutations in the Fd/FNR system may contribute to ART resistance (Kimata-Ariga & Morihisa, 2021). Isoprenoids are protein prenylation substrates, and prenylated proteins are required for vesicular transport to the digestive vacuole, including hemoglobin transport, in which K13 is thought to play an important role (Birnbaum et al., 2020; Mok et al., 2021). Hemoglobin degradation products are required for ART activation, and defects in hemoglobin trafficking cause ART resistance (Mukherjee et al., 2022). Therefore, loss of Fd/FNR function caused by fd mutation or chemical inhibition may enhance the effect of the k13 mutation on the hemoglobin trafficking pathway, resulting in enhanced ART resistance. However, fd/fnr mutations are constrained, because this redox system is essential (Swift et al., 2022). Moreover, Fd/FNR is a validated antimalarial drug target (Qidwai et al., 2014; Suwito et al., 2014). The interaction between recombinant PfFd and PfFNR proteins is inhibited in vitro by methoxyamino chalcone derivatives, of which the most potent compound, C3 (Fig. 1), exhibits 50% inhibition at 100 µM (Suwito et al., 2014).

In addition to Fd, iron is a cofactor for enzymes such as aconitase, oxidoreductases, and ribonucleotide reductase. Hemoglobin, myoglobin, and cytochromes contain an iron-prosthetic heme group. Iron also acts as a chemical catalyst for the generation of reactive oxygen species (ROS) via the Fenton and Haber–Weiss reactions. Iron chelators, such as desferrioxamine (DFO), 1,2-dimethyl-3-hydroxypyridin-4-one, deferiprone (DFP), and deferasirox (DFX) are clinically used for the treatment of patients with iron overload. Importantly, DFO, DFP, DFX, and other iron chelators, including 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) and deferiprone-resveratrol hybrid (DFP-RVT) possess antimalarial activity by interfering with iron uptake, depleting the intraerythrocytic labile iron pool in parasites and expediting the phagocytosis of ingested parasites (Cabantchik, Moody-Haupt & Gordeuk, 1999; Chuljerm et al., 2021; Gordeuk, Thuma & Brittenham, 1994; Maneekesorn et al., 2021; Thipubon et al., 2015). Some antimalarial drugs such as quinine, mefloquine, and artesunate are antagonized by DFP (Pattanapanyasat et al., 2001).

In combination therapy, two different drugs are used to increase the efficacy of treatment, and the interaction between these drugs can be assessed by isobologram analysis (Huang et al., 2019). Drugs with a connection in their mechanisms of action, such as those acting on the same metabolic pathway, can exhibit synergistic or antagonistic interactions. Iron chelators are antagonistic to ART and related compounds (Antoine et al., 2014; Suwito et al., 2014), which can be explained by the iron-dependent activation of ART (Meshnick et al., 1993; Pattanapanyasat et al., 2001; Xie et al., 2016; Klonis, Creek & Tilley, 2013). Although the putative loss of function in the fd-D193Y mutant is associated with ART resistance, it does not affect the survival of P. falciparum parasites exposed to DHA (Kampoun et al., 2022; Stokes et al., 2021). Therefore, it is unclear whether the parasite genetic background, in particular, the allelic status of the fd and k13 genes, affects the antimalarial potency of compounds targeting Fd/FNR and/or the interaction of these compounds with DHA. We hypothesized that the antimalarial potency of the chalcone derivative C3 and its interaction with DHA are affected by the genetic background of the parasite. Therefore, we evaluated the potencies of C3, DFP, DFP-RVT, and CM1, and the combinations of these compounds with DHA against P. falciparum wild-type (WT) and genome-edited parasites with k13 and fd mutations. The iron chelators were used as reference ART antagonists for assessing the interaction of C3 with DHA. This study provides information regarding DHA interactions with compounds that target Fd/FNR.

Materials and Methods

Results

Single compound sensitivity test of P. falciparum

The growth inhibitory properties of DHA, C3, DFP, DFP-RVT, and CM1 were assessed in P. falciparum parental strain 3D7 and the transgenic parasites fd^D193Y_3D7, k13^C580Y_3D7, and k13^C580Yfd^D193Y_3D7 (Kampoun et al., 2022). The potency of the compounds varied as follows: DHA > DFP-RVT > C3 > DFP ≈ CM1, in which the IC₅₀ 95% confidence intervals for DHA, DFP-RVT, C3, DFP, and CM1 were 4–5 nM, 8–10 µM, 21–25 µM, 32–41 µM, and 32–38 µM, respectively. However, the IC₅₀ values of the compounds were not significantly different between the 3D7 parental strain and the transgenic strains with k13 and/or fd mutations (Fig. 2).

Test of pharmacological interaction between DHA and test compounds in P. falciparum

The interactions between DHA and the test compounds were assessed as the fractional inhibition concentration (ΣFIC) index, and the mean ΣFICs of the combinations were compared between fd wild-type (fd^WT) and fd-mutated (fd^MT) parasites using Welch’s t-test (Fig. 3). An antagonistic effect was indicated when ΣFIC > 1.1, an additive effect was indicated when 0.9 < ΣFIC < 1.1, and synergism was indicated when ΣFIC < 0.9, using the criteria established by previous studies of antimalarial interactions (Chou, 2006; Crisafulli et al., 2021). For the DHA and C3 combinations, the mean ΣFICs were moderately antagonistic, which were 1.24, 1.32, 1.21, and 1.23 in the parental 3D7, fd^MT_3D7, k13^MT_3D7, and k13^MTfd^MT_3D7 mutants, respectively. Moreover, no significant differences in mean ΣFIC were observed between fd^WT and fd^MT parasites. All combinations of DHA with iron chelator compounds were antagonistic, although no significant difference was observed between the fd^WT and fd^MT parasites.

Discussion

ART resistance is modulated by several factors, including the genetic background and pharmacological interactions with other antimalarials. For example, the crt and mdr1 genes affect parasite response to Ca²⁺/Na⁺ channel blockers and their interactions with DHA (Eastman et al., 2016). Here, we investigated the interaction of C3, an inhibitor of the PfFd-PfFNR protein interaction, with DHA in parasites with different fd and k13 genetic backgrounds. C3 demonstrated moderate antagonistic activity toward DHA. Our hypothesis that inhibition of the Fd/FNR redox system in the apicoplast by C3 antagonizes DHA is in line with a recent report that other apicoplast-targeting antimalarials also antagonize DHA (Crisafulli et al., 2021). It should be noted that the degree of DHA antagonism by C3 cannot be directly compared with apicoplast targeting drugs reported previously (Crisafulli et al., 2021) owing to substantial methodological differences. Defects in the key apicoplast functions of isoprenoid metabolism and protein prenylation are thought to antagonize ART by the consequent reduction in hemoglobin trafficking (Crisafulli et al., 2021). Reduction in hemoglobin trafficking reduces the level of free heme from hemoglobin digestion, which is the main catalyst for ART activation. These findings caution against the use of ART partner drugs that inhibit Fd-FNR interaction and other apicoplast targets, which may reduce ART efficacy in malaria chemotherapy.

The antimalarial potency of the Fd-FNR inhibitor C3 was modest (IC₅₀ 95% confidence interval 23–25 µM) and did not differ between 3D7 and genome-edited parasites, including those with the fd D193Y mutation. From the study of C3 docked with Fd, C3 was predicted to bind Fd at a site distant from the D193Y mutation (Suwito et al., 2014), which might explain why no difference in antimalarial potency was observed between the fd^WT and fd^MT parasites. In contrast, the antimalarial effect of C3 could be due to the inhibition of targets other than Fd, as the antimalarial IC₅₀ is markedly lower than the concentration of C3 required for 50% inhibition of the interaction between recombinant Fd and FNR proteins (100 µM) (Suwito et al., 2014). The potential for promiscuous targeting by the chalcone derivative C3 is plausible because other chalcone derivatives are known to be promiscuous-targeting antimalarials, such as licochalcone A, the first identified anti-malarial chalcone derivative, which inhibits P. falciparum mitochondrial complexes II and III, and possibly the erythrocyte membrane (Mi-Ichi et al., 2005; Ziegler et al., 2004). Moreover, phenylurenyl chalcones, which are effective against quinoline-resistant P. falciparum, inhibit parasite cysteine proteases involved in hemoglobin degradation and hemozoin formation (Domínguez et al., 2005), and chalcone-chloroquine-based hybrids exert antimalarial activity by inhibiting hemozoin formation (Guantai et al., 2011; Sashidhara et al., 2012). In this study, the tested iron chelators showed modest antimalarial activity, similar to previous reports (Chuljerm et al., 2021; Ferrer et al., 2012; Maneekesorn et al., 2021; Thipubon et al., 2015). No differences in sensitivity to DHA were observed among the parasites tested, although it should be noted that the ART-resistance phenotype manifested in k13 mutants is not detectable by the growth inhibition assay employed in this study (Ariey et al., 2014).

Importantly, the interactions of C3 with DHA in different parasite backgrounds were moderately antagonistic; hence, inhibition of the Fd-FNR interaction by C3 could antagonize DHA. Nevertheless, there was no significant difference between the fd^WT and fd^MT parasites, even in the parasite that contains both fd and k13 mutations. Therefore, the antimalarial mode of action of C3 remains unclear. As expected, the iron chelators antagonized the antimalarial activity of DHA. As no significant differences in the mean ΣFICs between fd^WT and fd^MT parasites were observed, it can be concluded that this fd mutation does not affect the antagonistic interaction between DHA and iron chelators. Presumably, fd mutations associated with the loss of Fd function have a negligible impact on the iron pool targeted by iron chelators. The effect of the fd mutation on the interaction with DHA might be smaller than that of C3, such that it could not be detected by our experimental approach. Alternative approaches are required detect subtle interactions between DHA and apicoplast-targeting drugs. Apicoplast-targeting drugs typically exhibit a delayed-death antimalarial effect (Kennedy et al., 2019), such that interactions with the much faster-acting but short-lived DHA may not be detectable in assays with treatment conducted over one parasite replication cycle, as used in our study. To account for the different rates of killing, interaction assays can be performed by treating with apicoplast inhibitors for up to 72 h before pulsing with DHA (Crisafulli et al., 2021). Another possibility that should be considered is that the fd-D193Y mutation has no detectable effect on DHA interaction with other drugs, but is present at a high frequency among ART-resistant parasites because of positive selection to compensate for loss of fitness caused by k13 mutation, or genetic hitchhiking with k13 resistance mutations.

Conclusions

The putative Fd-FNR inhibitor C3 demonstrated antimalarial activity comparable to that of iron chelators DFP, DFP-RVT, and CM1. No difference in sensitivity was observed among parasites with mutations in the fd and k13 genes compared with the parental P. falciparum 3D7 strain for any compound tested. In combination with DHA, C3 showed moderate antagonistic interactions, and iron chelators were antagonistic. Other approaches are required to understand the effect of the fd mutation on Fd function and how this affects the interaction of C3 with ART.

Supplemental Information