Analysis of the interaction of antimalarial agents with Plasmodium falciparum Glutathione Reductase through molecular mechanical calculations

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H. do C Ferreira, L. R. Pinto, B. A. Oliveira, L. V. Daniel, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3952252/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 May, 2024 Read the published version in Journal of Molecular Modeling → Version 1 posted 4 You are reading this latest preprint version Abstract Malaria remains a significant global health challenge, with emerging resistance to current treatments necessitating the development of novel therapeutic strategies. P. falciparum Glutathione Reductase (PfGR) plays a critical role in the defense mechanisms of malaria parasites against oxidative stress. In this study, we investigate the potential of targeting PfGR with conventional antimalarial drugs and dual drugs combining aminoquinoline derivatives with GR inhibitors using molecular docking and molecular dynamics simulations. Our findings reveal promising interactions between PfGR and antimalarial drugs, with the naphthoquinone Atovaquone (ATV) demonstrating particularly high affinity and potential dual-mode binding with the enzyme active site and cavity. Furthermore, dual drugs exhibit enhanced binding affinity compared to reference inhibitors, suggesting their efficacy in inhibiting PfGR. Insights into their interaction mechanisms and structural dynamics are described. Overall, this research provides valuable insights into the potential of targeting PfGR and encourages further exploration of its role in the mechanisms of action of antimalarial drugs, including dual drugs, to enhance antiparasitic efficacy. Malaria Plasmodium falciparum antimalarials Glutathione Reductase PfGR Molecular Docking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Malaria is a condition resulting from the infection by protozoa of the genus Plasmodium ( P ). These microorganisms can affect birds, reptiles, and mammals, including humans, and there are approximately 200 species of Plasmodium in circulation [ 1 ]. Among these species, five are capable of infecting humans and causing the disease: P. falciparum , P. vivax , P. malariae , P. ovale , and, more recently, P. cynomolgi and P. simium . Although the latter two were initially reported in non-human primates, there are records of naturally acquired infection cases in humans [ 2 – 4 ]. According to the World Health Organization (WHO), the total number of disease cases reached 249 million in 2023. The majority of cases (82%) and deaths (95%) occurred in the African Region, followed by the Southeast Asia Region (cases 10% and deaths 3%). Recent records indicate that the major contributors to Malaria worldwide are the species P. falciparum and P. vivax , with the former responsible for the highest number of fatalities, particularly in the African continent [ 5 ]. Regarding Brazil, the Amazon region is indeed considered the endemic epicenter of Malaria in the country, accounting for 99% of recorded autochthonous cases. Outside this area, the majority of reported cases originate from endemic states or countries, representing over 80% of cases. In terms of analyses conducted by the Ministry of Health in 2023, there was a decline in Malaria notifications between 2010 and 2016. However, in 2017, there was a significant increase of 38.7% compared to the previous year, as highlighted in Fig. 1 below. In the subsequent years, a reduction in notifications was observed; however, the Ministry of Health raised alarms due to an unexpected increase of approximately 27.8% in 2020, in cases of Malaria associated with P. falciparum or mixed P. falciparum / P. vivax Malaria (red dots in Fig. 1 ) [ 6 , 7 ] Malaria is transmitted to the vertebrate host through the bite of female mosquitoes of the genus Anopheles. The sporozoites from the salivary glands infect the human host through the insect's bite. In the liver tissues, the sporozoites multiply through asexual reproduction, forming thousands of merozoites. Released into the bloodstream, these invade red blood cells and initiate the asexual erythrocytic phase, causing disease symptoms in the human host after 48 hours of infection. Some of the merozoites may develop into male or female gametocytes. When a mosquito bites an infected person, the gametocytes are ingested and mature in its digestive system in around 24 hours. Thus, mosquitoes become ready to infect a new host, completing the transmission cycle [ 8 , 9 ]. During the asexual erythrocytic phase, the parasite digests between 60% and 80% of hemoglobin, transporting it to its digestive vacuole (DV) to process it into an inert and non-toxic material known as hemozoin, which does not affect its survival [ 10 ]. In this same phase, the parasite is exposed to oxidative stress generated by the host's immune system to combat the infection, as well as by the heme group and other decomposition products of hemoglobin [ 11 ]. Glutathione (GSH) plays a fundamental role in the defense of Malaria parasites against oxidative stress. This tripeptide acts as an antioxidant, aiding the parasite in neutralizing reactive oxygen species both directly and through reactions catalyzed by glutathione peroxidase and glutathione S-transferase [ 12 ]. Thus, high levels of reduced GSH are maintained in the erythrocytes of infected hosts (asexual erythrocytic phase) thanks to the action of the enzyme Glutathione reductase (GR) [ 13 ]. GR is a homodimer that functions as a disulfide oxidoreductase and utilizes the cofactors FAD (prosthetic group) and NADPH to reduce one molar equivalent of GSSG to two molar equivalents of GSH (GSSG + NADPH + → NADP + + 2GSH) [ 13 , 14 ]. The crystal structure of P. falciparum GR (PfGR) was reported in 2003 by Sarma et al. Similar to human GR (hGR), each subunit of PfGR (Fig. 2 ) has two cysteine residues in the active site (Cys39 and Cys44) that mediate electron transfer [ 15 ]. In addition to the active redox pair Cys39/Cys44, the active site includes the cofactor FAD, which is reduced by NADPH after binding to the oxidized enzyme (2NADPH + FAD → 2NADP + + FADH 2 + 2e - ). Once reduced, the isoalloxazine ring of FADH - packs against the persulfide, forming a charge transfer complex, reducing the Cys39-Cys44 disulfide. Thus, oxidized glutathione (GSSG) binds to the active site to be reduced (GSH), and the cysteine redox pair reforms in the disulfide form. Isoalloxazine and 1,4-naphthoquinone derivatives, methylene blue, ajoene, among others, are considered strong competitive inhibitors of both human (hGR) and parasite (PfGR) enzymes, binding to their active sites [ 16 – 18 ]. Studies on the mechanisms of inhibitors such as methylene blue (MB) suggest that it acts as a "subversive substrate" in the enzyme's active site, oppositely altering its physiological function. The enzyme catalyzes the reduction of methylene blue by FADPH after reduction by NADPH. The corresponding resulting reduced species (Leucomethylene blue) is a more efficient auto-oxidant, oxidized by O 2 . From a cellular pharmacological perspective, each reaction cycle catalyzed by the GR-MB complex leads to the consumption of NADPH and O 2 , and the production of parasitotoxic reactive oxygen species, predominantly H 2 O 2 [ 16 ]. The most pronounced differences between the two enzymes (hGR and PfGR) are found in the cavity region at the dimer interface (Fig. 2 ), causing changes in the electrostatic properties of this region [ 15 ]. In addition to the active site, this cavity is also considered a binding site for non-competitive enzyme inhibitors, known as the allosteric site. Unlike the active site, the interface cavity does not confer selectivity regarding ligand binding [ 19 , 20 ]. In recent years, this region has also been studied for the selective design of drugs [ 21 ]. Inhibitors of PfGR such as Xanthane (6-hydroxy-3-oxo-3H-xanthene-9-propanoic acid) and Menadione (2-methyl-1,4-naphthoquinone) bind to this cavity, potentially causing non-competitive enzyme inhibition [ 19 ]. Some studies indicate that MB can also act as a non-competitive inhibitor of GR and PfGR, probably by binding to the cavitiy of the enzyme [ 22 ]. Studies have shown that erythrocytes deficient in GR were able to fulfill their physiological functions, albeit with a shorter lifespan. However, they did not serve as host cells for P. falciparum . In fact, the depletion of GSH by GR inhibitors in erythrocytes infected with P. falciparum produces a drastic antiplasmodial effect [ 12 ]. Thus, the search for effective inhibitors of PfGR has become a promising strategy for the development of new and effective antimalarial drugs, aiming to overcome current concerns about the disease, given the emergence of strains resistant to first-line treatment (combined therapy with artemisinin). In addition to the defense of Malaria parasites against oxidative stress, studies have highlighted the relationship between GSH and PfGR with the development of resistance to antimalarial drugs. This is supported by the fact that GSH levels increase in mice infected with CQ-resistant strains. Dubois et al demonstrated that, despite CQ treatment not influencing intracellular GSH levels in erythrocytes infected with resistant or sensitive strains, the activity of the enzymes GR and GPx (Glutathione peroxidase) significantly decreases [ 12 ]. Consistently, the depletion of GSH by L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH synthesis, in resistant strains of P. falciparum , restores sensitivity to CQ and its analogs [ 23 ]. In response to the challenges posed by parasite resistance and adverse effects, various antimalarial drugs have been developed, targeting different stages of the parasite's life cycle [ 24 ]. These drugs, classified into classes such as aminoquinolines (e.g., chloroquine (CQ), amodiaquine (AQ), primaquine (PQ), mefloquine (MQ)), naphthoquinones (e.g., atovaquone (ATV)), antifolates (e.g., proguanil), and artemisinin derivatives, aim to combat malaria effectively [ 25 ]. Existing research suggests that 4-aminoquinoline antimalarials and artemisinin derivatives function during the intraerythrocytic stage by binding to the heme group, preventing its sequestration into hemozoin (Hz) [ 26 , 27 ]. 8-aminoquinolines like PQ are believed to induce oxidative stress by accumulating H 2 O 2 in the liver, leading to Plasmodium parasite toxicity [ 28 ] Despite these valuable findings, details of this mechanism of action are not well understood to date. PfGR, a disulfide reductase enzyme, has not been extensively studied as a potential target for these drugs. Motivated by this gap, our study explores the in silico interaction of quinoline-derived drugs and 1,4-naphthoquinones with the PfGR enzyme through molecular mechanical calculations. Additionally, we investigate double-drugs proposed by Davioud-Charvet et al. [ 29 ], combining quinoline-based alcohols with derivatives of 1,4-naphthoquinones, to understand their efficacy in inhibiting PfGR activities. Our discussion, based on binding energy results and graphical analysis of drug-receptor interactions, provides insights valuable for designing potent inhibitors for this essential enzyme in Malaria parasites. 2. Methodology Molecular docking studies were conducted to determine the binding affinity and interactions between candidate inhibitors and the PfGR enzyme (Fig. 2 ) and some results were better discussed via a molecular dynamics simulation. The aminoquinolines CQ, AQ, PQ, MQ, and the naphthoquinone ATV were selected among commercial drugs available for malaria treatment as ligands for the present study. Additionally, we address the double-drugs strategy proposed by Davioud-Charvet et al. [ 29 ], which combines quinoline-based drugs comprising known antimalarial activity with 1,4-naphthoquinones (known as GR inhibitors), linked by a labile ester bond (Fig. 3 ). The structural characteristics were defined among the library of studied compounds that exhibited greater affinity with PfGR. The [2-(3-methyl)naphthoquinolyl]alcanoic acids of menadione and plumbagin had the strongest inhibitory activity, where the most active of this last series led to more than 80% inhibition of both hGR and PfGR [ 29 ]. In this way, the double-drugs 1 – 3 represented in Fig. 3 were also selected as ligands for the present study. The ligands 1c , 3a - c were designed by the authors of the present study. The proposed ligands 3a - c , combine the inhibitor Menadione (MD) directly with quinoline drugs, unlike ligands 1 and 2 , which have an ester bridge with a variable number of carbon atoms in the aliphatic part (n) connecting both residues as depicted in Fig. 3 . The ligands represented in Fig. 3 were optimized, and characterized as minima on the potential energy surface in aqueous phase at the Hartree-Fock level, using the 3-21G basis set in the ORCA 5.0.3 program [ 30 ]. The solvent effect was accounted for using the CPCM method [ 31 ]. The choice of a more basic method is justified by the subsequent treatment by molecular mechanics methods since all structural properties are parameterized by the force field, and the initial structures only provide a reference for the program without requiring an advanced description of the electronic part. The crystal structure of the PfGR enzyme was obtained from the Protein Data Bank (PDB) under the code 1NOF [ 32 ] and edited using Discovery Studio software [ 33 ]. Water molecules and the ligand were removed to prepare the system for molecular docking. Concerning the enzyme's cofactors, FADH 2 was kept near the active site of the biomolecule, which was reconstructed based on the incomplete structure present in the original file. 2.1 Molecular Docking Molecular docking simulations were conducted for all ligands using the software package Autodock 4 version 4.2.6. [ 34 ]. Two regions were selected for structural testing, the enzyme's active site (site 1) and the intersubunit cavity (site 2), as depicted in Fig. 2 . Both were determined at specific locations by fixing a rectangular box, measuring 60Å-70Å-60Å and centered at x:73.371; y:67.404; z:80.181 for site 1, and measuring 50Å-100Å-60Å and centered at x:62.671; y:38.441; z:88.822 for site 2, with a spacing of 0.4 Å. Lamarckian algorithms were employed to obtain 20 target-ligand interaction poses in a population of 250 structures. Images of the poses were generated using Discovery Studio Visualizer 2019 software [ 33 ] and Chimera 1.17.3. [ 35 ]. 2.2 Molecular Dynamics The GAFF2 [ 36 , 37 ] force field was employed for the parameterization of biomolecules. It was used some explicit water molecules as solvent in a periodic boundaries condition of a truncated octahedron box, and the solvent was instantiated as TIP3P [ 38 ] with a 15.0 angstroms radius. The simulation protocol comprised two successive minimization steps: the initial step involved steepest descent cycles (1000) followed by cycles of conjugated gradients (1500). During the first minimization step, a restraint force constant of 500 kcal mol⁻¹ Å⁻² was applied to the solute, while in the second step, no restraint was imposed, allowing the entire system to undergo unconstrained minimization. Subsequently, six intercalated heating and equilibrium steps were conducted, each involving a temperature rise of 50 K (but the last, which increase 60K all the way up to 310K). These steps were executed under constant volume periodic boundaries (NVT) over 2800 cycles (2fs in time), with frames of equilibrium (2fs) implemented under constant pressure periodic boundary conditions (NTP) at a mean pressure of 1 bar. The production phase extended over 100 ns, comprising frames with a temporal interval of 2 fs between each frame and a cutoff distance of 6.0 Å for non-bonded interactions. The simulation was conducted under constant pressure and temperature (NPT) conditions, with the temperature maintained at approximately 310K through the implementation of a Langevin thermostat, and the pressure regulated at 1 bar using the Barendsen barostat [ 39 , 40 ], in order to simulate physiological condition. The SHAKE [ 41 ] algorithm was activated to restrict hydrogen stretching during the simulation. Molecular dynamics energy evaluations were performed using MM-GBSA profile, accessible through the MM-PBSA.py script [ 42 ]. All molecular mechanics simulations were performed in Amber 16 [ 43 , 44 ]. 3. Results and discussion In general, the geometric parameters of ligand structures (CQ, AQ, PQ, MQ, and ATV), optimized at the HF level were in agreement with the solid structures available on the CCDC Cambridge website. Bond lengths were overestimated in aqueous solution, with a maximum error of 1.7% and a maximum deviation of 0.15 Å in relation to the solid state. The error falls within the margin of the HF method, partly due to the solvent effect that tends to increase bond lengths in aqueous solution. Angular parameters also show satisfactory agreement with experimental values, with a maximum error of 5.8%. 3.1 Evaluation of conventional antimalarial drugs as PfGR Inhibitors Initially, molecular docking studies were conducted with the drugs CQ, AQ, PQ, MQ, and ATV, compounds of pharmacological significance, particularly in the treatment of malaria. CQ and AQ are weak diprotic bases; therefore, at physiological pH (∼7.2), they can exist in non-protonated, monoprotonated, and diprotonated forms [ 45 ]. Thus, the effect of protonation on these two drugs was investigated. 3.1.1 Interaction studies in the active site (site 1). The molecular docking data at the enzyme's active site are reported in Table 1 . The binding energies and estimated constants of Table 1 indicate a higher affinity of the studied antimalarials when compared to reference inhibitors MD and MB. All the studied drugs exhibited a binding energy score (∆G int ) lower than − 6.0 kcal mol − 1 (except for CQ in the neutral form). Despite the neutral CQ showing the lowest scoring of the (∆G int =-5.89 kcal mol − 1 ), among the series of antimalarials studied, this value is quite close to reference inhibitors MD (-5.53 kcal mol − 1 ) and MB (-6.08 kcal mol − 1 ). Table 1 Binding affinity scores of commercial antimalarials with the active site of PfGR enzyme. The residues involved in interactions and antiplasmodial activities are also included. Compound ∆G int (kcal/mol) K i (µM) #contacts/residues involved in the interaction IC 50 (nM) Ref Hydrogen bonds Hydrophobic Electrost. 3D7 W2 CQ CQH CQH 2 -5.89 -6.31 -6.71 48.1 23.9 12.0 1/Val383 1/Pro381 2/Pro381,Asp458 11/FAD,Cys44, Val45,Lys48, Leu352,Pro354, Pro381,Val383. 7/ Pro381,Val383, Ile393,Val461, Ala465. 6/Tyr185,Leu352,Pro381,Thr382, Val383, Phe385. 0 0 1/Glu459 18 12–15 459 571 [ 46 ] [ 47 ] AQ AQH AQH 2 -6.96 -6.85 -7.39 7.89 9.45 3.80 2/Asp458,Gln462 4/Gln462,Asp458 4/Pro381,Val383,Asp458,Glu459 6/Tyr185,Leu352,Pro354,Val383, Asp458,Glu459, Gln462. 7/FAD,Leu352, Pro354,Val383, Pro381 6/Tyr185, Leu352,Val383, Phe385. 0 1/Glu459 2/Lys48, Glu459 18 9.7 86.2 6.4 [ 46 ] [ 47 ] PQ -6.33 104.1 3/Glu32,Val383,Asp458,Glu459 7/ FAD,Leu352, Val461,Pro381, Val383. 0 104.1 1117 [ 46 ] MQ (R,S) -6.59 14.7 2/Gln462, FAD 11/ FAD,Tyr185, Val461, Val383, Pro354, Leu352, Pro381. 0 15 3.5 [ 48 ] ATV -8,10 1.15 3/Val383, FAD 9/ FAD, Cys44, Val45, Lys48, Ile49,Leu352, Pro354. 0 2.4 2.1 [ 49 ] MD -5.53 88.6 (82.2) * 1/Gln462 4/Pro354,Pro381,Val383. 0 ‒ ‒ [ 50 ] MB -6.08 35.01 (42.2) * 1/FAD 0 0 ‒ ‒ [ 50 ] *Experimental K m values for MD and MB reduction by PfGR enzyme. In this case, both inhibitors act as redox-cyclers subversive substrates. 3D7 is a chloroquine susceptible strain, whereas W2 is a chloroquine resistant strain. In its most stable state, neutral chloroquine (CQ) (in orange; Fig. 4 a) adopts an extended conformation. The quinoline ring is perpendicular to the isoalloxazine ring, engaging in a π-π T-shaped interaction (4.6 Å) and an N-H···Ph hydrogen bond (2.5 Å) with the drug's pyridine π electron cloud (Fig. 4 a). H-bonds with Val383 assist in positioning the quinoline ring toward FADH 2 (Fig. 4 a). Moreover, the diethyl-pentane-amine group approaches Cys44, essential for the enzyme's electron transfer intermediate, through carbon-hydrogen bonds and hydrophobic interactions (Fig. 4 a). As expected, increasing protonation in CQ leads to more H-bond formation, reflected in the elevated score (Table 1 , Figure S1 ). In CQH and CQH 2 forms, a more closed conformation is observed, especially in the monoprotonated form with a higher RMSD value of 3.23 Å compared to the neutral (1.90 Å) and diprotonated (1.94 Å) forms. In the diprotonated form (green; Fig. 4 a), the quinoline ring points toward the enzyme surface, interacting with residues in the α-helix. Conversely, in the CQH form (navy blue), the structure inverts, directing the quinoline ring toward the interface and interacting with residues in the β-sheets (Fig. 4 a). Like CQH 2 , AQ's quinoline ring approaches the FADH 2 while its diethylamino-phenol group moves toward the interface region (Fig. 4 b). Only the monoprotonated form interacts with FADH 2 , establishing weak π-π stacked interactions between the flavin and quinoline rings and a π-alkyl interaction involving the drug's chloride (Figure S2). The diprotonated form AQH 2 scores slightly better than the neutral and monoprotonated forms, displaying the lowest binding energy (-7.39 kcal mol-1). This is attributed to the formation of strong H-bonds between the diethylamino-phenol group with residues Asp458, Glu459, and Gln462 (⁓2.0 Å) of the first α-helix in this interface (Fig. 4 b; Table 1 ). Moreover, the electrostatic contribution increased, resulting from interactions of the diethylaminomethyl-phenol group with residues Lys48 and Glu459 in the interface (Table 1 ; Fig. 4 b) and a better fit into the cavity of this region (Fig. 4 b). Protonation has a more pronounced effect on the lead drug CQ than its derivative AQ, causing significant conformational changes and larger scoring alterations (Table 1 ). The 8-aminoquinoline PQ adopts a conformation similar to CQ and AQ. The methoxy group of the quinoline ring interacts weakly with FADH 2 through a π-alkyl bond (4.1 Å). The alkyl portion with the amine function heads towards the protein interface, forming H-bonds and hydrophobic interactions with the same residues involved in interactions with CQ and AQ (Table 1 ). The MQ has two stereogenic centers, but its erythro racemic mixture ((11S,12R) and (11R,12S)) is used clinically [ 51 ]. The drug's stereochemistry was considered for the docking study. Both MQ enantiomers exhibited similar interaction energy values at the PfGR active site. However, (R,S)-MQ showed a slightly better score, boasting an interaction energy of -6.59 kcal mol-1 and an inhibition constant of 14.74 µM (Table 1 ). In the lowest energy orientation, the piperidine approaches FADH 2 , forming a robust H-bond (1.89 Å) between the oxygen of FADH 2 and the nitrogen of the piperidine ring of MQ. Simultaneously, the quinoline portion with two –CF 3 groups addresses the enzyme interface, showcasing notable halogen bonding interactions F∙∙O (2.4 Å) and F∙∙N (2.9 Å), especially with Pro381(Figure S3). The hydroxy-1,4-naphthoquinone ATV, in docking analysis, exhibits a ∆Gint value of -8.10 kcal mol-1 (best scoring), showcasing the highest affinity for the enzyme's active site. The estimated inhibition constant value (1.15 µM) is in agreement with reported values for 1,4-naphthoquinone in PfGR inhibition (2.2 µM) and hGR (1.3 µM) [ 17 ]. Similar to neutral CQ, ATV's naphthoquinone ring is oriented inward, perpendicular to the isoalloxazine ring (Fig. 4 c). In fact, the same interactions are found: an H-bond N-H···Ph and a π-π (T-shaped) interaction of 4.63 Å between the isoalloxazine and quinoline rings. However, an extra weak amino-hydroxide H-bond (N-H∙∙OH; 3.08 Å) stabilizes the ligand-receptor arrangement (see Fig. 4 c). More H-bonds with Val383 (Table 1 ) are formed in this system, aiding in positioning the NQ ring toward FADH 2 . Moreover, the chlorophenyl-cyclohexyl group adopts a conformation against the plane of the NQ ring, weakly interacting with initial residues of the α-helix, including Cys44. This arrangement strengthens interactions at site 1, positioning the ligand close to FADH 2 and the active redox pair Cys39/Cys44, with ATV showing stronger interactions than CQ. In the docking analysis for the MB inhibitor (Fig. 5 ), the best-scoring orientations are closely similar to the positioning of the quinoline rings of CQ and the 1,4-NQ of ATV in the FADH 2 binding site (Fig. 4 a and 4 c). Similar aromatic ring interactions (π-π, T-shaped) with FADH 2 and Val383 from the β-sheet are observed (Figs. 4 a, c, and 5 ). In contrast, MD positions itself farther from FADH 2 (⁓4.9 Å) and weakly interacts with Val383, resulting in a lower score than MB (-5.65 kcal mol -1 ). The Michaelis constants (K m ) for PfGR inhibition, obtained by following NADPH oxidation (42.2 µM for MB and 82.2 µM for Menadione), are in agreement with those estimated in the docking study (35.5 µM and 88.6 µM, respectively; Table 1 ) [ 50 ]. According to the vdW surface of the enzyme in Figs. 4 a and 4 c, it is noted that CQ and ATV dock similarly to the active site of the enzyme. It is observed that neither of the two ligands penetrate the cavity above FADH 2 (Figs. 4 a and 4 c). In the case of CQ, the presence of a substituent bulkier larger than chloride could fit better into this cavity, potentially providing extra stability to the drug-receptor complex. Analogously for ATV, with a substituent in positions 7 or 8 of the NQ ring. This cavity is partially filled by the dimethylamino substituent in the reference inhibitor MB, according to the second-best binding pose in this active site (pose 2; Fig. 5 ). It is worth highlighting that 1,4-naphthoquinones are oxidant redox cyclers and can act as acceptors of electrons from different flavoproteins like glutathione-disulfide reductase. The reduction of these compounds by these latter enzymes results in the formation of semiquinone radicals or quinone dianion. These species lead to the generation of superoxide and peroxide through oxygen reduction and, ultimately, the regeneration of naphthoquinone [ 52 ]. Nevertheless, previous studies have emphasized that the reduction potential of ATV is low for efficient two-electron reduction under intracellular conditions (⁓ -0.26 V vs NADP) [ 18 ]. In contrast, the two-electron reduction potential for methylene blue is estimated at ⁓ -0.01 V and ⁓ -0.25 V for MD at pH 7. In order to estimate the tendency to reduce the antimalarial drugs, the standard reduction potential (E o ) relative to the NADPH/NADP + redox couple at pH = 7 was calculated for the antimalarial conventional drugs together with the inhibitors MD and MB (Fig. 3 ). The E o was calculated in aqueous solution at level B3LYP/6–31 + G(d) [ 53 , 54 ] (see more details in Supplementary Materials). Although the estimated E o values of ATV, MD and MB were underestimated (Fig. 6 ), the reduction trend is in agreement with the reported data. Overall, the reduction potential increased in the order PQ < AQ < AQH < MQ < CQH 2 < AQH 2 < MD < ATV < CQH < CQ < MB. MB exhibits the highest reduction potential in the studied series (-0.34 V), followed by CQ (-0.63 V) and ATV (-0.76 V) whose E o value is pretty close to that estimated for the inhibitor MD (-0.81 V) as showed experimentally [ 18 ]. MQ exhibited a redox stability similar to CQH 2 , while PQ was the most stable drug with the lowest potential in the series (Fig. 6 ). The chloroquine reduction potential decreases significantly for the diprotonated form (CQH 2 ) (-1.38 V), where the electron density is more concentrated over the quinoline ring for this full protonation form, as evidenced by the molecular electrostatic potential maps (MEP) of Figure S4. A similar reduction is observed for AQH 2 , with a slightly higher E o (-1.13 V). For both reduced species, the greatest charge variation is observed on the 4-amino pyridine group, confirming its active participation in the reduction. The N-C 2 bond length increases while C 2 -C 3 decreases, showing the loss of aromaticity and the formation of a structure with a localized double bond on the pyridine ring as in the quinolidine anion structure. Distortions in the planar quinoline ring geometry are evident in these reduced structures (Figures S4 and S5). CQ and CQH exhibited a greater tendency to reduction (E o ⁓ 0.64 V) than their analogs AQ and AQH (E o < -2.0 V). After CQ and CQH reduction, the C 7 ‒Cl bond is broken, observing a high charge density on the quinoline ring's C 7 and the leaving chloride, where the minimum MEP is found (Figures S4). In contrast, chlorine remains attached to the quinoline ring after the reduction of AQ and AQH, with no significant distortion of planarity observed. The electron density is distributed mainly on the p-hydroxyanilino and pyridine aromatic rings (Figure S5), disfavoring the reduction in relation to CQ and CQH 2 . Concerning ATV and MD, the formation of the quinone dianion occurs after a two-electron reduction, leading to structural modifications over the 1,4-dione ring. Similar structural changes over the quinoline ring are observed after the reduction of MQ and PQ (Figure S6). Such electrochemical results suggest that ATV and CQ may not be as efficient "subversive substrates" as MB; however, they exhibit slightly higher E o values to MD, indicating similar redox features to this inhibitor, which is considered a moderate redox-cycler drug [ 17 ]. Their similar redox characteristics to the inhibitor MD, along with their observed docking mode in the enzyme's active site, are interesting. The ligand-receptor arrangement promotes contacts necessary to position the ligand in a more lipophilic region near FADH 2 and the active redox pair Cys39/Cys44 (Fig. 4 a,c). These last residues are crucial for the enzyme's redox reactions. If the ligand interacts in this vital pathway, it could potentially inhibit enzyme activity, possibly competing with GSSG. However, interactions with FADH 2 and Cys44 are weaker for CQ, and protonation disfavors its reduction, impacting their efficacy as redox-cyclers. 3.1.2 Interaction studies in the intersubunit cavity (site 2). In addition to its active site, the interface region's cavity is considered another binding site of the PfGR enzyme. The interaction of the same ligands in Fig. 2 with the intersubunit cavity (site 2) was analyzed, and the results are presented in Table 2 . The docking evaluation results again reveal ATV to have the highest affinity for this site 2, with a free binding energy of -9.28 kcal mol -1 and an inhibition constant of 156.51 nM (Table 2 ). It is noteworthy that all the ligands analyzed increased their interaction energy values compared to site 1 and outperformed the scores for the inhibitors MD, Xanthane, and MB, except for PQ (Table 2 ). Nevertheless, a consistent trend in the stability of the drug-PfGR complex at the active site is observed in the cavity (ATV > AQ > MQ⁓CQ > PQ). These results suggest a higher affinity of antimalarial drugs for the cavity when compared to the active site. Table 2 Binding affinity scores of commercial antimalarials with the homodimer intersubunit cavity of PfGR. The residues involved in interactions and antiplasmodial activities are also included. Compound ∆G int (kcal/ mol) K i (µM) #contacts/residues involved in the interaction IC 50 (nM) Ref Hydrogen bonds Hydrophobic Eletrost. 3D7 -S W2 -R CQ CQH CQH 2 -6.38 -6.78 -6.34 20.90 10.66 22.41 1/Glu432. 3/Ser55, Asn456,Glu432. 3/Asp58,Asn62, Glu432. 8/ Ile426, Lys431,Leu455, Pro389,Pro388, His387,Phe421. 6/ Phe51, Ile59, Pro389 Leu455. 5/Asp58,Phe421,Try424,Leu45. 1/Glu432 3/Asp58, His387, Glu432. 2/ Asp58, Glu432. 18 12–15 459 571 [ 46 ] [ 47 ] AQ AQH AQH 2 -7.83 -7.27 -8.61 1.83 4.69 0.49 4/Asp225, Lys228 2/Ser55, Glu432 3/Ser55, Asn62,Glu432. 7/His387,Pro389,Phe421,Tyr424,Ile426,Leu45. 2/ Ile426,Lys431. 3/ Ile426,Lys431, Tyr424. 2/Glu432 3/Asp58, Glu432 4/Asp58, Glu432 18 9.7 86.2 6.4 [ 46 ] [ 47 ] PQ -4.66 380.8 4/Ser55, Asn456,Glu432. 7/Phe51,Ile59, Leu455,His387, Pro388, Pro389. 3/His387, Glu432 104.1 1117 [ 46 ] MQ (S,R) -6.82 9.95 7/ Ser55,Ile59, Glu432,Asn456. 4/His387, Pro389, Leu455. 2/His387 15 3.5 [ 48 ] ATV -9.28 0.16 4/Asp58, Arg196,Asn229. 3/Phe51,His387, Leu455. 2/ Asp58, Glu432 2.4 2.1 [ 49 ] MD -5.65 72.74 1/Asn456 4/Phe51, His387, Leu455. 3/His387, Glu432 ‒ ‒ ‒ Xantane -5.81 54.72 1/Leu419 6/Leu419, Pro485,Thr486, Ala487. 0 ‒ ‒ ‒ MB -6.41 19.87 1/Leu419 3/Leu419. 0 ‒ ‒ ‒ IC 50 data for PfGR inhibition by conventional antimalarial drugs are unavailable in the literature. Davioud-Charve et al. explored this property for ATV and analogs, reporting capabilities below 25 µM [ 55 ]. However, precise IC 50 values were hindered by compound precipitation in solution at doses exceeding 25 µM. To compensate for this absence of data, in vitro antiplasmodial activities against CQ-susceptible and resistant strains were included in Tables 1 and 2 . These results demonstrate a notable correlation with docking interaction energies, where drugs with stronger antiplasmodial activity exhibit lower binding energy (better score), particularly in their interaction with the enzyme cavity (site 2). The correlation in this second binding site reveals a correlation coefficient, R 2 ⁓0.75 (Figure S7), observed in both CQ-sensitive and resistant strains. All antimalarial drugs, including the MD inhibitor, dock within the same region of the cavity (Fig. 7 ). The docking takes place in more hydrophilic between the final residues of the enzyme's largest α-helix from the FADH 2 binding domain, linking the cavity with the active site (in blue, Fig. 7 ), and the sequence of parallel β-sheets in the interface (in red, Fig. 7 ). ATV and AQ have the ability to reach the innermost part of the enzyme by interacting with residues in the NADPH-binding domain region (in gray, Fig. 7 ) and the connecting loop between the last two β-sheets of the interface. Conversely, the inhibitor Xantane and MB are positioned at the monomer interface, proximate to the last α-helix and the β-sheet of this region (Figure S8). A similar binding position of inhibitor Xantane is found at the hGR dimer crystal structure [ 15 ]. In general, the complexation of the drugs with the cavity is generally stabilized by a greater number of H-bonds and electrostatic interactions due to the hydrophilic character of the region (Table 2 ). Concerning chloroquine, the monoprotonated form CQH demonstrates the most favorable interaction with the cavity (∆G bind = -6.78 kcal mol -1 ; Table 2 ). In contrast to the active site, CQH positions its quinoline ring towards the enzyme surface, engaging in H-bonds with residues from the β-sheets of the interface (Asn456) and the largest α-helix (Ser55) (Fig. 7 ). Π-sigma interactions with Leu455 and π-cation interactions with His387 contribute to the quinoline ring stabilization. Although the aliphatic chain has minimal interaction with the cavity, charge attractions occur between Glu432 and the protonated nitrogen of the tertiary amine within the cavity (Fig. 8 a). Notably, the monoprotonated form establishes the highest number of contacts with the enzyme (Table 2 ), predominantly through its well-fitted quinoline ring on the cavity's surface (Fig. 8 a), enhancing the stability of the PfGR-CQH complex. In the protonated forms, the amodiaquine quinoline ring deepens into the cavity's inner part with the diethylamino-phenol group towards the interface's last two β-sheets (see Figs. 7 and 8 b). In contrast, the neutral form of amodiaquine assumes a different orientation, placing its quinoline ring on the enzyme's surface. Simultaneously, the diethylamino-phenol group shifts towards the cavity's interior, establishing H-bond interactions with Lys228 and Asp225 (see Table 2 and Figure S9). The AQH 2 form maintains its extended conformation upon interacting with the cavity, exhibiting minimal structural deformations. Its RMSD value is significantly smaller at 1.78 Å compared to AQH (4.70 Å) and AQ (4.48 Å). Both protonated structures' OH and secondary NH groups act as H-bond acceptors with residues Ser55 and Glu432 (⁓2.1 Å). Moreover, the additional proton from the quinoline ring in AQH 2 engages in an H-bond with Asn62 (2.3 Å), a terminal residue of the α-helix (Fig. 8 b). Protonation considerably increases electrostatic energy, resulting in a 0.81 kcal mol − 1 increase between the neutral and di-protonated forms. Notably, protonated states exhibit a higher number of charge attractions between the diethylamino-phenol group and Asp58 and Glu432 (Table 2 and Fig. 8 b). These interactions significantly contribute to the better binding score of AQH 2 (∆G bind = -8.61) compared to the neutral form (∆Gint = -7.83). The effect of protonation on chloroquine and amodiaquine becomes more pronounced in this secondary binding site, characterized by a more lipophilic nature. For this second site, the (S,R) enantiomer of MQ exhibited high score than (R,S) counterpart. The conformation adopted by (S,R)-MQ enhances contact and forms numerous hydrogen bonds with cavity residues, significantly contributing to the free interaction energy. Similar to CQH and neutral AQ, the quinoline ring of (S,R)-MQ, with its two –CF 3 groups, is oriented towards the surface (Fig. 7 ). Here, fluorine acts as a hydrogen bond acceptor with Asn456 of the interface and Ser55 of the α-helix (Fig. 8 c). Additionally, the –CF 3 substituents engage in halogen interactions. The 2-piperidyl-methanol group also contributes to stabilizing the conformation through hydrogen bonds with Ser55 and Glu432 (Fig. 8 c). However, the ligand structure of (S,R)-MQ does not conform to the cavity shape or align with the site compared to CQH and ATV (see vdW surface in Fig. 8 c). Docking results highlight ATV's tighter binding to the enzyme's cavity compared to the other studied drugs. The 1,4-NQ ring of ATV penetrates the cavity, reaching its innermost part (Fig. 7 ). The carbonyl oxygen serves as a hydrogen bond acceptor with internal residues Arg196 and Asn229, while the hydroxyl is directed towards the major α-helix, forming a robust hydrogen bond with Asp58 (OH∙∙∙O = CO; 2.0 Å) (Fig. 8 d). Notably, residues such as Asp58, Asn62, His65, and Arg196 in PfGR, as identified by Sarma et al., contribute to altering the electrostatic properties of the cavity compared to the analogous human enzyme (hGR) [ 15 ]. Moreover, the NQ ring is stabilized by electrostatic interactions (π-ion), particularly with residues Asp58 (4.87 Å) and Glu432 (2.97 Å) (Fig. 8 d), influencing the drug's positioning within the cavity. The same interactions are observed for the naphthoquinone ring of the reference drug Menadione; However, the charge attraction is weaker with Glu432 (3.67 Å) (Figure S10), and the lack of the extra polar group hydroxide as in ATV disfavor significantly the H-bond formation inside the cavity. Furthermore, the ATV's chlorophenyl-cyclohexyl group stretches across site 2, occupying a substantial cavity region and contributing to its heightened stability (see vdW surface Fig. 8 d). ATV stood out as the drug with the highest score for both investigated binding sites, showcasing as the best candidate to inhibit PfGR between the series of conventional antimalarial drugs studied. The potent antimalarial activity of ATV against both sensitive and resistant parasite strains might involve the inhibition of PfGR, complementing its established action mechanism, specifically, inhibiting the mitochondrial bc1 complex of P. falciparum and disrupting the mitochondrial electron transport chain [ 56 ]. This inhibition is likely attributed to the drug's binding in both the active site and the cavity within the interface region, exhibiting a dual docking mode. 3.2 Antimalarial dual drugs based on inhibitors of PfGR 3.2.1 Analysis via molecular docking simulations Table 3 presents the energetic data obtained from molecular docking simulations, along with experimental ED 50 and inhibition percentage data for dual drug compounds [ 29 ]. Compounds 1a , 1b , and 1c are all naphthoquinones that share the same number of carbons in the 3-ester linker, comprising different O-R substituents. While in 1a , naphthoquinone position 3 is linked to a pentoic acid, 1b and 1c are occupied by chloroquine and amodiaquine, respectively. An amodiaquine-like substituent is employed in compounds 1 ( d-f ), varying the length of the linking ester between the naphthoquinone motif and the aminoquinoline ring. The influence of an -OH group (Plumbagin-based compounds) in the naphthoquinone skeleton was also evaluated through compounds 2a and 2b , which are direct analogs of 1e and 1f , respectively. Additionally, the direct naphthoquinone-aminoquinoline dual drugs are under the names of 1 ( a-c ), which comprise ( 3a ) chloroquine, ( 3b ) amodiaquine, and ( 3c ) the amodiaquine-like compound. These dual drugs showed high stability toward chemical hydrolysis in aqueous solutions and under physiologic conditions [ 29 ]. Table 3 Estimated binding energy estimated by the molecular mechanics analysis for the different inhibitors in each studied site along with calculated inhibition constant and experimental ED 50 and inhibition percentage. Compound Site 1 Site 2 ED 50 FcB1R (µM) % inhibition of P. falciparum * Binding energy (kcal/mol) Inhibition constant (Ki/µM) Binding energy (kcal/mol) Inhibition constant (Ki/µM) 1a -6.74 11.53 -7.22 5.11 3.5 [0.5] a - 1b -4.43 564.28 -6.34 22.69 0.107 - 1c -7.41 3.67 -6.72 11.82 - - 1d -9.77 0.069 -9.11 0.210 0.144 54 1e -9.09 0.217 -9.62 0.088 0.047 82 1f -7.93(-57.42) 1.54 -9.58(-54.82) 0.096 0.023 87 2a -9.00 0.253 -8.65 0.455 0.0287 - 2b -7.74 2.12 -8.32 0.799 0.056 - 3a -7.94 1.52 -7.56 2.88 - - 3b -8.79 0.361 -8.55 0.542 - - 3c -8.67(-32.74) 0.437 -9.28(-45.62) 0.157 - - MD -5.53(-18.68) 88.6 -5.65(-15.40) 72.74 - - ( ) indicates average values of the binding free energies obtained from the 100 ns of production in the dynamics simulation, via MM-GBSA profile. a Experimental IC 50 value for PfGR inhibition [ 29 ] *Inhibition assays at 25 µM of compound [ 29 ] The best molecular docking binding energy achieved for site 1 comprises compound 1d (n = 2 / -9.77 kcal mol − 1 ), with the shortest alkane chain among the ester-linkers. In the same group, the binding energy reduced with the increase of carbons in the ester bridge: compound 1e (n = 4 / -9.09 kcal mol − 1 ) and 1f (n = 5 / -7.93 kcal mol − 1 ). On the other hand, 1e and 1f showed a slightly better score than 1d , whose binding energy did not vary significantly (⁓ 9.60 kcal mol − 1 ) for site 2. A similar tendency is observed experimentally, where both P. falciparum inhibition and ED 50 increase with the ester length; nonetheless, such an increase of n = 4 for n = 5 produces slight biological effects (Table 3 ). This might me explained with the differences in the permeability in the cellular membrane as hydrophobicity may increase with the length of the chain allowing more of the compound to reach the active target thus presenting higher toxicity to the parasite cell. Furthermore, among the compounds with five carbons in the alkane chain, 1b [the double drug containing chloroquine] presented an ED 50 of 0.107 µM. However, the docking interaction energy was only − 4.43 kcal mol − 1 in the protein's active site. In this instance, the discordance suggests that the conjugation strategy involving an NQ alkanoic acid might instigate a synergistic effect, leading to the accumulation of the ester in the parasite's food vacuole. This accumulation amplifies the interaction with heme groups, thus impeding the formation of inert hemozoin crystals, rather than obstructing the biological function of PfGR. 1b presented − 6.34 kcal mol − 1 interaction energy in the allosteric site, which is in the mean of the other compounds in the group. In the plumbagin compounds ( 2a and 2b ), both performed well in the molecular docking tests interacting with site 2 with energies of -8.65 and − 8.32 kcal mol − 1 , respectively. 2a was the most favored in relation to the active site (-9.00 kcal mol − 1 ) what might infer the lower ED 50 concentration in relation to 2b . Again, there is a direct connection between the reported values of ED 50 and the calculated K i for the compounds. Compounds 1f and 3c differ only by the linking chain that connects the naphthoquinone group with the quinolinic condensed rings. While the first is connected through an ester functional, the second is bound via a simple alkane chain. 1f demonstrated activity in the 0.0023 µM of ED 50 , which is concise to the 0.096 µM inhibition constant based on the − 9.58 kcal mol − 1 interaction with the allosteric site 2. Thus, for compound 1f the simulations agreed with experimental in vitro and in vivo tests [ 29 ] that demonstrated 1f as the most promising inhibitor among the ones studied. Besides, 3d displayed an interaction energy with site 2 of -9.28 kcal mol − 1 , and is indicated, thus, as a possible candidate to biological tests to investigate its antiparasitic properties further. Compared to the currently employed inhibitor MD, all studied compounds (but 1b in site 1) displayed lower binding energy, indicating they might be more effective PfGR inhibitors. This highlights the potential of combining drugs to design better analogs that positively impact some treatments' efficiency. Molecular dynamics simulations of the dual drugs 1f and 3c were performed for the best pose in docking, in order to explore the binding modes at thermodynamic conditions and validate the docking results. 3.2.2 Complementary molecular dynamics simulations The molecular dynamics simulations allowed a more detailed analysis of compounds 1f and 3c , mainly about the reference MD. The estimated average binding free energies were also reported in Table 3 in parenthesis, where data point out the dual drugs with a significantly greater binding affinity than inhibitor MD. The thermodynamic analysis in the realm of molecular docking had predicted a better affinity of 3c than 1f for site 1. This binding energy was corrected by the molecular dynamics GBSA profile which predicted an interaction energetics of -57.42 kcal mol − 1 , which is almost twice as favored as 3c (-32.74 kcal mol − 1 ) (Table 3 ). However, regarding site 2, this interaction energy difference lowers to a 9.2 kcal mol − 1 ( 1f : -54.82 kcal mol − 1 ; 3c : -45.62 kcal mol − 1 ), presenting a more modest variation, which is closer to the data obtained from molecular docking analyses. Along the molecular simulation trajectory, some insightful conformations were observable. At first, 1f interaction on site 1 was predicted to occur mainly via the residue Val383 (as observed for the conventional antimalarials and the inhibitor MB), which acts as an arm that appropriately allocates the ligand in the binding position. That supposition was confirmed by the molecular dynamics, which described the ester oxygen interacting uninterruptedly with the amine group in the protein residue. In the first 3ns of the simulation, the conformation obtained in the molecular docking for this binding mode rotated in the esters' C-O bond, resulting in a new conformation. Figure 9 a indicates the interaction poses on both molecular docking and dynamics. Whereas in the docking-obtained structure, the naphthoquinone part of the ligand went towards the NADPH-binding domain, and the amodiaquine part was found near the FADH 2 binding domain, in the molecular dynamics, after 3ns, the rotation of the ester group lead both drug skeleton to a more hydrophobic region. Differently, ligand 3c keeps the interaction profile observed in the molecular docking simulation on site 1, and the only observable contrast in the molecular dynamics is a movement away from the FADH 2 . This movement allows the ligand to move to a more hydrophilic a part of the protein surface, which is stabilized by an H-bonds between the carboxylate in NQ and the residue GLN462. Figure 9 b demonstrates the binding mode described above. That difference in binding mode between 1f and 3c may infer the importance of the ester group, which interacts directly with FADH 2 in the case of 1f and keeps the drug positioned optimally in the active site of the PfGR. The lack of a hydrogen acceptor group in the alkane chain of 3c makes it translate to a less effective area, thus reducing the GBSA interaction energy ( 1f : -57.42 kcal mol − 1 ; 3c : -32.74 kcal mol − 1 ; Table 3 ). On the other hand, 1f interacted with the cavity of the enzyme (site 2) mainly via the interface area. Here, the linking chain was merely a better tool to optimally position both terminal drug structures. The long chain allows both terminal drugs to interact better with the interface region and fit more suitably with the enzyme shape (Fig. 10 a,b). The idea for testing the interaction in this cavity is to study the viability of a protein dimerization blockage once this is the area that connects both monomers in the dimer unit. Additionally, as the cavity is more hydrophilic than the active site, the increase in the number of polar groups (such as the ester) may favor the binding energy as is the case of 1f over 3c (Fig. 10 b,c). This energetic behavior on site 2 is also shown in Table 3 ( 1f : -54.85 kcal mol − 1 ; 3c :-45.62 kcal mol − 1 ). Besides, 3c interaction geometry did not change in relation to the molecular docking simulation. In the case of site 2, the shorter linking chain of 3c did not affected significantly in the interaction mode, and a longer linker ( 1f ) only led to a slightly better proximity with the protein residues. Some deviation analysis through an RMSD approach indicates that, in general, the protein residues have a reduction in structural freedom when interacting with the drugs compared to a simulation comprising only the protein and its cofactor. 1f interaction in the active site demonstrates an RMSD mean of 3.84 Å, whereas 3c in the same location accounted for a mean of 4.80 Å. This difference infers the binding effectiveness of each compound in pfGR and may indicate the importance of a more flexible and polar linking chain (1f: 5 carbons-ester long; 3c: 2 carbons-alkane long). Similarly, a RMSD analysis in the MD and reference resulted in 4.61Å and 5.95 Å, respectively. Regarding the interaction influence over the FADH 2 RMSD, 1f causes both the major deviations and for longer than 3c (Figure S11). This might be caused by the shorter proximity between 1f and the cofactor, once 3c , as discussed earlier, tends to move away from the FADH 2 domain. Furthermore, when interacting in the cavity (site 2), a contrary behavior was observed: 1f reduced the protein RMSD by 4.29 Å compared to a reduction of 3.61 Å achieved by 3c . MD had a slightly better performance than the one for the active site and displayed a protein RMSD of 4.45 Å. All RMSD graphs are displayed in Figure S11 in the supplementary information. Similarly, a solvent-accessible surface area analysis (SASA) demonstrated a mean area available to the solvent of 25767.91 Å2 on the protein during the reference simulation. This average was lower for the compound interacting on site 2 (1f: 23859.96 Å2; 3c: 23097.26 Å2) than for site 1 (1f: 24075.94 Å2; 3c: 23954.61 Å2). However, MD behaved differently and the SASA for site 1 (24210.24 Å2) was lower than the one achieved interacting with site 2 (24435.59 Å2). All SASA graphs are displayed on Figure S12 in the supplementary information. A root mean square of atomic fluctuation (RMSF) was also evaluated, and the analysis of the residues along both sites of interest demonstrates the binding effect of the drugs based on the fluctuation of atoms in the protein. All drugs can decrease RMSF in the protein in comparison to the free protein simulations. The highest decrease was, in general, achieved by both compounds 1f and 3c , which was better than MD in most studied frames. This very same pattern appeared on site 2 RMSF analysis. Although dual-drugs 1f and 3c performed quite similarly in both sites, 1f binding was slightly more effective in the active site than 3c . On the other hand, 3c performed slightly better on site 2, according to the RMSF results. All RMSF graphs are displayed in Figures S13-14 in the supplementary information. In summary, it was not observed a significative change in the interacting pose, which demonstrates the affinity of those simulated dual-drugs and PfGR. Figure S13 in the supplemental material illustrates that even though there is a fluctuation in both CQ condensed rings and MD motifs, the overall binding conformation is kept, and the drugs do not rotate inside the binging sites. Conclusions The molecular docking assessment of conventional antimalarial drugs as PfGR inhibitors revealed that all drugs exhibited higher affinity than reference inhibitors MD and MB, particularly at the enzyme's active site. ATV emerged as a promising candidate, displaying the highest affinity for both the active site and the cavity in the interface region, suggesting a dual docking mode. The docking results also showed a moderate correlation with in vitro antiplasmodial activities, suggesting that the potent antimalarial activity of ATV might involve the inhibition of PfGR. ATV and CQ demonstrated a binding mode similar to inhibitor MB at the enzyme's active site, interacting with the cofactor FADH 2 through H-bonds and π-π interactions. Valine383 plays an essential role assisting the drug binding to the active site, acting as an "anchor" residue and keeping it close to the cofactor FADH 2 . Electrochemical analysis suggested that ATV and CQ, despite being less efficient than MB, could act as moderate "subversive substrates" at the active site. Exploring the enzyme's cavity highlighted distinct binding modes, with ATV and AQ exhibiting notable affinity. Their polar groups of naphtoquinone/quinoline rings play a crucial role as robust H-bond acceptors, while their substituent groups contribute significantly to the ligand's precise adjustment within the site. Moreover, according to molecular mechanism analysis, dual drugs combining aminoquinoline derivatives and GR inhibitors exhibited significantly greater binding affinity than the reference MD. Results provided insights into their interaction mechanisms, where the presence of the aliphatic ester bond (linker) is essential for effective binding with the enzyme's active site. Analysis of protein structural dynamics indicated reduced freedom of protein residues when interacting with the dual drugs 1f and 3c , highlighting their effectiveness in stabilizing the protein structure. The linker ester with a long chain does not play an important role in the dual-drug binding to the enzyme's cavity; nonetheless, it provides flexibility for both terminal drugs to interact strongly with the interface region and better fit the enzyme shape. Overall, the study confirms the strong affinity of these dual-drugs for PfGR, with stable binding poses maintained throughout the molecular dynamics simulations. In summary, 1f showed to be a good alternative as a dual drug due to the good binding modes reported for both enzyme sites. The study encourages further experiments to investigate the role of PfGR or other disulfide reductases in the mechanisms of action of conventional antimalarial drugs and dual-drugs and their contribution to antiparasitic efficacy. Declarations Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contribution This study was conceptualized and written by Dr. GYSD. FHCF, BAO, LVD, and LRP conducted the molecular dockings. FHCF also performed molecular dynamics calculations and contributed to writing. MN supervised the work and revised the manuscript. All authors approved the final version. Acknowledgement The authors thank the Minas Gerais State Agency for Research and Development FAPEMIG (BPD-00777-22), the National Council for Scientific and Technological Development CNPq and the Coordination of Superior Level Staff Improvement CAPES (Finance Code 001) for supporting this work. The authors also thank SDumont/LNCC and NEQC(UFJF) for the computational resources for calculations and D. Quintanilha for helping generate some images. FHCF would additionally like to thank CeMEAI/EULER and Repesq/UFJF for the computational resources. References Rich SM, Ayala FJ (2006) Evolutionary Origins of Human Malaria Parasites. In: Malaria: Genetic and Evolutionary Aspects. Springer US, Boston, MA, pp 125–146 Singh B, Daneshvar C (2013) Human Infections and Detection of Plasmodium knowlesi. Clin Microbiol Rev 26:165–184. https://doi.org/10.1128/CMR.00079-12 Ta TH, Hisam S, Lanza M, et al (2014) First case of a naturally acquired human infection with Plasmodium cynomolgi. Malar J 13:68. https://doi.org/10.1186/1475-2875-13-68 Imwong M, Madmanee W, Suwannasin K, et al (2019) Asymptomatic Natural Human Infections with the Simian Malaria Parasites Plasmodium cynomolgi and Plasmodium knowlesi. J Infect Dis 219:695–702. https://doi.org/10.1093/infdis/jiy519 World Health Organization (2023) World malaria report 2023. https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2023 . Boletim epidemiológico da malária vol 55: Dia da Malária nas Américas – um panorama da malária no Brasil em 2022 e no primeiro semestre de 2023. https://www.gov.br/saude/pt-br/centrais-deconteudo/publicacoes/boletins/epidemiologic epidemiologicos/edicoes/2024/boletim-epidemiologico-volume-55-no-01/ Epidemiológico B, Especial N Biblioteca Virtual em Saúde do Ministério da Saúde Boletim Epidemiológico. Cowman AF, Healer J, Marapana D, Marsh K (2016) Malaria: Biology and Disease. Cell 167:610–624. https://doi.org/10.1016/j.cell.2016.07.055 Biot C, Castro W, Botté CY, Navarro M (2012) The therapeutic potential of metal-based antimalarial agents: Implications for the mechanism of action. Dalton Transactions 41:6335. https://doi.org/10.1039/c2dt12247b de Villiers KA, Egan TJ (2021) Heme Detoxification in the Malaria Parasite: A Target for Antimalarial Drug Development. Acc Chem Res 54:2649–2659. https://doi.org/10.1021/acs.accounts.1c00154 Vasquez M, Zuniga M, Rodriguez A (2021) Oxidative Stress and Pathogenesis in Malaria. Front Cell Infect Microbiol 11:. https://doi.org/10.3389/fcimb.2021.768182 Dubois VL, Platel DFN, Pauly G, Tribouleyduret J (1995) Plasmodium berghei : Implication of Intracellular Glutathione and Its Related Enzyme in Chloroquine Resistance in vivo . Exp Parasitol 81:117–124. https://doi.org/10.1006/expr.1995.1099 Jortzik E, Becker K (2012) Thioredoxin and glutathione systems in Plasmodium falciparum . International Journal of Medical Microbiology 302:187–194. https://doi.org/10.1016/j.ijmm.2012.07.007 Huber PC, Almeida WP, Fátima  de (2008) Glutationa e enzimas relacionadas: papel biológico e importância em processos patológicos. Quim Nova 31:1170–1179. https://doi.org/10.1590/S0100-40422008000500046 Sarma GN, Savvides SN, Becker K, et al (2003) Glutathione Reductase of the Malarial Parasite Plasmodium falciparum : Crystal Structure and Inhibitor Development. J Mol Biol 328:893–907. https://doi.org/10.1016/S0022-2836(03)00347-4 Buchholz K, Schirmer RH, Eubel JK, et al (2008) Interactions of Methylene Blue with Human Disulfide Reductases and Their Orthologues from Plasmodium falciparum . Antimicrob Agents Chemother 52:183–191. https://doi.org/10.1128/AAC.00773-07 Morin C, Besset T, Moutet J-C, et al (2008) The aza-analogues of 1,4-naphthoquinones are potent substrates and inhibitors of plasmodial thioredoxin and glutathione reductases and of human erythrocyte glutathione reductase. Org Biomol Chem 6:2731. https://doi.org/10.1039/b802649c Ehrhardt K, Davioud-Charvet E, Ke H, et al (2013) The Antimalarial Activities of Methylene Blue and the 1,4-Naphthoquinone 3-[4-(Trifluoromethyl)Benzyl]-Menadione Are Not Due to Inhibition of the Mitochondrial Electron Transport Chain. Antimicrob Agents Chemother 57:2114–2120. https://doi.org/10.1128/AAC.02248-12 Iribarne F, González M, Cerecetto H, et al (2007) Interaction energies of nitrofurans with trypanothione reductase and glutathione reductase studied by molecular docking. Journal of Molecular Structure: THEOCHEM 818:7–22. https://doi.org/10.1016/j.theochem.2007.04.035 Iribarne F, Paulino M, Aguilera S, Tapia O (2009) Assaying phenothiazine derivatives as trypanothione reductase and glutathione reductase inhibitors by theoretical docking and Molecular Dynamics studies. J Mol Graph Model 28:371–381. https://doi.org/10.1016/j.jmgm.2009.09.003 Tyagi C, Bathke J, Goyal S, et al (2015) Targeting the intersubunit cavity of Plasmodium falciparum glutathione reductase by a novel natural inhibitor: Computational and experimental evidence. Int J Biochem Cell Biol 61:72–80. https://doi.org/10.1016/j.biocel.2015.01.014 Färber PM, Arscott LD, Williams CH, et al (1998) Recombinant Plasmodium falciparum glutathione reductase is inhibited by the antimalarial dye methylene blue. FEBS Lett 422:311–314. https://doi.org/10.1016/S0014-5793(98)00031-3 Ginsburg H, Famin O, Zhang J, Krugliak M (1998) Inhibition of glutathione-dependent degradation of heme by chloroquine and amodiaquine as a possible basis for their antimalarial mode of action. Biochem Pharmacol 56:1305–1313. https://doi.org/10.1016/S0006-2952(98)00184-1 Shibeshi MA, Kifle ZD, Atnafie SA (2020) Antimalarial Drug Resistance and Novel Targets for Antimalarial Drug Discovery. Infect Drug Resist Volume 13:4047–4060. https://doi.org/10.2147/IDR.S279433 Tibon NS, Ng CH, Cheong SL (2020) Current progress in antimalarial pharmacotherapy and multi-target drug discovery. Eur J Med Chem 188:111983. https://doi.org/10.1016/j.ejmech.2019.111983 Sullivan DJ, Gluzman IY, Russell DG, Goldberg DE (1996) On the molecular mechanism of chloroquine’s antimalarial action. Proceedings of the National Academy of Sciences 93:11865–11870. https://doi.org/10.1073/pnas.93.21.11865 Buller R, Peterson ML, Almarsson Ö, Leiserowitz L (2002) Quinoline Binding Site on Malaria Pigment Crystal: A Rational Pathway for Antimalaria Drug Design. Cryst Growth Des 2:553–562. https://doi.org/10.1021/cg025550i Camarda G, Jirawatcharadech P, Priestley RS, et al (2019) Antimalarial activity of primaquine operates via a two-step biochemical relay. Nat Commun 10:3226. https://doi.org/10.1038/s41467-019-11239-0 Davioud-Charvet E, Delarue S, Biot C, et al (2001) A Prodrug Form of a Plasmodium falciparum Glutathione Reductase Inhibitor Conjugated with a 4-Anilinoquinoline. J Med Chem 44:4268–4276. https://doi.org/10.1021/jm010268g Neese F (2022) Software update: The ORCA program system—Version 5.0. WIREs Computational Molecular Science 12:. https://doi.org/10.1002/wcms.1606 Barone V, Cossi M (1998) Quantum Calculation of Molecular Energies and Energy Gradients in Solution by a Conductor Solvent Model. J Phys Chem A 102:1995–2001. https://doi.org/10.1021/jp9716997 Larson SB, Day J, Barba de la Rosa AP, et al (2003) First Crystallographic Structure of a Xylanase from Glycoside Hydrolase Family 5: Implications for Catalysis. Biochemistry 42:8411–8422. https://doi.org/10.1021/bi034144c (2019) Dassault Systèmes BIOVIA. Discovery Studio Visualizer v.20.1.0.19295: San Diego. Morris GM, Huey R, Lindstrom W, et al (2009) AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem 30:2785–2791. https://doi.org/10.1002/jcc.21256 Pettersen EF, Goddard TD, Huang CC, et al (2004) UCSF Chimera—A visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612. https://doi.org/10.1002/jcc.20084 Vassetti D, Pagliai M, Procacci P (2019) Assessment of GAFF2 and OPLS-AA General Force Fields in Combination with the Water Models TIP3P, SPCE, and OPC3 for the Solvation Free Energy of Druglike Organic Molecules. J Chem Theory Comput 15:1983–1995. https://doi.org/10.1021/acs.jctc.8b01039 He X, Man VH, Yang W, et al (2020) A fast and high-quality charge model for the next generation general AMBER force field. J Chem Phys 153:. https://doi.org/10.1063/5.0019056 Jorgensen WL, Chandrasekhar J, Madura JD, et al (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys 79:926–935. https://doi.org/10.1063/1.445869 Berendsen HJC, Postma JPM, van Gunsteren WF, et al (1984) Molecular dynamics with coupling to an external bath. J Chem Phys 81:3684–3690. https://doi.org/10.1063/1.448118 Uberuaga BP, Anghel M, Voter AF (2004) Synchronization of trajectories in canonical molecular-dynamics simulations: Observation, explanation, and exploitation. J Chem Phys 120:6363–6374. https://doi.org/10.1063/1.1667473 van Gunsteren WF, Berendsen HJC (1977) Algorithms for macromolecular dynamics and constraint dynamics. Mol Phys 34:1311–1327. https://doi.org/10.1080/00268977700102571 Miller BR, McGee TD, Swails JM, et al (2012) MMPBSA.py: An Efficient Program for End-State Free Energy Calculations. J Chem Theory Comput 8:3314–3321. https://doi.org/10.1021/ct300418h Case DA, Cheatham TE, Darden T, et al (2005) The Amber biomolecular simulation programs. J Comput Chem 26:1668–1688. https://doi.org/10.1002/jcc.20290 Salomon-Ferrer R, Case DA, Walker RC (2013) An overview of the Amber biomolecular simulation package. WIREs Computational Molecular Science 3:198–210. https://doi.org/10.1002/wcms.1121 Yayon A, Cabantchik ZI, Ginsburg H (1984) Identification of the acidic compartment of Plasmodium falciparum -infected human erythrocytes as the target of the antimalarial drug chloroquine. EMBO J 3:2695–2700. https://doi.org/10.1002/j.1460-2075.1984.tb02195.x De Souza Pereira C, Quadros HC, Aboagye SY, et al (2022) A Hybrid of Amodiaquine and Primaquine Linked by Gold(I) Is a Multistage Antimalarial Agent Targeting Heme Detoxification and Thiol Redox Homeostasis. Pharmaceutics 14:1251. https://doi.org/10.3390/pharmaceutics14061251 Friebolin W, Jannack B, Wenzel N, et al (2008) Antimalarial Dual Drugs Based on Potent Inhibitors of Glutathione Reductase from Plasmodium falciparum . J Med Chem 51:1260–1277. https://doi.org/10.1021/jm7009292 P Villareal WJ (2017) Complexos fosfíncos de Platina(II) e Paládio(II): atividade farmacológica e interação com o DNA e com a Ferriprotoporfirina Daniel L, Karam A, Hebert C, et al (2023) Metal(triphenylphosphine)-Atovaquone complexes: Synthesis, antimalarial activity, and suppression of heme detoxification. [Manuscript submitted for publication] Müller T, Johann L, Jannack B, et al (2011) Glutathione Reductase-Catalyzed Cascade of Redox Reactions to Bioactivate Potent Antimalarial 1,4-Naphthoquinones – A New Strategy to Combat Malarial Parasites. J Am Chem Soc 133:11557–11571. https://doi.org/10.1021/ja201729z Basco L, Gillotin C, Gimenez F, et al (1992) In vitro activity of the enantiomers of mefloquine, halofantrine and enpiroline against Plasmodium falciparum . Br J Clin Pharmacol 33:517–520. https://doi.org/10.1111/j.1365-2125.1992.tb04081.x Belorgey D, Antoine Lanfranchi D, Davioud-Charvet E (2013) 1,4-Naphthoquinones and Other NADPH-Dependent Glutathione Reductase- Catalyzed Redox Cyclers as Antimalarial Agents. Curr Pharm Des 19:2512–2528. https://doi.org/10.2174/1381612811319140003 Becke AD (1993) Density-functional thermochemistry. I. The effect of the exchange‐only gradient correction. J Chem Phys 98:5648–5652. https://doi.org/10.1063/1.462066 W. J. Hehre, R. Ditchfield and J. A. Pople (1972) Self–Consistent Molecular Orbital Methods. XII. Further Extensions of Gaussian–Type Basis Sets for Use in Molecular Orbital Studies of Organic Molecules. J. Chem. Phys. 56, 2257. https://doi.org/10.1063/1.1677527 Lanfranchi DA, Belorgey D, Müller T, et al (2012) Exploring the trifluoromenadione core as a template to design antimalarial redox-active agents interacting with glutathione reductase. Org Biomol Chem 10:4795. https://doi.org/10.1039/c2ob25229e Birth D, Kao W-C, Hunte C (2014) Structural analysis of atovaquone-inhibited cytochrome bc1 complex reveals the molecular basis of antimalarial drug action. Nat Commun 5:4029. https://doi.org/10.1038/ncomms5029 Additional Declarations No competing interests reported. Supplementary Files SIpapermanuscript2024finalversion.docx Cite Share Download PDF Status: Published Journal Publication published 23 May, 2024 Read the published version in Journal of Molecular Modeling → Version 1 posted Editorial decision: Revision requested 14 Feb, 2024 Editor assigned by journal 14 Feb, 2024 Submission checks completed at journal 13 Feb, 2024 First submitted to journal 12 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3952252","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":272832822,"identity":"a5a18c95-f205-40b8-9ca2-40aaec1a557a","order_by":0,"name":"F. H. do C Ferreira","email":"","orcid":"","institution":"Universidade Federal de Juiz de Fora","correspondingAuthor":false,"prefix":"","firstName":"F.","middleName":"H. do C","lastName":"Ferreira","suffix":""},{"id":272832823,"identity":"0c04b080-b837-4c01-9b47-efca689c0304","order_by":1,"name":"L. R. Pinto","email":"","orcid":"","institution":"Universidade Federal de Juiz de Fora","correspondingAuthor":false,"prefix":"","firstName":"L.","middleName":"R.","lastName":"Pinto","suffix":""},{"id":272832824,"identity":"86feed11-5914-457e-abaa-2d40e93861b2","order_by":2,"name":"B. A. Oliveira","email":"","orcid":"","institution":"Universidade Federal de Juiz de Fora","correspondingAuthor":false,"prefix":"","firstName":"B.","middleName":"A.","lastName":"Oliveira","suffix":""},{"id":272832825,"identity":"384ea6d4-69c3-4318-9b88-4137aabd0b9d","order_by":3,"name":"L. V. Daniel","email":"","orcid":"","institution":"Universidade Federal de Juiz de Fora","correspondingAuthor":false,"prefix":"","firstName":"L.","middleName":"V.","lastName":"Daniel","suffix":""},{"id":272832826,"identity":"168126e6-3b01-4939-b06f-06f477d8e138","order_by":4,"name":"M. Navarro","email":"","orcid":"","institution":"Universidade Federal de Juiz de Fora","correspondingAuthor":false,"prefix":"","firstName":"M.","middleName":"","lastName":"Navarro","suffix":""},{"id":272832827,"identity":"3f77871b-4b1a-400b-b808-7e72046ac867","order_by":5,"name":"G. Y. Sánchez Delgado","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYJACCYYCIHmA+cABEI+94eADZsJaDEBa2BLAWngOHDYgVguPAQNECzN+LfIR6Q9vfDColec73vPxwI8KhsQexsMMzIV7cGsxvJFjbDnD4LjhzDNnNxzsOQPUwgDUMuMZHi0zctikeQyOMW64kbvhMGMbQ+J+hvMHmHkO4NOS/kz6j8Ex+w03ch4cZvwHtQWfFnmJBDNpBoOaRKAWhsOMDURoMeB5Y2zZY3AgeeaZYwYHe45JGIO0HJ6Bz5Z2YIj9qKiz7Tve/PjDjxob2R6Jw4yPC/DZciEBRB2G8SWA6AADHg1AW/rB0nVIQvwN+DSMglEwCkbBCAQATGVfYYuYcFQAAAAASUVORK5CYII=","orcid":"","institution":"Universidade Federal de Juiz de Fora","correspondingAuthor":true,"prefix":"","firstName":"G.","middleName":"Y. Sánchez","lastName":"Delgado","suffix":""}],"badges":[],"createdAt":"2024-02-13 00:19:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3952252/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3952252/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00894-024-05968-3","type":"published","date":"2024-05-23T09:08:53+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51159786,"identity":"edc4dbf2-51ad-428b-aede-966baa7db6fb","added_by":"auto","created_at":"2024-02-15 07:04:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":23475,"visible":true,"origin":"","legend":"\u003cp\u003eReported Malaria Cases in Brazil from 2010 to 2022. The red dots represent the cases of Malaria associated with \u003cem\u003eP. falciparum\u003c/em\u003e or mixed \u003cem\u003eP. falciparum\u003c/em\u003e/\u003cem\u003eP. vivax\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-3952252/v1/f5ef3124313dbf790042c12e.png"},{"id":51159788,"identity":"4ec590b9-956a-48f5-9065-1a49d9475ce7","added_by":"auto","created_at":"2024-02-15 07:04:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":426322,"visible":true,"origin":"","legend":"\u003cp\u003eCrystallographic structure of Glutathione Reductase from \u003cem\u003ePlasmodium falciparum\u003c/em\u003e (subunit; PDB 1ONF) indicating the active site and its key components, along with the cavity located at the enzyme interface\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-3952252/v1/3d32df1d1f42885fa1ec88b5.png"},{"id":51160228,"identity":"70e6d3c1-5fd9-4756-9ec6-ce7bbead66d7","added_by":"auto","created_at":"2024-02-15 07:12:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":118078,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular structures of conventional antimalarials and double-drugs combining quinoline-based with 1,4-naphthoquinones studied in the present work\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-3952252/v1/dcfba03b635c077dea3c4127.png"},{"id":51160230,"identity":"f1813825-4d23-4f78-a7db-d2ae7eda07b9","added_by":"auto","created_at":"2024-02-15 07:12:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1094213,"visible":true,"origin":"","legend":"\u003cp\u003eBinding mode of CQ, AQ, and ATV after docking at the active site of PfGR. 3D structures of the drug-receptor complexes are presented, highlighting the drug conformation and interactions with the main residues in direct contact with the ligand. The van der Waals surface of the enzyme is also shown.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-3952252/v1/7b6698f933e9c7be3ecce6a9.png"},{"id":51159791,"identity":"f9f5acf0-271a-40ea-8781-d07c7705e296","added_by":"auto","created_at":"2024-02-15 07:04:33","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":836794,"visible":true,"origin":"","legend":"\u003cp\u003eMethylene blue binding modes with the active site of PfGR. 3D structures of the drug-receptor complexes are presented, showing the drug conformation and interactions with the main residues in direct contact with the ligand. The van der Waals surface of the enzyme is also presented.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-3952252/v1/d9ccd9f6ca2cb51707113282.png"},{"id":51159790,"identity":"0612e73c-bc83-4927-9dd4-c525818ab2cb","added_by":"auto","created_at":"2024-02-15 07:04:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":27056,"visible":true,"origin":"","legend":"\u003cp\u003ePredicted reduction potentials of antimalarial drugs vs NADPH/NADP\u003csup\u003e+\u003c/sup\u003e couple redox.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-3952252/v1/ca112356694eaed8025a341c.png"},{"id":51159794,"identity":"01037577-4f4b-4ee6-9285-affe1f71dad6","added_by":"auto","created_at":"2024-02-15 07:04:33","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":517058,"visible":true,"origin":"","legend":"\u003cp\u003eComplexes PfGR-antimalarials in their most favorable arrangement in the enzyme cavity.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-3952252/v1/1f6e1d1fe837935e07c7952c.png"},{"id":51160229,"identity":"64289f94-05dd-4055-918b-43b2eab6131f","added_by":"auto","created_at":"2024-02-15 07:12:33","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":763162,"visible":true,"origin":"","legend":"\u003cp\u003eBinding mode of CQH (a), AQH\u003csub\u003e2 \u003c/sub\u003e(b), MQ (c) and ATV (d) after docking into the PfGR cavity. 3D structures of the drug-receptor complexes are presented, showing the drug conformation and interactions with the main residues in direct contact with the ligand. The van der Waals surface of the enzyme is also presented.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-3952252/v1/16abb9487e7beacb4880f142.png"},{"id":51160231,"identity":"13f51a97-cae4-4341-b5f9-50a44a00eddb","added_by":"auto","created_at":"2024-02-15 07:12:33","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":455895,"visible":true,"origin":"","legend":"\u003cp\u003eBinding mode of (a) \u003cstrong\u003e1f \u003c/strong\u003eand (b) \u003cstrong\u003e3c\u003c/strong\u003e in the molecular docking and molecular dynamics simulation in the enzyme active site (site 1). The van der Waals surface of the enzyme is also presented.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-3952252/v1/0e353550eaa448f158f55bed.png"},{"id":51159793,"identity":"2858e074-3db4-47db-8c08-12e3d2ea1df4","added_by":"auto","created_at":"2024-02-15 07:04:33","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":336304,"visible":true,"origin":"","legend":"\u003cp\u003eBinding mode of \u003cstrong\u003e1f \u003c/strong\u003eand \u003cstrong\u003e3c\u003c/strong\u003e in the molecular docking and molecular dynamics simulation in the enzyme cavity (site 2) (a). 3D structures of the \u003cstrong\u003e1f\u003c/strong\u003e-PfGR (b) and \u003cstrong\u003e3c\u003c/strong\u003e-PfGR (c) complexes are presented, showing the drug conformation and interactions with the main residues in direct contact with the ligand. The van der Waals surface of the enzyme is also presented.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-3952252/v1/e8adf925874b773d6292c5c7.png"},{"id":57836980,"identity":"5b1dd471-55fc-4908-a346-f35a428d8015","added_by":"auto","created_at":"2024-06-06 09:09:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6534918,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3952252/v1/65611987-9ed9-45da-a331-252b3f984251.pdf"},{"id":51159797,"identity":"b6a7c60c-c36e-4ebb-80d7-6484d517369a","added_by":"auto","created_at":"2024-02-15 07:04:34","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":13855710,"visible":true,"origin":"","legend":"","description":"","filename":"SIpapermanuscript2024finalversion.docx","url":"https://assets-eu.researchsquare.com/files/rs-3952252/v1/51b805fae50a665503f848c0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Analysis of the interaction of antimalarial agents with Plasmodium falciparum Glutathione Reductase through molecular mechanical calculations","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMalaria is a condition resulting from the infection by protozoa of the genus \u003cem\u003ePlasmodium\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e). These microorganisms can affect birds, reptiles, and mammals, including humans, and there are approximately 200 species of \u003cem\u003ePlasmodium\u003c/em\u003e in circulation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among these species, five are capable of infecting humans and causing the disease: \u003cem\u003eP. falciparum\u003c/em\u003e, \u003cem\u003eP. vivax\u003c/em\u003e, \u003cem\u003eP. malariae\u003c/em\u003e, \u003cem\u003eP. ovale\u003c/em\u003e, and, more recently, \u003cem\u003eP. cynomolgi\u003c/em\u003e and \u003cem\u003eP. simium\u003c/em\u003e. Although the latter two were initially reported in non-human primates, there are records of naturally acquired infection cases in humans [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to the World Health Organization (WHO), the total number of disease cases reached 249\u0026nbsp;million in 2023. The majority of cases (82%) and deaths (95%) occurred in the African Region, followed by the Southeast Asia Region (cases 10% and deaths 3%). Recent records indicate that the major contributors to Malaria worldwide are the species \u003cem\u003eP. falciparum\u003c/em\u003e and \u003cem\u003eP. vivax\u003c/em\u003e, with the former responsible for the highest number of fatalities, particularly in the African continent [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Regarding Brazil, the Amazon region is indeed considered the endemic epicenter of Malaria in the country, accounting for 99% of recorded autochthonous cases. Outside this area, the majority of reported cases originate from endemic states or countries, representing over 80% of cases. In terms of analyses conducted by the Ministry of Health in 2023, there was a decline in Malaria notifications between 2010 and 2016. However, in 2017, there was a significant increase of 38.7% compared to the previous year, as highlighted in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e below. In the subsequent years, a reduction in notifications was observed; however, the Ministry of Health raised alarms due to an unexpected increase of approximately 27.8% in 2020, in cases of Malaria associated with \u003cem\u003eP. falciparum\u003c/em\u003e or mixed \u003cem\u003eP. falciparum\u003c/em\u003e/\u003cem\u003eP. vivax\u003c/em\u003e Malaria (red dots in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMalaria is transmitted to the vertebrate host through the bite of female mosquitoes of the genus Anopheles. The sporozoites from the salivary glands infect the human host through the insect's bite. In the liver tissues, the sporozoites multiply through asexual reproduction, forming thousands of merozoites. Released into the bloodstream, these invade red blood cells and initiate the asexual erythrocytic phase, causing disease symptoms in the human host after 48 hours of infection. Some of the merozoites may develop into male or female gametocytes. When a mosquito bites an infected person, the gametocytes are ingested and mature in its digestive system in around 24 hours. Thus, mosquitoes become ready to infect a new host, completing the transmission cycle [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. During the asexual erythrocytic phase, the parasite digests between 60% and 80% of hemoglobin, transporting it to its digestive vacuole (DV) to process it into an inert and non-toxic material known as hemozoin, which does not affect its survival [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In this same phase, the parasite is exposed to oxidative stress generated by the host's immune system to combat the infection, as well as by the heme group and other decomposition products of hemoglobin [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGlutathione (GSH) plays a fundamental role in the defense of Malaria parasites against oxidative stress. This tripeptide acts as an antioxidant, aiding the parasite in neutralizing reactive oxygen species both directly and through reactions catalyzed by glutathione peroxidase and glutathione S-transferase [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Thus, high levels of reduced GSH are maintained in the erythrocytes of infected hosts (asexual erythrocytic phase) thanks to the action of the enzyme Glutathione reductase (GR) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. GR is a homodimer that functions as a disulfide oxidoreductase and utilizes the cofactors FAD (prosthetic group) and NADPH to reduce one molar equivalent of GSSG to two molar equivalents of GSH (GSSG\u0026thinsp;+\u0026thinsp;NADPH\u003csup\u003e+\u003c/sup\u003e \u0026rarr; NADP\u003csup\u003e+\u003c/sup\u003e + 2GSH) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe crystal structure of \u003cem\u003eP. falciparum\u003c/em\u003e GR (PfGR) was reported in 2003 by Sarma et al. Similar to human GR (hGR), each subunit of PfGR (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) has two cysteine residues in the active site (Cys39 and Cys44) that mediate electron transfer [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition to the active redox pair Cys39/Cys44, the active site includes the cofactor FAD, which is reduced by NADPH after binding to the oxidized enzyme (2NADPH\u0026thinsp;+\u0026thinsp;FAD \u0026rarr; 2NADP\u003csup\u003e+\u003c/sup\u003e + FADH\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;2e\u003csup\u003e-\u003c/sup\u003e). Once reduced, the isoalloxazine ring of FADH\u003csup\u003e-\u003c/sup\u003e packs against the persulfide, forming a charge transfer complex, reducing the Cys39-Cys44 disulfide. Thus, oxidized glutathione (GSSG) binds to the active site to be reduced (GSH), and the cysteine redox pair reforms in the disulfide form. Isoalloxazine and 1,4-naphthoquinone derivatives, methylene blue, ajoene, among others, are considered strong competitive inhibitors of both human (hGR) and parasite (PfGR) enzymes, binding to their active sites [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Studies on the mechanisms of inhibitors such as methylene blue (MB) suggest that it acts as a \"subversive substrate\" in the enzyme's active site, oppositely altering its physiological function. The enzyme catalyzes the reduction of methylene blue by FADPH after reduction by NADPH. The corresponding resulting reduced species (Leucomethylene blue) is a more efficient auto-oxidant, oxidized by O\u003csub\u003e2\u003c/sub\u003e. From a cellular pharmacological perspective, each reaction cycle catalyzed by the GR-MB complex leads to the consumption of NADPH and O\u003csub\u003e2\u003c/sub\u003e, and the production of parasitotoxic reactive oxygen species, predominantly H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe most pronounced differences between the two enzymes (hGR and PfGR) are found in the cavity region at the dimer interface (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), causing changes in the electrostatic properties of this region [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition to the active site, this cavity is also considered a binding site for non-competitive enzyme inhibitors, known as the allosteric site. Unlike the active site, the interface cavity does not confer selectivity regarding ligand binding [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In recent years, this region has also been studied for the selective design of drugs [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Inhibitors of PfGR such as Xanthane (6-hydroxy-3-oxo-3H-xanthene-9-propanoic acid) and Menadione (2-methyl-1,4-naphthoquinone) bind to this cavity, potentially causing non-competitive enzyme inhibition [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Some studies indicate that MB can also act as a non-competitive inhibitor of GR and PfGR, probably by binding to the cavitiy of the enzyme [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStudies have shown that erythrocytes deficient in GR were able to fulfill their physiological functions, albeit with a shorter lifespan. However, they did not serve as host cells for \u003cem\u003eP. falciparum\u003c/em\u003e. In fact, the depletion of GSH by GR inhibitors in erythrocytes infected with \u003cem\u003eP. falciparum\u003c/em\u003e produces a drastic antiplasmodial effect [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Thus, the search for effective inhibitors of PfGR has become a promising strategy for the development of new and effective antimalarial drugs, aiming to overcome current concerns about the disease, given the emergence of strains resistant to first-line treatment (combined therapy with artemisinin). In addition to the defense of Malaria parasites against oxidative stress, studies have highlighted the relationship between GSH and PfGR with the development of resistance to antimalarial drugs. This is supported by the fact that GSH levels increase in mice infected with CQ-resistant strains. Dubois et al demonstrated that, despite CQ treatment not influencing intracellular GSH levels in erythrocytes infected with resistant or sensitive strains, the activity of the enzymes GR and GPx (Glutathione peroxidase) significantly decreases [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Consistently, the depletion of GSH by L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH synthesis, in resistant strains of \u003cem\u003eP. falciparum\u003c/em\u003e, restores sensitivity to CQ and its analogs [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn response to the challenges posed by parasite resistance and adverse effects, various antimalarial drugs have been developed, targeting different stages of the parasite's life cycle [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. These drugs, classified into classes such as aminoquinolines (e.g., chloroquine (CQ), amodiaquine (AQ), primaquine (PQ), mefloquine (MQ)), naphthoquinones (e.g., atovaquone (ATV)), antifolates (e.g., proguanil), and artemisinin derivatives, aim to combat malaria effectively [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Existing research suggests that 4-aminoquinoline antimalarials and artemisinin derivatives function during the intraerythrocytic stage by binding to the heme group, preventing its sequestration into hemozoin (Hz) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. 8-aminoquinolines like PQ are believed to induce oxidative stress by accumulating H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the liver, leading to \u003cem\u003ePlasmodium\u003c/em\u003e parasite toxicity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] Despite these valuable findings, details of this mechanism of action are not well understood to date. PfGR, a disulfide reductase enzyme, has not been extensively studied as a potential target for these drugs. Motivated by this gap, our study explores the in silico interaction of quinoline-derived drugs and 1,4-naphthoquinones with the PfGR enzyme through molecular mechanical calculations. Additionally, we investigate double-drugs proposed by Davioud-Charvet et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], combining quinoline-based alcohols with derivatives of 1,4-naphthoquinones, to understand their efficacy in inhibiting PfGR activities. Our discussion, based on binding energy results and graphical analysis of drug-receptor interactions, provides insights valuable for designing potent inhibitors for this essential enzyme in Malaria parasites.\u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cp\u003eMolecular docking studies were conducted to determine the binding affinity and interactions between candidate inhibitors and the PfGR enzyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and some results were better discussed via a molecular dynamics simulation. The aminoquinolines CQ, AQ, PQ, MQ, and the naphthoquinone ATV were selected among commercial drugs available for malaria treatment as ligands for the present study. Additionally, we address the double-drugs strategy proposed by Davioud-Charvet et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], which combines quinoline-based drugs comprising known antimalarial activity with 1,4-naphthoquinones (known as GR inhibitors), linked by a labile ester bond (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The structural characteristics were defined among the library of studied compounds that exhibited greater affinity with PfGR. The [2-(3-methyl)naphthoquinolyl]alcanoic acids of menadione and plumbagin had the strongest inhibitory activity, where the most active of this last series led to more than 80% inhibition of both hGR and PfGR [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In this way, the double-drugs \u003cb\u003e1\u003c/b\u003e\u0026ndash;\u003cb\u003e3\u003c/b\u003e represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e were also selected as ligands for the present study. The ligands \u003cb\u003e1c\u003c/b\u003e, \u003cb\u003e3a\u003c/b\u003e-\u003cb\u003ec\u003c/b\u003e were designed by the authors of the present study. The proposed ligands \u003cb\u003e3a\u003c/b\u003e-\u003cb\u003ec\u003c/b\u003e, combine the inhibitor Menadione (MD) directly with quinoline drugs, unlike ligands \u003cb\u003e1\u003c/b\u003e and \u003cb\u003e2\u003c/b\u003e, which have an ester bridge with a variable number of carbon atoms in the aliphatic part (n) connecting both residues as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe ligands represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e were optimized, and characterized as minima on the potential energy surface in aqueous phase at the Hartree-Fock level, using the 3-21G basis set in the ORCA 5.0.3 program [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The solvent effect was accounted for using the CPCM method [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The choice of a more basic method is justified by the subsequent treatment by molecular mechanics methods since all structural properties are parameterized by the force field, and the initial structures only provide a reference for the program without requiring an advanced description of the electronic part.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe crystal structure of the PfGR enzyme was obtained from the Protein Data Bank (PDB) under the code 1NOF [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] and edited using Discovery Studio software [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Water molecules and the ligand were removed to prepare the system for molecular docking. Concerning the enzyme's cofactors, FADH\u003csub\u003e2\u003c/sub\u003e was kept near the active site of the biomolecule, which was reconstructed based on the incomplete structure present in the original file.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Molecular Docking\u003c/h2\u003e \u003cp\u003eMolecular docking simulations were conducted for all ligands using the software package Autodock 4 version 4.2.6. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Two regions were selected for structural testing, the enzyme's active site (site 1) and the intersubunit cavity (site 2), as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Both were determined at specific locations by fixing a rectangular box, measuring 60\u0026Aring;-70\u0026Aring;-60\u0026Aring; and centered at x:73.371; y:67.404; z:80.181 for site 1, and measuring 50\u0026Aring;-100\u0026Aring;-60\u0026Aring; and centered at x:62.671; y:38.441; z:88.822 for site 2, with a spacing of 0.4 \u0026Aring;. Lamarckian algorithms were employed to obtain 20 target-ligand interaction poses in a population of 250 structures. Images of the poses were generated using Discovery Studio Visualizer 2019 software [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and Chimera 1.17.3. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Molecular Dynamics\u003c/h2\u003e \u003cp\u003eThe GAFF2 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] force field was employed for the parameterization of biomolecules. It was used some explicit water molecules as solvent in a periodic boundaries condition of a truncated octahedron box, and the solvent was instantiated as TIP3P [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] with a 15.0 angstroms radius. The simulation protocol comprised two successive minimization steps: the initial step involved steepest descent cycles (1000) followed by cycles of conjugated gradients (1500). During the first minimization step, a restraint force constant of 500 kcal mol⁻\u0026sup1; \u0026Aring;⁻\u0026sup2; was applied to the solute, while in the second step, no restraint was imposed, allowing the entire system to undergo unconstrained minimization.\u003c/p\u003e \u003cp\u003eSubsequently, six intercalated heating and equilibrium steps were conducted, each involving a temperature rise of 50 K (but the last, which increase 60K all the way up to 310K). These steps were executed under constant volume periodic boundaries (NVT) over 2800 cycles (2fs in time), with frames of equilibrium (2fs) implemented under constant pressure periodic boundary conditions (NTP) at a mean pressure of 1 bar. The production phase extended over 100 ns, comprising frames with a temporal interval of 2 fs between each frame and a cutoff distance of 6.0 \u0026Aring; for non-bonded interactions. The simulation was conducted under constant pressure and temperature (NPT) conditions, with the temperature maintained at approximately 310K through the implementation of a Langevin thermostat, and the pressure regulated at 1 bar using the Barendsen barostat [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], in order to simulate physiological condition. The SHAKE [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] algorithm was activated to restrict hydrogen stretching during the simulation. Molecular dynamics energy evaluations were performed using MM-GBSA profile, accessible through the MM-PBSA.py script [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. All molecular mechanics simulations were performed in Amber 16 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003eIn general, the geometric parameters of ligand structures (CQ, AQ, PQ, MQ, and ATV), optimized at the HF level were in agreement with the solid structures available on the CCDC Cambridge website. Bond lengths were overestimated in aqueous solution, with a maximum error of 1.7% and a maximum deviation of 0.15 Å in relation to the solid state. The error falls within the margin of the HF method, partly due to the solvent effect that tends to increase bond lengths in aqueous solution. Angular parameters also show satisfactory agreement with experimental values, with a maximum error of 5.8%.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Evaluation of conventional antimalarial drugs as PfGR Inhibitors\u003c/h2\u003e \u003cp\u003eInitially, molecular docking studies were conducted with the drugs CQ, AQ, PQ, MQ, and ATV, compounds of pharmacological significance, particularly in the treatment of malaria. CQ and AQ are weak diprotic bases; therefore, at physiological pH (∼7.2), they can exist in non-protonated, monoprotonated, and diprotonated forms [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Thus, the effect of protonation on these two drugs was investigated.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 Interaction studies in the active site (site 1).\u003c/h2\u003e \u003cp\u003eThe molecular docking data at the enzyme's active site are reported in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The binding energies and estimated constants of Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e indicate a higher affinity of the studied antimalarials when compared to reference inhibitors MD and MB. All the studied drugs exhibited a binding energy score (∆G\u003csub\u003eint\u003c/sub\u003e) lower than − 6.0 kcal mol\u003csup\u003e− 1\u003c/sup\u003e (except for CQ in the neutral form). Despite the neutral CQ showing the lowest scoring of the (∆G\u003csub\u003eint\u003c/sub\u003e =-5.89 kcal mol\u003csup\u003e− 1\u003c/sup\u003e), among the series of antimalarials studied, this value is quite close to reference inhibitors MD (-5.53 kcal mol\u003csup\u003e− 1\u003c/sup\u003e) and MB (-6.08 kcal mol\u003csup\u003e− 1\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBinding affinity scores of commercial antimalarials with the active site of PfGR enzyme. The residues involved in interactions and antiplasmodial activities are also included.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e∆G\u003csub\u003eint\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(kcal/mol)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eK\u003csub\u003ei\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(µM)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e#contacts/residues involved in the interaction\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u0026nbsp;(nM)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eRef\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHydrogen bonds\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHydrophobic\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eElectrost.\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e3D7\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eW2\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCQ\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eCQH\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eCQH\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-5.89\u003c/p\u003e \u003cp\u003e-6.31\u003c/p\u003e \u003cp\u003e-6.71\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e48.1\u003c/p\u003e \u003cp\u003e23.9\u003c/p\u003e \u003cp\u003e12.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1/Val383\u003c/p\u003e \u003cp\u003e1/Pro381\u003c/p\u003e \u003cp\u003e2/Pro381,Asp458\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11/FAD,Cys44,\u003c/p\u003e \u003cp\u003eVal45,Lys48,\u003c/p\u003e \u003cp\u003eLeu352,Pro354, Pro381,Val383.\u003c/p\u003e \u003cp\u003e7/ Pro381,Val383, Ile393,Val461,\u003c/p\u003e \u003cp\u003eAla465.\u003c/p\u003e \u003cp\u003e6/Tyr185,Leu352,Pro381,Thr382,\u003c/p\u003e \u003cp\u003eVal383, Phe385.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e1/Glu459\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e18\u003c/p\u003e \u003cp\u003e12–15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e459\u003c/p\u003e \u003cp\u003e571\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAQ\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eAQH\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eAQH\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-6.96\u003c/p\u003e \u003cp\u003e-6.85\u003c/p\u003e \u003cp\u003e-7.39\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.89\u003c/p\u003e \u003cp\u003e9.45\u003c/p\u003e \u003cp\u003e3.80\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2/Asp458,Gln462\u003c/p\u003e \u003cp\u003e4/Gln462,Asp458\u003c/p\u003e \u003cp\u003e4/Pro381,Val383,Asp458,Glu459\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6/Tyr185,Leu352,Pro354,Val383,\u003c/p\u003e \u003cp\u003eAsp458,Glu459,\u003c/p\u003e \u003cp\u003eGln462.\u003c/p\u003e \u003cp\u003e7/FAD,Leu352, Pro354,Val383, Pro381\u003c/p\u003e \u003cp\u003e6/Tyr185, Leu352,Val383,\u003c/p\u003e \u003cp\u003ePhe385.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003cp\u003e1/Glu459\u003c/p\u003e \u003cp\u003e2/Lys48,\u003c/p\u003e \u003cp\u003eGlu459\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e18\u003c/p\u003e \u003cp\u003e9.7\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e86.2\u003c/p\u003e \u003cp\u003e6.4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePQ\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-6.33\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e104.1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3/Glu32,Val383,Asp458,Glu459\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7/ FAD,Leu352, Val461,Pro381,\u003c/p\u003e \u003cp\u003eVal383.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e104.1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1117\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMQ\u003c/b\u003e (R,S)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-6.59\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.7\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2/Gln462, FAD\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11/ FAD,Tyr185, Val461, Val383, Pro354, Leu352, Pro381.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eATV\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-8,10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3/Val383, FAD\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9/ FAD, Cys44, Val45, Lys48, Ile49,Leu352, Pro354.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-5.53\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88.6\u003c/p\u003e \u003cp\u003e(82.2)\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1/Gln462\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4/Pro354,Pro381,Val383.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e‒\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e‒\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-6.08\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.01 (42.2)\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1/FAD\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e‒\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e‒\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e*Experimental K\u003csub\u003em\u003c/sub\u003e values for MD and MB reduction by PfGR enzyme. In this case, both inhibitors act as redox-cyclers subversive substrates. 3D7 is a chloroquine susceptible strain, whereas W2 is a chloroquine resistant strain.\u003c/p\u003e \u003cp\u003eIn its most stable state, neutral chloroquine (CQ) (in orange; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) adopts an extended conformation. The quinoline ring is perpendicular to the isoalloxazine ring, engaging in a π-π T-shaped interaction (4.6 Å) and an N-H···Ph hydrogen bond (2.5 Å) with the drug's pyridine π electron cloud (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). H-bonds with Val383 assist in positioning the quinoline ring toward FADH\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Moreover, the diethyl-pentane-amine group approaches Cys44, essential for the enzyme's electron transfer intermediate, through carbon-hydrogen bonds and hydrophobic interactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). As expected, increasing protonation in CQ leads to more H-bond formation, reflected in the elevated score (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In CQH and CQH\u003csub\u003e2\u003c/sub\u003e forms, a more closed conformation is observed, especially in the monoprotonated form with a higher RMSD value of 3.23 Å compared to the neutral (1.90 Å) and diprotonated (1.94 Å) forms. In the diprotonated form (green; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), the quinoline ring points toward the enzyme surface, interacting with residues in the α-helix. Conversely, in the CQH form (navy blue), the structure inverts, directing the quinoline ring toward the interface and interacting with residues in the β-sheets (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLike CQH\u003csub\u003e2\u003c/sub\u003e, AQ's quinoline ring approaches the FADH\u003csub\u003e2\u003c/sub\u003e while its diethylamino-phenol group moves toward the interface region (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Only the monoprotonated form interacts with FADH\u003csub\u003e2\u003c/sub\u003e, establishing weak π-π stacked interactions between the flavin and quinoline rings and a π-alkyl interaction involving the drug's chloride (Figure S2).\u003c/p\u003e \u003cp\u003eThe diprotonated form AQH\u003csub\u003e2\u003c/sub\u003e scores slightly better than the neutral and monoprotonated forms, displaying the lowest binding energy (-7.39 kcal mol-1). This is attributed to the formation of strong H-bonds between the diethylamino-phenol group with residues Asp458, Glu459, and Gln462 (⁓2.0 Å) of the first α-helix in this interface (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Moreover, the electrostatic contribution increased, resulting from interactions of the diethylaminomethyl-phenol group with residues Lys48 and Glu459 in the interface (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb) and a better fit into the cavity of this region (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Protonation has a more pronounced effect on the lead drug CQ than its derivative AQ, causing significant conformational changes and larger scoring alterations (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The 8-aminoquinoline PQ adopts a conformation similar to CQ and AQ. The methoxy group of the quinoline ring interacts weakly with FADH\u003csub\u003e2\u003c/sub\u003e through a π-alkyl bond (4.1 Å). The alkyl portion with the amine function heads towards the protein interface, forming H-bonds and hydrophobic interactions with the same residues involved in interactions with CQ and AQ (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe MQ has two stereogenic centers, but its erythro racemic mixture ((11S,12R) and (11R,12S)) is used clinically [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The drug's stereochemistry was considered for the docking study. Both MQ enantiomers exhibited similar interaction energy values at the PfGR active site. However, (R,S)-MQ showed a slightly better score, boasting an interaction energy of -6.59 kcal mol-1 and an inhibition constant of 14.74 µM (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In the lowest energy orientation, the piperidine approaches FADH\u003csub\u003e2\u003c/sub\u003e, forming a robust H-bond (1.89 Å) between the oxygen of FADH\u003csub\u003e2\u003c/sub\u003e and the nitrogen of the piperidine ring of MQ. Simultaneously, the quinoline portion with two –CF\u003csub\u003e3\u003c/sub\u003e groups addresses the enzyme interface, showcasing notable halogen bonding interactions F∙∙O (2.4 Å) and F∙∙N (2.9 Å), especially with Pro381(Figure S3).\u003c/p\u003e \u003cp\u003eThe hydroxy-1,4-naphthoquinone ATV, in docking analysis, exhibits a ∆Gint value of -8.10 kcal mol-1 (best scoring), showcasing the highest affinity for the enzyme's active site. The estimated inhibition constant value (1.15 µM) is in agreement with reported values for 1,4-naphthoquinone in PfGR inhibition (2.2 µM) and hGR (1.3 µM) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Similar to neutral CQ, ATV's naphthoquinone ring is oriented inward, perpendicular to the isoalloxazine ring (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). In fact, the same interactions are found: an H-bond N-H···Ph and a π-π (T-shaped) interaction of 4.63 Å between the isoalloxazine and quinoline rings. However, an extra weak amino-hydroxide H-bond (N-H∙∙OH; 3.08 Å) stabilizes the ligand-receptor arrangement (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). More H-bonds with Val383 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) are formed in this system, aiding in positioning the NQ ring toward FADH\u003csub\u003e2\u003c/sub\u003e. Moreover, the chlorophenyl-cyclohexyl group adopts a conformation against the plane of the NQ ring, weakly interacting with initial residues of the α-helix, including Cys44. This arrangement strengthens interactions at site 1, positioning the ligand close to FADH\u003csub\u003e2\u003c/sub\u003e and the active redox pair Cys39/Cys44, with ATV showing stronger interactions than CQ.\u003c/p\u003e \u003cp\u003eIn the docking analysis for the MB inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), the best-scoring orientations are closely similar to the positioning of the quinoline rings of CQ and the 1,4-NQ of ATV in the FADH\u003csub\u003e2\u003c/sub\u003e binding site (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Similar aromatic ring interactions (π-π, T-shaped) with FADH\u003csub\u003e2\u003c/sub\u003e and Val383 from the β-sheet are observed (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, c, and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In contrast, MD positions itself farther from FADH\u003csub\u003e2\u003c/sub\u003e (⁓4.9 Å) and weakly interacts with Val383, resulting in a lower score than MB (-5.65 kcal mol\u003csup\u003e-1\u003c/sup\u003e). The Michaelis constants (K\u003csub\u003em\u003c/sub\u003e) for PfGR inhibition, obtained by following NADPH oxidation (42.2 µM for MB and 82.2 µM for Menadione), are in agreement with those estimated in the docking study (35.5 µM and 88.6 µM, respectively; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to the vdW surface of the enzyme in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, it is noted that CQ and ATV dock similarly to the active site of the enzyme. It is observed that neither of the two ligands penetrate the cavity above FADH\u003csub\u003e2\u003c/sub\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). In the case of CQ, the presence of a substituent bulkier larger than chloride could fit better into this cavity, potentially providing extra stability to the drug-receptor complex. Analogously for ATV, with a substituent in positions 7 or 8 of the NQ ring. This cavity is partially filled by the dimethylamino substituent in the reference inhibitor MB, according to the second-best binding pose in this active site (pose 2; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIt is worth highlighting that 1,4-naphthoquinones are oxidant redox cyclers and can act as acceptors of electrons from different flavoproteins like glutathione-disulfide reductase. The reduction of these compounds by these latter enzymes results in the formation of semiquinone radicals or quinone dianion. These species lead to the generation of superoxide and peroxide through oxygen reduction and, ultimately, the regeneration of naphthoquinone [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Nevertheless, previous studies have emphasized that the reduction potential of ATV is low for efficient two-electron reduction under intracellular conditions (⁓ -0.26 V vs NADP) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In contrast, the two-electron reduction potential for methylene blue is estimated at ⁓ -0.01 V and ⁓ -0.25 V for MD at pH 7.\u003c/p\u003e \u003cp\u003eIn order to estimate the tendency to reduce the antimalarial drugs, the standard reduction potential (E\u003csup\u003eo\u003c/sup\u003e) relative to the NADPH/NADP\u003csup\u003e+\u003c/sup\u003e redox couple at pH = 7 was calculated for the antimalarial conventional drugs together with the inhibitors MD and MB (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The E\u003csup\u003eo\u003c/sup\u003e was calculated in aqueous solution at level B3LYP/6–31 + G(d) [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] (see more details in Supplementary Materials).\u003c/p\u003e \u003cp\u003eAlthough the estimated E\u003csup\u003eo\u003c/sup\u003e values of ATV, MD and MB were underestimated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), the reduction trend is in agreement with the reported data. Overall, the reduction potential increased in the order PQ \u0026lt; AQ \u0026lt; AQH \u0026lt; MQ \u0026lt; CQH\u003csub\u003e2\u003c/sub\u003e \u0026lt; AQH\u003csub\u003e2\u003c/sub\u003e \u0026lt; MD \u0026lt; ATV \u0026lt; CQH \u0026lt; CQ \u0026lt; MB. MB exhibits the highest reduction potential in the studied series (-0.34 V), followed by CQ (-0.63 V) and ATV (-0.76 V) whose E\u003csup\u003eo\u003c/sup\u003e value is pretty close to that estimated for the inhibitor MD (-0.81 V) as showed experimentally [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. MQ exhibited a redox stability similar to CQH\u003csub\u003e2\u003c/sub\u003e, while PQ was the most stable drug with the lowest potential in the series (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe chloroquine reduction potential decreases significantly for the diprotonated form (CQH\u003csub\u003e2\u003c/sub\u003e) (-1.38 V), where the electron density is more concentrated over the quinoline ring for this full protonation form, as evidenced by the molecular electrostatic potential maps (MEP) of Figure S4. A similar reduction is observed for AQH\u003csub\u003e2\u003c/sub\u003e, with a slightly higher E\u003csup\u003eo\u003c/sup\u003e (-1.13 V). For both reduced species, the greatest charge variation is observed on the 4-amino pyridine group, confirming its active participation in the reduction. The N-C\u003csup\u003e2\u003c/sup\u003e bond length increases while C\u003csup\u003e2\u003c/sup\u003e-C\u003csup\u003e3\u003c/sup\u003e decreases, showing the loss of aromaticity and the formation of a structure with a localized double bond on the pyridine ring as in the quinolidine anion structure. Distortions in the planar quinoline ring geometry are evident in these reduced structures (Figures S4 and S5).\u003c/p\u003e \u003cp\u003eCQ and CQH exhibited a greater tendency to reduction (E\u003csup\u003eo\u003c/sup\u003e⁓ 0.64 V) than their analogs AQ and AQH (E\u003csup\u003eo\u003c/sup\u003e \u0026lt; -2.0 V). After CQ and CQH reduction, the C\u003csup\u003e7\u003c/sup\u003e‒Cl bond is broken, observing a high charge density on the quinoline ring's C\u003csup\u003e7\u003c/sup\u003e and the leaving chloride, where the minimum MEP is found (Figures S4). In contrast, chlorine remains attached to the quinoline ring after the reduction of AQ and AQH, with no significant distortion of planarity observed. The electron density is distributed mainly on the p-hydroxyanilino and pyridine aromatic rings (Figure S5), disfavoring the reduction in relation to CQ and CQH\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eConcerning ATV and MD, the formation of the quinone dianion occurs after a two-electron reduction, leading to structural modifications over the 1,4-dione ring. Similar structural changes over the quinoline ring are observed after the reduction of MQ and PQ (Figure S6).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSuch electrochemical results suggest that ATV and CQ may not be as efficient \"subversive substrates\" as MB; however, they exhibit slightly higher E\u003csup\u003eo\u003c/sup\u003e values to MD, indicating similar redox features to this inhibitor, which is considered a moderate redox-cycler drug [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Their similar redox characteristics to the inhibitor MD, along with their observed docking mode in the enzyme's active site, are interesting. The ligand-receptor arrangement promotes contacts necessary to position the ligand in a more lipophilic region near FADH\u003csub\u003e2\u003c/sub\u003e and the active redox pair Cys39/Cys44 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,c). These last residues are crucial for the enzyme's redox reactions. If the ligand interacts in this vital pathway, it could potentially inhibit enzyme activity, possibly competing with GSSG. However, interactions with FADH\u003csub\u003e2\u003c/sub\u003e and Cys44 are weaker for CQ, and protonation disfavors its reduction, impacting their efficacy as redox-cyclers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 Interaction studies in the intersubunit cavity (site 2).\u003c/h2\u003e \u003cp\u003eIn addition to its active site, the interface region's cavity is considered another binding site of the PfGR enzyme. The interaction of the same ligands in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e with the intersubunit cavity (site 2) was analyzed, and the results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The docking evaluation results again reveal ATV to have the highest affinity for this site 2, with a free binding energy of -9.28 kcal mol\u003csup\u003e-1\u003c/sup\u003e and an inhibition constant of 156.51 nM (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). It is noteworthy that all the ligands analyzed increased their interaction energy values compared to site 1 and outperformed the scores for the inhibitors MD, Xanthane, and MB, except for PQ (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Nevertheless, a consistent trend in the stability of the drug-PfGR complex at the active site is observed in the cavity (ATV \u0026gt; AQ \u0026gt; MQ⁓CQ \u0026gt; PQ). These results suggest a higher affinity of antimalarial drugs for the cavity when compared to the active site.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBinding affinity scores of commercial antimalarials with the homodimer intersubunit cavity of PfGR. The residues involved in interactions and antiplasmodial activities are also included.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e∆G\u003csub\u003eint\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(kcal/\u003c/p\u003e \u003cp\u003emol)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eK\u003csub\u003ei\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e(µM)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c7\" namest=\"c4\"\u003e \u003cp\u003e#contacts/residues involved in the interaction\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u0026nbsp;(nM)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c10\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRef\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eHydrogen bonds\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHydrophobic\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEletrost.\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e3D7\u003c/b\u003e\u003c/p\u003e \u003cp\u003e-S\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eW2\u003c/b\u003e\u003c/p\u003e \u003cp\u003e-R\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCQ\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eCQH\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eCQH\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-6.38\u003c/p\u003e \u003cp\u003e-6.78\u003c/p\u003e \u003cp\u003e-6.34\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e20.90\u003c/p\u003e \u003cp\u003e10.66\u003c/p\u003e \u003cp\u003e22.41\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1/Glu432.\u003c/p\u003e \u003cp\u003e3/Ser55, Asn456,Glu432.\u003c/p\u003e \u003cp\u003e3/Asp58,Asn62, Glu432.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8/ Ile426, Lys431,Leu455,\u003c/p\u003e \u003cp\u003ePro389,Pro388,\u003c/p\u003e \u003cp\u003eHis387,Phe421.\u003c/p\u003e \u003cp\u003e6/ Phe51,\u0026nbsp;Ile59,\u003c/p\u003e \u003cp\u003ePro389 Leu455.\u003c/p\u003e \u003cp\u003e5/Asp58,Phe421,Try424,Leu45.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1/Glu432\u003c/p\u003e \u003cp\u003e3/Asp58,\u003c/p\u003e \u003cp\u003eHis387,\u003c/p\u003e \u003cp\u003eGlu432.\u003c/p\u003e \u003cp\u003e2/ Asp58,\u003c/p\u003e \u003cp\u003eGlu432.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e18\u003c/p\u003e \u003cp\u003e12–15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e459\u003c/p\u003e \u003cp\u003e571\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAQ\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eAQH\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eAQH\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-7.83\u003c/p\u003e \u003cp\u003e-7.27\u003c/p\u003e \u003cp\u003e-8.61\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e1.83\u003c/p\u003e \u003cp\u003e4.69\u003c/p\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4/Asp225,\u003c/p\u003e \u003cp\u003eLys228\u003c/p\u003e \u003cp\u003e2/Ser55, Glu432\u003c/p\u003e \u003cp\u003e3/Ser55,\u003c/p\u003e \u003cp\u003eAsn62,Glu432.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7/His387,Pro389,Phe421,Tyr424,Ile426,Leu45.\u003c/p\u003e \u003cp\u003e2/ Ile426,Lys431.\u003c/p\u003e \u003cp\u003e3/ Ile426,Lys431,\u003c/p\u003e \u003cp\u003eTyr424.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2/Glu432\u003c/p\u003e \u003cp\u003e3/Asp58,\u003c/p\u003e \u003cp\u003eGlu432\u003c/p\u003e \u003cp\u003e4/Asp58,\u003c/p\u003e \u003cp\u003eGlu432\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e18\u003c/p\u003e \u003cp\u003e9.7\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e86.2\u003c/p\u003e \u003cp\u003e6.4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePQ\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-4.66\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e380.8\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4/Ser55,\u003c/p\u003e \u003cp\u003eAsn456,Glu432.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7/Phe51,Ile59,\u003c/p\u003e \u003cp\u003eLeu455,His387,\u003c/p\u003e \u003cp\u003ePro388, Pro389.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3/His387,\u003c/p\u003e \u003cp\u003eGlu432\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e104.1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1117\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMQ\u003c/b\u003e (S,R)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-6.82\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e9.95\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7/ Ser55,Ile59,\u003c/p\u003e \u003cp\u003eGlu432,Asn456.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4/His387, Pro389, Leu455.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2/His387\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eATV\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-9.28\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4/Asp58,\u003c/p\u003e \u003cp\u003eArg196,Asn229.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3/Phe51,His387,\u003c/p\u003e \u003cp\u003eLeu455.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2/ Asp58, Glu432\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-5.65\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e72.74\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1/Asn456\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4/Phe51, His387, Leu455.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3/His387,\u003c/p\u003e \u003cp\u003eGlu432\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e‒\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e‒\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e‒\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eXantane\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-5.81\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e54.72\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1/Leu419\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6/Leu419,\u003c/p\u003e \u003cp\u003ePro485,Thr486, Ala487.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e‒\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e‒\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e‒\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMB\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-6.41\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e19.87\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1/Leu419\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3/Leu419.\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e‒\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e‒\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e‒\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e data for PfGR inhibition by conventional antimalarial drugs are unavailable in the literature. Davioud-Charve et al. explored this property for ATV and analogs, reporting capabilities below 25 µM [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. However, precise IC\u003csub\u003e50\u003c/sub\u003e values were hindered by compound precipitation in solution at doses exceeding 25 µM. To compensate for this absence of data, \u003cem\u003ein vitro\u003c/em\u003e antiplasmodial activities against CQ-susceptible and resistant strains were included in Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. These results demonstrate a notable correlation with docking interaction energies, where drugs with stronger antiplasmodial activity exhibit lower binding energy (better score), particularly in their interaction with the enzyme cavity (site 2). The correlation in this second binding site reveals a correlation coefficient, R\u003csup\u003e2\u003c/sup\u003e ⁓0.75 (Figure S7), observed in both CQ-sensitive and resistant strains.\u003c/p\u003e \u003cp\u003eAll antimalarial drugs, including the MD inhibitor, dock within the same region of the cavity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The docking takes place in more hydrophilic between the final residues of the enzyme's largest α-helix from the FADH\u003csub\u003e2\u003c/sub\u003e binding domain, linking the cavity with the active site (in blue, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), and the sequence of parallel β-sheets in the interface (in red, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). ATV and AQ have the ability to reach the innermost part of the enzyme by interacting with residues in the NADPH-binding domain region (in gray, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) and the connecting loop between the last two β-sheets of the interface. Conversely, the inhibitor Xantane and MB are positioned at the monomer interface, proximate to the last α-helix and the β-sheet of this region (Figure S8). A similar binding position of inhibitor Xantane is found at the hGR dimer crystal structure [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn general, the complexation of the drugs with the cavity is generally stabilized by a greater number of H-bonds and electrostatic interactions due to the hydrophilic character of the region (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Concerning chloroquine, the monoprotonated form CQH demonstrates the most favorable interaction with the cavity (∆G\u003csub\u003ebind\u003c/sub\u003e= -6.78 kcal mol\u003csup\u003e-1\u003c/sup\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In contrast to the active site, CQH positions its quinoline ring towards the enzyme surface, engaging in H-bonds with residues from the β-sheets of the interface (Asn456) and the largest α-helix (Ser55) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Π-sigma interactions with Leu455 and π-cation interactions with His387 contribute to the quinoline ring stabilization. Although the aliphatic chain has minimal interaction with the cavity, charge attractions occur between Glu432 and the protonated nitrogen of the tertiary amine within the cavity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Notably, the monoprotonated form establishes the highest number of contacts with the enzyme (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), predominantly through its well-fitted quinoline ring on the cavity's surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea), enhancing the stability of the PfGR-CQH complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the protonated forms, the amodiaquine quinoline ring deepens into the cavity's inner part with the diethylamino-phenol group towards the interface's last two β-sheets (see Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). In contrast, the neutral form of amodiaquine assumes a different orientation, placing its quinoline ring on the enzyme's surface. Simultaneously, the diethylamino-phenol group shifts towards the cavity's interior, establishing H-bond interactions with Lys228 and Asp225 (see Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Figure S9).\u003c/p\u003e \u003cp\u003eThe AQH\u003csub\u003e2\u003c/sub\u003e form maintains its extended conformation upon interacting with the cavity, exhibiting minimal structural deformations. Its RMSD value is significantly smaller at 1.78 Å compared to AQH (4.70 Å) and AQ (4.48 Å). Both protonated structures' OH and secondary NH groups act as H-bond acceptors with residues Ser55 and Glu432 (⁓2.1 Å). Moreover, the additional proton from the quinoline ring in AQH\u003csub\u003e2\u003c/sub\u003e engages in an H-bond with Asn62 (2.3 Å), a terminal residue of the α-helix (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eProtonation considerably increases electrostatic energy, resulting in a 0.81 kcal mol\u003csup\u003e− 1\u003c/sup\u003e increase between the neutral and di-protonated forms. Notably, protonated states exhibit a higher number of charge attractions between the diethylamino-phenol group and Asp58 and Glu432 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). These interactions significantly contribute to the better binding score of AQH\u003csub\u003e2\u003c/sub\u003e (∆G\u003csub\u003ebind\u003c/sub\u003e = -8.61) compared to the neutral form (∆Gint = -7.83). The effect of protonation on chloroquine and amodiaquine becomes more pronounced in this secondary binding site, characterized by a more lipophilic nature.\u003c/p\u003e \u003cp\u003eFor this second site, the (S,R) enantiomer of MQ exhibited high score than (R,S) counterpart. The conformation adopted by (S,R)-MQ enhances contact and forms numerous hydrogen bonds with cavity residues, significantly contributing to the free interaction energy. Similar to CQH and neutral AQ, the quinoline ring of (S,R)-MQ, with its two –CF\u003csub\u003e3\u003c/sub\u003e groups, is oriented towards the surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Here, fluorine acts as a hydrogen bond acceptor with Asn456 of the interface and Ser55 of the α-helix (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec). Additionally, the –CF\u003csub\u003e3\u003c/sub\u003e substituents engage in halogen interactions. The 2-piperidyl-methanol group also contributes to stabilizing the conformation through hydrogen bonds with Ser55 and Glu432 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec). However, the ligand structure of (S,R)-MQ does not conform to the cavity shape or align with the site compared to CQH and ATV (see vdW surface in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eDocking results highlight ATV's tighter binding to the enzyme's cavity compared to the other studied drugs. The 1,4-NQ ring of ATV penetrates the cavity, reaching its innermost part (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The carbonyl oxygen serves as a hydrogen bond acceptor with internal residues Arg196 and Asn229, while the hydroxyl is directed towards the major α-helix, forming a robust hydrogen bond with Asp58 (OH∙∙∙O = CO; 2.0 Å) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). Notably, residues such as Asp58, Asn62, His65, and Arg196 in PfGR, as identified by Sarma et al., contribute to altering the electrostatic properties of the cavity compared to the analogous human enzyme (hGR) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Moreover, the NQ ring is stabilized by electrostatic interactions (π-ion), particularly with residues Asp58 (4.87 Å) and Glu432 (2.97 Å) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed), influencing the drug's positioning within the cavity. The same interactions are observed for the naphthoquinone ring of the reference drug Menadione; However, the charge attraction is weaker with Glu432 (3.67 Å) (Figure S10), and the lack of the extra polar group hydroxide as in ATV disfavor significantly the H-bond formation inside the cavity. Furthermore, the ATV's chlorophenyl-cyclohexyl group stretches across site 2, occupying a substantial cavity region and contributing to its heightened stability (see vdW surface Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eATV stood out as the drug with the highest score for both investigated binding sites, showcasing as the best candidate to inhibit PfGR between the series of conventional antimalarial drugs studied. The potent antimalarial activity of ATV against both sensitive and resistant parasite strains might involve the inhibition of PfGR, complementing its established action mechanism, specifically, inhibiting the mitochondrial bc1 complex of \u003cem\u003eP. falciparum\u003c/em\u003e and disrupting the mitochondrial electron transport chain [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. This inhibition is likely attributed to the drug's binding in both the active site and the cavity within the interface region, exhibiting a dual docking mode.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Antimalarial dual drugs based on inhibitors of PfGR\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1 Analysis via molecular docking simulations\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents the energetic data obtained from molecular docking simulations, along with experimental ED\u003csub\u003e50\u003c/sub\u003e and inhibition percentage data for dual drug compounds [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Compounds \u003cb\u003e1a\u003c/b\u003e, \u003cb\u003e1b\u003c/b\u003e, and \u003cb\u003e1c\u003c/b\u003e are all naphthoquinones that share the same number of carbons in the 3-ester linker, comprising different O-R substituents. While in \u003cb\u003e1a\u003c/b\u003e, naphthoquinone position 3 is linked to a pentoic acid, \u003cb\u003e1b\u003c/b\u003e and \u003cb\u003e1c\u003c/b\u003e are occupied by chloroquine and amodiaquine, respectively. An amodiaquine-like substituent is employed in compounds \u003cb\u003e1\u003c/b\u003e(\u003cb\u003ed-f\u003c/b\u003e), varying the length of the linking ester between the naphthoquinone motif and the aminoquinoline ring. The influence of an -OH group (Plumbagin-based compounds) in the naphthoquinone skeleton was also evaluated through compounds \u003cb\u003e2a\u003c/b\u003e and \u003cb\u003e2b\u003c/b\u003e, which are direct analogs of \u003cb\u003e1e\u003c/b\u003e and \u003cb\u003e1f\u003c/b\u003e, respectively. Additionally, the direct naphthoquinone-aminoquinoline dual drugs are under the names of \u003cb\u003e1\u003c/b\u003e(\u003cb\u003ea-c\u003c/b\u003e), which comprise (\u003cb\u003e3a\u003c/b\u003e) chloroquine, (\u003cb\u003e3b\u003c/b\u003e) amodiaquine, and (\u003cb\u003e3c\u003c/b\u003e) the amodiaquine-like compound. These dual drugs showed high stability toward chemical hydrolysis in aqueous solutions and under physiologic conditions [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEstimated binding energy estimated by the molecular mechanics analysis for the different inhibitors in each studied site along with calculated inhibition constant and experimental ED\u003csub\u003e50\u003c/sub\u003e and inhibition percentage.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eSite 1\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eSite 2\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eED\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003cp\u003eFcB1R (µM)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e% inhibition of \u003cem\u003eP. falciparum\u003c/em\u003e\u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBinding energy\u003c/p\u003e \u003cp\u003e(kcal/mol)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInhibition constant (Ki/µM)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBinding energy\u003c/p\u003e \u003cp\u003e(kcal/mol)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInhibition constant (Ki/µM)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1a\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-6.74\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.53\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-7.22\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.11\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.5 [0.5]\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1b\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-4.43\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e564.28\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-6.34\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e22.69\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.107\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-7.41\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.67\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-6.72\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.82\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1d\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-9.77\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.069\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-9.11\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.210\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.144\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1e\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-9.09\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.217\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-9.62\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.088\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.047\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1f\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-7.93(-57.42)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.54\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-9.58(-54.82)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.096\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.023\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2a\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-9.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.253\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-8.65\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.455\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0287\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2b\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-7.74\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.12\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-8.32\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.799\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.056\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3a\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-7.94\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.52\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-7.56\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.88\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3b\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-8.79\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.361\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-8.55\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.542\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-8.67(-32.74)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.437\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-9.28(-45.62)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.157\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMD\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-5.53(-18.68)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e88.6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-5.65(-15.40)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e72.74\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e( ) indicates average values of the binding free energies obtained from the 100 ns of production in the dynamics simulation, via MM-GBSA profile.\u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003eExperimental IC\u003csub\u003e50\u003c/sub\u003e value for PfGR inhibition [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e*Inhibition assays at 25 µM of compound [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eThe best molecular docking binding energy achieved for site 1 comprises compound \u003cb\u003e1d\u003c/b\u003e (n = 2 / -9.77 kcal mol\u003csup\u003e− 1\u003c/sup\u003e), with the shortest alkane chain among the ester-linkers. In the same group, the binding energy reduced with the increase of carbons in the ester bridge: compound \u003cb\u003e1e\u003c/b\u003e (n = 4 / -9.09 kcal mol\u003csup\u003e− 1\u003c/sup\u003e) and \u003cb\u003e1f\u003c/b\u003e (n = 5 / -7.93 kcal mol\u003csup\u003e− 1\u003c/sup\u003e).\u003c/p\u003e \u003cp\u003eOn the other hand, \u003cb\u003e1e\u003c/b\u003e and \u003cb\u003e1f\u003c/b\u003e showed a slightly better score than \u003cb\u003e1d\u003c/b\u003e, whose binding energy did not vary significantly (⁓ 9.60 kcal mol\u003csup\u003e− 1\u003c/sup\u003e) for site 2. A similar tendency is observed experimentally, where both \u003cem\u003eP. falciparum\u003c/em\u003e inhibition and ED\u003csub\u003e50\u003c/sub\u003e increase with the ester length; nonetheless, such an increase of n = 4 for n = 5 produces slight biological effects (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This might me explained with the differences in the permeability in the cellular membrane as hydrophobicity may increase with the length of the chain allowing more of the compound to reach the active target thus presenting higher toxicity to the parasite cell.\u003c/p\u003e \u003cp\u003eFurthermore, among the compounds with five carbons in the alkane chain, \u003cb\u003e1b\u003c/b\u003e [the double drug containing chloroquine] presented an ED\u003csub\u003e50\u003c/sub\u003e of 0.107 µM. However, the docking interaction energy was only − 4.43 kcal mol\u003csup\u003e− 1\u003c/sup\u003e in the protein's active site. In this instance, the discordance suggests that the conjugation strategy involving an NQ alkanoic acid might instigate a synergistic effect, leading to the accumulation of the ester in the parasite's food vacuole. This accumulation amplifies the interaction with heme groups, thus impeding the formation of inert hemozoin crystals, rather than obstructing the biological function of PfGR. \u003cb\u003e1b\u003c/b\u003e presented − 6.34 kcal mol\u003csup\u003e− 1\u003c/sup\u003e interaction energy in the allosteric site, which is in the mean of the other compounds in the group.\u003c/p\u003e \u003cp\u003eIn the plumbagin compounds (\u003cb\u003e2a\u003c/b\u003e and \u003cb\u003e2b\u003c/b\u003e), both performed well in the molecular docking tests interacting with site 2 with energies of -8.65 and − 8.32 kcal mol\u003csup\u003e− 1\u003c/sup\u003e, respectively. \u003cb\u003e2a\u003c/b\u003e was the most favored in relation to the active site (-9.00 kcal mol\u003csup\u003e− 1\u003c/sup\u003e) what might infer the lower ED\u003csub\u003e50\u003c/sub\u003e concentration in relation to \u003cb\u003e2b\u003c/b\u003e. Again, there is a direct connection between the reported values of ED\u003csub\u003e50\u003c/sub\u003e and the calculated K\u003csub\u003ei\u003c/sub\u003e for the compounds.\u003c/p\u003e \u003cp\u003eCompounds \u003cb\u003e1f\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e differ only by the linking chain that connects the naphthoquinone group with the quinolinic condensed rings. While the first is connected through an ester functional, the second is bound via a simple alkane chain. \u003cb\u003e1f\u003c/b\u003e demonstrated activity in the 0.0023 µM of ED\u003csub\u003e50\u003c/sub\u003e, which is concise to the 0.096 µM inhibition constant based on the − 9.58 kcal mol\u003csup\u003e− 1\u003c/sup\u003e interaction with the allosteric site 2. Thus, for compound \u003cb\u003e1f\u003c/b\u003e the simulations agreed with experimental \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e tests [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] that demonstrated \u003cb\u003e1f\u003c/b\u003e as the most promising inhibitor among the ones studied.\u003c/p\u003e \u003cp\u003eBesides, \u003cb\u003e3d\u003c/b\u003e displayed an interaction energy with site 2 of -9.28 kcal mol\u003csup\u003e− 1\u003c/sup\u003e, and is indicated, thus, as a possible candidate to biological tests to investigate its antiparasitic properties further. Compared to the currently employed inhibitor MD, all studied compounds (but \u003cb\u003e1b\u003c/b\u003e in site 1) displayed lower binding energy, indicating they might be more effective PfGR inhibitors. This highlights the potential of combining drugs to design better analogs that positively impact some treatments' efficiency.\u003c/p\u003e \u003cp\u003eMolecular dynamics simulations of the dual drugs \u003cb\u003e1f\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e were performed for the best pose in docking, in order to explore the binding modes at thermodynamic conditions and validate the docking results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2 Complementary molecular dynamics simulations\u003c/h2\u003e \u003cp\u003eThe molecular dynamics simulations allowed a more detailed analysis of compounds \u003cb\u003e1f\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e, mainly about the reference MD. The estimated average binding free energies were also reported in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e in parenthesis, where data point out the dual drugs with a significantly greater binding affinity than inhibitor MD. The thermodynamic analysis in the realm of molecular docking had predicted a better affinity of \u003cb\u003e3c\u003c/b\u003e than \u003cb\u003e1f\u003c/b\u003e for site 1. This binding energy was corrected by the molecular dynamics GBSA profile which predicted an interaction energetics of -57.42 kcal mol\u003csup\u003e− 1\u003c/sup\u003e, which is almost twice as favored as \u003cb\u003e3c\u003c/b\u003e (-32.74 kcal mol\u003csup\u003e− 1\u003c/sup\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, regarding site 2, this interaction energy difference lowers to a 9.2 kcal mol\u003csup\u003e− 1\u003c/sup\u003e (\u003cb\u003e1f\u003c/b\u003e: -54.82 kcal mol\u003csup\u003e− 1\u003c/sup\u003e; \u003cb\u003e3c\u003c/b\u003e: -45.62 kcal mol\u003csup\u003e− 1\u003c/sup\u003e), presenting a more modest variation, which is closer to the data obtained from molecular docking analyses.\u003c/p\u003e \u003cp\u003eAlong the molecular simulation trajectory, some insightful conformations were observable. At first, \u003cb\u003e1f\u003c/b\u003e interaction on site 1 was predicted to occur mainly via the residue Val383 (as observed for the conventional antimalarials and the inhibitor MB), which acts as an arm that appropriately allocates the ligand in the binding position. That supposition was confirmed by the molecular dynamics, which described the ester oxygen interacting uninterruptedly with the amine group in the protein residue. In the first 3ns of the simulation, the conformation obtained in the molecular docking for this binding mode rotated in the esters' C-O bond, resulting in a new conformation. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea indicates the interaction poses on both molecular docking and dynamics. Whereas in the docking-obtained structure, the naphthoquinone part of the ligand went towards the NADPH-binding domain, and the amodiaquine part was found near the FADH\u003csub\u003e2\u003c/sub\u003e binding domain, in the molecular dynamics, after 3ns, the rotation of the ester group lead both drug skeleton to a more hydrophobic region.\u003c/p\u003e \u003cp\u003eDifferently, ligand \u003cb\u003e3c\u003c/b\u003e keeps the interaction profile observed in the molecular docking simulation on site 1, and the only observable contrast in the molecular dynamics is a movement away from the FADH\u003csub\u003e2\u003c/sub\u003e. This movement allows the ligand to move to a more hydrophilic a part of the protein surface, which is stabilized by an H-bonds between the carboxylate in NQ and the residue GLN462. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb demonstrates the binding mode described above. That difference in binding mode between \u003cb\u003e1f\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e may infer the importance of the ester group, which interacts directly with FADH\u003csub\u003e2\u003c/sub\u003e in the case of \u003cb\u003e1f\u003c/b\u003e and keeps the drug positioned optimally in the active site of the PfGR. The lack of a hydrogen acceptor group in the alkane chain of \u003cb\u003e3c\u003c/b\u003e makes it translate to a less effective area, thus reducing the GBSA interaction energy (\u003cb\u003e1f\u003c/b\u003e: -57.42 kcal mol\u003csup\u003e− 1\u003c/sup\u003e; \u003cb\u003e3c\u003c/b\u003e: -32.74 kcal mol\u003csup\u003e− 1\u003c/sup\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn the other hand, \u003cb\u003e1f\u003c/b\u003e interacted with the cavity of the enzyme (site 2) mainly via the interface area. Here, the linking chain was merely a better tool to optimally position both terminal drug structures. The long chain allows both terminal drugs to interact better with the interface region and fit more suitably with the enzyme shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea,b). The idea for testing the interaction in this cavity is to study the viability of a protein dimerization blockage once this is the area that connects both monomers in the dimer unit. Additionally, as the cavity is more hydrophilic than the active site, the increase in the number of polar groups (such as the ester) may favor the binding energy as is the case of \u003cb\u003e1f\u003c/b\u003e over \u003cb\u003e3c\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eb,c). This energetic behavior on site 2 is also shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (\u003cb\u003e1f\u003c/b\u003e: -54.85 kcal mol\u003csup\u003e− 1\u003c/sup\u003e; \u003cb\u003e3c\u003c/b\u003e:-45.62 kcal mol\u003csup\u003e− 1\u003c/sup\u003e). Besides, \u003cb\u003e3c\u003c/b\u003e interaction geometry did not change in relation to the molecular docking simulation. In the case of site 2, the shorter linking chain of \u003cb\u003e3c\u003c/b\u003e did not affected significantly in the interaction mode, and a longer linker (\u003cb\u003e1f\u003c/b\u003e) only led to a slightly better proximity with the protein residues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSome deviation analysis through an RMSD approach indicates that, in general, the protein residues have a reduction in structural freedom when interacting with the drugs compared to a simulation comprising only the protein and its cofactor. \u003cb\u003e1f\u003c/b\u003e interaction in the active site demonstrates an RMSD mean of 3.84 Å, whereas 3c in the same location accounted for a mean of 4.80 Å. This difference infers the binding effectiveness of each compound in pfGR and may indicate the importance of a more flexible and polar linking chain (1f: 5 carbons-ester long; 3c: 2 carbons-alkane long). Similarly, a RMSD analysis in the MD and reference resulted in 4.61Å and 5.95 Å, respectively. Regarding the interaction influence over the FADH\u003csub\u003e2\u003c/sub\u003e RMSD, \u003cb\u003e1f\u003c/b\u003e causes both the major deviations and for longer than \u003cb\u003e3c\u003c/b\u003e (Figure S11). This might be caused by the shorter proximity between \u003cb\u003e1f\u003c/b\u003e and the cofactor, once \u003cb\u003e3c\u003c/b\u003e, as discussed earlier, tends to move away from the FADH\u003csub\u003e2\u003c/sub\u003e domain.\u003c/p\u003e \u003cp\u003eFurthermore, when interacting in the cavity (site 2), a contrary behavior was observed: \u003cb\u003e1f\u003c/b\u003e reduced the protein RMSD by 4.29 Å compared to a reduction of 3.61 Å achieved by \u003cb\u003e3c\u003c/b\u003e. MD had a slightly better performance than the one for the active site and displayed a protein RMSD of 4.45 Å. All RMSD graphs are displayed in Figure S11 in the supplementary information. Similarly, a solvent-accessible surface area analysis (SASA) demonstrated a mean area available to the solvent of 25767.91 Å2 on the protein during the reference simulation. This average was lower for the compound interacting on site 2 (1f: 23859.96 Å2; 3c: 23097.26 Å2) than for site 1 (1f: 24075.94 Å2; 3c: 23954.61 Å2). However, MD behaved differently and the SASA for site 1 (24210.24 Å2) was lower than the one achieved interacting with site 2 (24435.59 Å2). All SASA graphs are displayed on Figure S12 in the supplementary information.\u003c/p\u003e \u003cp\u003eA root mean square of atomic fluctuation (RMSF) was also evaluated, and the analysis of the residues along both sites of interest demonstrates the binding effect of the drugs based on the fluctuation of atoms in the protein. All drugs can decrease RMSF in the protein in comparison to the free protein simulations. The highest decrease was, in general, achieved by both compounds \u003cb\u003e1f\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e, which was better than MD in most studied frames. This very same pattern appeared on site 2 RMSF analysis. Although dual-drugs \u003cb\u003e1f\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e performed quite similarly in both sites, \u003cb\u003e1f\u003c/b\u003e binding was slightly more effective in the active site than \u003cb\u003e3c\u003c/b\u003e. On the other hand, \u003cb\u003e3c\u003c/b\u003e performed slightly better on site 2, according to the RMSF results. All RMSF graphs are displayed in Figures S13-14 in the supplementary information.\u003c/p\u003e \u003cp\u003eIn summary, it was not observed a significative change in the interacting pose, which demonstrates the affinity of those simulated dual-drugs and PfGR. Figure S13 in the supplemental material illustrates that even though there is a fluctuation in both CQ condensed rings and MD motifs, the overall binding conformation is kept, and the drugs do not rotate inside the binging sites.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe molecular docking assessment of conventional antimalarial drugs as PfGR inhibitors revealed that all drugs exhibited higher affinity than reference inhibitors MD and MB, particularly at the enzyme's active site. ATV emerged as a promising candidate, displaying the highest affinity for both the active site and the cavity in the interface region, suggesting a dual docking mode. The docking results also showed a moderate correlation with \u003cem\u003ein vitro\u003c/em\u003e antiplasmodial activities, suggesting that the potent antimalarial activity of ATV might involve the inhibition of PfGR. ATV and CQ demonstrated a binding mode similar to inhibitor MB at the enzyme's active site, interacting with the cofactor FADH\u003csub\u003e2\u003c/sub\u003e through H-bonds and π-π interactions. Valine383 plays an essential role assisting the drug binding to the active site, acting as an \"anchor\" residue and keeping it close to the cofactor FADH\u003csub\u003e2\u003c/sub\u003e. Electrochemical analysis suggested that ATV and CQ, despite being less efficient than MB, could act as moderate \"subversive substrates\" at the active site.\u003c/p\u003e\u003cp\u003eExploring the enzyme's cavity highlighted distinct binding modes, with ATV and AQ exhibiting notable affinity. Their polar groups of naphtoquinone/quinoline rings play a crucial role as robust H-bond acceptors, while their substituent groups contribute significantly to the ligand's precise adjustment within the site.\u003c/p\u003e\u003cp\u003eMoreover, according to molecular mechanism analysis, dual drugs combining aminoquinoline derivatives and GR inhibitors exhibited significantly greater binding affinity than the reference MD. Results provided insights into their interaction mechanisms, where the presence of the aliphatic ester bond (linker) is essential for effective binding with the enzyme's active site. Analysis of protein structural dynamics indicated reduced freedom of protein residues when interacting with the dual drugs \u003cb\u003e1f\u003c/b\u003e and \u003cb\u003e3c\u003c/b\u003e, highlighting their effectiveness in stabilizing the protein structure. The linker ester with a long chain does not play an important role in the dual-drug binding to the enzyme's cavity; nonetheless, it provides flexibility for both terminal drugs to interact strongly with the interface region and better fit the enzyme shape. Overall, the study confirms the strong affinity of these dual-drugs for PfGR, with stable binding poses maintained throughout the molecular dynamics simulations. In summary, \u003cb\u003e1f\u003c/b\u003e showed to be a good alternative as a dual drug due to the good binding modes reported for both enzyme sites. The study encourages further experiments to investigate the role of PfGR or other disulfide reductases in the mechanisms of action of conventional antimalarial drugs and dual-drugs and their contribution to antiparasitic efficacy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThis study was conceptualized and written by Dr. GYSD. FHCF, BAO, LVD, and LRP conducted the molecular dockings. FHCF also performed molecular dynamics calculations and contributed to writing. MN supervised the work and revised the manuscript. All authors approved the final version.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThe authors thank the Minas Gerais State Agency for Research and Development FAPEMIG (BPD-00777-22), the National Council for Scientific and Technological Development CNPq and the Coordination of Superior Level Staff Improvement CAPES (Finance Code 001) for supporting this work. The authors also thank SDumont/LNCC and NEQC(UFJF) for the computational resources for calculations and D. Quintanilha for helping generate some images. FHCF would additionally like to thank CeMEAI/EULER and Repesq/UFJF for the computational resources.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRich SM, Ayala FJ (2006) Evolutionary Origins of Human Malaria Parasites. In: Malaria: Genetic and Evolutionary Aspects. Springer US, Boston, MA, pp 125\u0026ndash;146\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh B, Daneshvar C (2013) Human Infections and Detection of Plasmodium knowlesi. Clin Microbiol Rev 26:165\u0026ndash;184. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/CMR.00079-12\u003c/span\u003e\u003cspan address=\"10.1128/CMR.00079-12\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTa TH, Hisam S, Lanza M, et al (2014) First case of a naturally acquired human infection with Plasmodium cynomolgi. Malar J 13:68. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1475-2875-13-68\u003c/span\u003e\u003cspan address=\"10.1186/1475-2875-13-68\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eImwong M, Madmanee W, Suwannasin K, et al (2019) Asymptomatic Natural Human Infections with the Simian Malaria Parasites Plasmodium cynomolgi and Plasmodium knowlesi. J Infect Dis 219:695\u0026ndash;702. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/infdis/jiy519\u003c/span\u003e\u003cspan address=\"10.1093/infdis/jiy519\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWorld Health Organization (2023) World malaria report 2023. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2023\u003c/span\u003e\u003cspan address=\"https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2023\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoletim epidemiol\u0026oacute;gico da mal\u0026aacute;ria vol 55: Dia da Mal\u0026aacute;ria nas Am\u0026eacute;ricas \u0026ndash; um panorama da mal\u0026aacute;ria no Brasil em 2022 e no primeiro semestre de 2023. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.gov.br/saude/pt-br/centrais-deconteudo/publicacoes/boletins/epidemiologic epidemiologicos/edicoes/2024/boletim-epidemiologico-volume-55-no-01/\u003c/span\u003e\u003cspan address=\"https://www.gov.br/saude/pt-br/centrais-deconteudo/publicacoes/boletins/epidemiologic epidemiologicos/edicoes/2024/boletim-epidemiologico-volume-55-no-01/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEpidemiol\u0026oacute;gico B, Especial N Biblioteca Virtual em Sa\u0026uacute;de do Minist\u0026eacute;rio da Sa\u0026uacute;de Boletim Epidemiol\u0026oacute;gico.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCowman AF, Healer J, Marapana D, Marsh K (2016) Malaria: Biology and Disease. Cell 167:610\u0026ndash;624. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2016.07.055\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2016.07.055\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBiot C, Castro W, Bott\u0026eacute; CY, Navarro M (2012) The therapeutic potential of metal-based antimalarial agents: Implications for the mechanism of action. Dalton Transactions 41:6335. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/c2dt12247b\u003c/span\u003e\u003cspan address=\"10.1039/c2dt12247b\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Villiers KA, Egan TJ (2021) Heme Detoxification in the Malaria Parasite: A Target for Antimalarial Drug Development. Acc Chem Res 54:2649\u0026ndash;2659. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.accounts.1c00154\u003c/span\u003e\u003cspan address=\"10.1021/acs.accounts.1c00154\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVasquez M, Zuniga M, Rodriguez A (2021) Oxidative Stress and Pathogenesis in Malaria. Front Cell Infect Microbiol 11:. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fcimb.2021.768182\u003c/span\u003e\u003cspan address=\"10.3389/fcimb.2021.768182\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDubois VL, Platel DFN, Pauly G, Tribouleyduret J (1995) \u003cem\u003ePlasmodium berghei\u003c/em\u003e: Implication of Intracellular Glutathione and Its Related Enzyme in Chloroquine Resistance \u003cem\u003ein vivo\u003c/em\u003e. Exp Parasitol 81:117\u0026ndash;124. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1006/expr.1995.1099\u003c/span\u003e\u003cspan address=\"10.1006/expr.1995.1099\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJortzik E, Becker K (2012) Thioredoxin and glutathione systems in \u003cem\u003ePlasmodium falciparum\u003c/em\u003e. International Journal of Medical Microbiology 302:187\u0026ndash;194. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ijmm.2012.07.007\u003c/span\u003e\u003cspan address=\"10.1016/j.ijmm.2012.07.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuber PC, Almeida WP, F\u0026aacute;tima \u0026Acirc; de (2008) Glutationa e enzimas relacionadas: papel biol\u0026oacute;gico e import\u0026acirc;ncia em processos patol\u0026oacute;gicos. Quim Nova 31:1170\u0026ndash;1179. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/S0100-40422008000500046\u003c/span\u003e\u003cspan address=\"10.1590/S0100-40422008000500046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarma GN, Savvides SN, Becker K, et al (2003) Glutathione Reductase of the Malarial Parasite \u003cem\u003ePlasmodium falciparum\u003c/em\u003e: Crystal Structure and Inhibitor Development. J Mol Biol 328:893\u0026ndash;907. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0022-2836(03)00347-4\u003c/span\u003e\u003cspan address=\"10.1016/S0022-2836(03)00347-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuchholz K, Schirmer RH, Eubel JK, et al (2008) Interactions of Methylene Blue with Human Disulfide Reductases and Their Orthologues from \u003cem\u003ePlasmodium falciparum\u003c/em\u003e. Antimicrob Agents Chemother 52:183\u0026ndash;191. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AAC.00773-07\u003c/span\u003e\u003cspan address=\"10.1128/AAC.00773-07\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorin C, Besset T, Moutet J-C, et al (2008) The aza-analogues of 1,4-naphthoquinones are potent substrates and inhibitors of plasmodial thioredoxin and glutathione reductases and of human erythrocyte glutathione reductase. Org Biomol Chem 6:2731. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/b802649c\u003c/span\u003e\u003cspan address=\"10.1039/b802649c\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEhrhardt K, Davioud-Charvet E, Ke H, et al (2013) The Antimalarial Activities of Methylene Blue and the 1,4-Naphthoquinone 3-[4-(Trifluoromethyl)Benzyl]-Menadione Are Not Due to Inhibition of the Mitochondrial Electron Transport Chain. Antimicrob Agents Chemother 57:2114\u0026ndash;2120. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/AAC.02248-12\u003c/span\u003e\u003cspan address=\"10.1128/AAC.02248-12\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIribarne F, Gonz\u0026aacute;lez M, Cerecetto H, et al (2007) Interaction energies of nitrofurans with trypanothione reductase and glutathione reductase studied by molecular docking. Journal of Molecular Structure: THEOCHEM 818:7\u0026ndash;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.theochem.2007.04.035\u003c/span\u003e\u003cspan address=\"10.1016/j.theochem.2007.04.035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIribarne F, Paulino M, Aguilera S, Tapia O (2009) Assaying phenothiazine derivatives as trypanothione reductase and glutathione reductase inhibitors by theoretical docking and Molecular Dynamics studies. J Mol Graph Model 28:371\u0026ndash;381. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jmgm.2009.09.003\u003c/span\u003e\u003cspan address=\"10.1016/j.jmgm.2009.09.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTyagi C, Bathke J, Goyal S, et al (2015) Targeting the intersubunit cavity of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e glutathione reductase by a novel natural inhibitor: Computational and experimental evidence. Int J Biochem Cell Biol 61:72\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biocel.2015.01.014\u003c/span\u003e\u003cspan address=\"10.1016/j.biocel.2015.01.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eF\u0026auml;rber PM, Arscott LD, Williams CH, et al (1998) Recombinant \u003cem\u003ePlasmodium falciparum\u003c/em\u003e glutathione reductase is inhibited by the antimalarial dye methylene blue. FEBS Lett 422:311\u0026ndash;314. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0014-5793(98)00031-3\u003c/span\u003e\u003cspan address=\"10.1016/S0014-5793(98)00031-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGinsburg H, Famin O, Zhang J, Krugliak M (1998) Inhibition of glutathione-dependent degradation of heme by chloroquine and amodiaquine as a possible basis for their antimalarial mode of action. Biochem Pharmacol 56:1305\u0026ndash;1313. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0006-2952(98)00184-1\u003c/span\u003e\u003cspan address=\"10.1016/S0006-2952(98)00184-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShibeshi MA, Kifle ZD, Atnafie SA (2020) Antimalarial Drug Resistance and Novel Targets for Antimalarial Drug Discovery. Infect Drug Resist Volume 13:4047\u0026ndash;4060. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/IDR.S279433\u003c/span\u003e\u003cspan address=\"10.2147/IDR.S279433\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTibon NS, Ng CH, Cheong SL (2020) Current progress in antimalarial pharmacotherapy and multi-target drug discovery. Eur J Med Chem 188:111983. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ejmech.2019.111983\u003c/span\u003e\u003cspan address=\"10.1016/j.ejmech.2019.111983\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSullivan DJ, Gluzman IY, Russell DG, Goldberg DE (1996) On the molecular mechanism of chloroquine\u0026rsquo;s antimalarial action. Proceedings of the National Academy of Sciences 93:11865\u0026ndash;11870. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.93.21.11865\u003c/span\u003e\u003cspan address=\"10.1073/pnas.93.21.11865\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuller R, Peterson ML, Almarsson \u0026Ouml;, Leiserowitz L (2002) Quinoline Binding Site on Malaria Pigment Crystal: A Rational Pathway for Antimalaria Drug Design. Cryst Growth Des 2:553\u0026ndash;562. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/cg025550i\u003c/span\u003e\u003cspan address=\"10.1021/cg025550i\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCamarda G, Jirawatcharadech P, Priestley RS, et al (2019) Antimalarial activity of primaquine operates via a two-step biochemical relay. Nat Commun 10:3226. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-019-11239-0\u003c/span\u003e\u003cspan address=\"10.1038/s41467-019-11239-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDavioud-Charvet E, Delarue S, Biot C, et al (2001) A Prodrug Form of a \u003cem\u003ePlasmodium falciparum\u003c/em\u003e Glutathione Reductase Inhibitor Conjugated with a 4-Anilinoquinoline. J Med Chem 44:4268\u0026ndash;4276. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jm010268g\u003c/span\u003e\u003cspan address=\"10.1021/jm010268g\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeese F (2022) Software update: The ORCA program system\u0026mdash;Version 5.0. WIREs Computational Molecular Science 12:. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/wcms.1606\u003c/span\u003e\u003cspan address=\"10.1002/wcms.1606\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarone V, Cossi M (1998) Quantum Calculation of Molecular Energies and Energy Gradients in Solution by a Conductor Solvent Model. J Phys Chem A 102:1995\u0026ndash;2001. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jp9716997\u003c/span\u003e\u003cspan address=\"10.1021/jp9716997\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLarson SB, Day J, Barba de la Rosa AP, et al (2003) First Crystallographic Structure of a Xylanase from Glycoside Hydrolase Family 5: Implications for Catalysis. Biochemistry 42:8411\u0026ndash;8422. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/bi034144c\u003c/span\u003e\u003cspan address=\"10.1021/bi034144c\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e(2019) Dassault Syst\u0026egrave;mes BIOVIA. Discovery Studio Visualizer v.20.1.0.19295: San Diego.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorris GM, Huey R, Lindstrom W, et al (2009) AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem 30:2785\u0026ndash;2791. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jcc.21256\u003c/span\u003e\u003cspan address=\"10.1002/jcc.21256\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePettersen EF, Goddard TD, Huang CC, et al (2004) UCSF Chimera\u0026mdash;A visualization system for exploratory research and analysis. J Comput Chem 25:1605\u0026ndash;1612. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jcc.20084\u003c/span\u003e\u003cspan address=\"10.1002/jcc.20084\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVassetti D, Pagliai M, Procacci P (2019) Assessment of GAFF2 and OPLS-AA General Force Fields in Combination with the Water Models TIP3P, SPCE, and OPC3 for the Solvation Free Energy of Druglike Organic Molecules. J Chem Theory Comput 15:1983\u0026ndash;1995. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jctc.8b01039\u003c/span\u003e\u003cspan address=\"10.1021/acs.jctc.8b01039\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe X, Man VH, Yang W, et al (2020) A fast and high-quality charge model for the next generation general AMBER force field. J Chem Phys 153:. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1063/5.0019056\u003c/span\u003e\u003cspan address=\"10.1063/5.0019056\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJorgensen WL, Chandrasekhar J, Madura JD, et al (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys 79:926\u0026ndash;935. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1063/1.445869\u003c/span\u003e\u003cspan address=\"10.1063/1.445869\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerendsen HJC, Postma JPM, van Gunsteren WF, et al (1984) Molecular dynamics with coupling to an external bath. J Chem Phys 81:3684\u0026ndash;3690. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1063/1.448118\u003c/span\u003e\u003cspan address=\"10.1063/1.448118\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUberuaga BP, Anghel M, Voter AF (2004) Synchronization of trajectories in canonical molecular-dynamics simulations: Observation, explanation, and exploitation. J Chem Phys 120:6363\u0026ndash;6374. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1063/1.1667473\u003c/span\u003e\u003cspan address=\"10.1063/1.1667473\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Gunsteren WF, Berendsen HJC (1977) Algorithms for macromolecular dynamics and constraint dynamics. Mol Phys 34:1311\u0026ndash;1327. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/00268977700102571\u003c/span\u003e\u003cspan address=\"10.1080/00268977700102571\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiller BR, McGee TD, Swails JM, et al (2012) MMPBSA.py: An Efficient Program for End-State Free Energy Calculations. J Chem Theory Comput 8:3314\u0026ndash;3321. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ct300418h\u003c/span\u003e\u003cspan address=\"10.1021/ct300418h\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCase DA, Cheatham TE, Darden T, et al (2005) The Amber biomolecular simulation programs. J Comput Chem 26:1668\u0026ndash;1688. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jcc.20290\u003c/span\u003e\u003cspan address=\"10.1002/jcc.20290\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalomon-Ferrer R, Case DA, Walker RC (2013) An overview of the Amber biomolecular simulation package. WIREs Computational Molecular Science 3:198\u0026ndash;210. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/wcms.1121\u003c/span\u003e\u003cspan address=\"10.1002/wcms.1121\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYayon A, Cabantchik ZI, Ginsburg H (1984) Identification of the acidic compartment of \u003cem\u003ePlasmodium falciparum\u003c/em\u003e-infected human erythrocytes as the target of the antimalarial drug chloroquine. EMBO J 3:2695\u0026ndash;2700. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/j.1460-2075.1984.tb02195.x\u003c/span\u003e\u003cspan address=\"10.1002/j.1460-2075.1984.tb02195.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Souza Pereira C, Quadros HC, Aboagye SY, et al (2022) A Hybrid of Amodiaquine and Primaquine Linked by Gold(I) Is a Multistage Antimalarial Agent Targeting Heme Detoxification and Thiol Redox Homeostasis. Pharmaceutics 14:1251. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/pharmaceutics14061251\u003c/span\u003e\u003cspan address=\"10.3390/pharmaceutics14061251\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFriebolin W, Jannack B, Wenzel N, et al (2008) Antimalarial Dual Drugs Based on Potent Inhibitors of Glutathione Reductase from \u003cem\u003ePlasmodium falciparum\u003c/em\u003e. J Med Chem 51:1260\u0026ndash;1277. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jm7009292\u003c/span\u003e\u003cspan address=\"10.1021/jm7009292\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP Villareal WJ (2017) Complexos fosf\u0026iacute;ncos de Platina(II) e Pal\u0026aacute;dio(II): atividade farmacol\u0026oacute;gica e intera\u0026ccedil;\u0026atilde;o com o DNA e com a Ferriprotoporfirina\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDaniel L, Karam A, Hebert C, et al (2023) Metal(triphenylphosphine)-Atovaquone complexes: Synthesis, antimalarial activity, and suppression of heme detoxification. [Manuscript submitted for publication]\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM\u0026uuml;ller T, Johann L, Jannack B, et al (2011) Glutathione Reductase-Catalyzed Cascade of Redox Reactions to Bioactivate Potent Antimalarial 1,4-Naphthoquinones \u0026ndash; A New Strategy to Combat Malarial Parasites. J Am Chem Soc 133:11557\u0026ndash;11571. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/ja201729z\u003c/span\u003e\u003cspan address=\"10.1021/ja201729z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBasco L, Gillotin C, Gimenez F, et al (1992) In vitro activity of the enantiomers of mefloquine, halofantrine and enpiroline against \u003cem\u003ePlasmodium falciparum\u003c/em\u003e. Br J Clin Pharmacol 33:517\u0026ndash;520. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-2125.1992.tb04081.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1365-2125.1992.tb04081.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelorgey D, Antoine Lanfranchi D, Davioud-Charvet E (2013) 1,4-Naphthoquinones and Other NADPH-Dependent Glutathione Reductase- Catalyzed Redox Cyclers as Antimalarial Agents. Curr Pharm Des 19:2512\u0026ndash;2528. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2174/1381612811319140003\u003c/span\u003e\u003cspan address=\"10.2174/1381612811319140003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBecke AD (1993) Density-functional thermochemistry. I. The effect of the exchange‐only gradient correction. J Chem Phys 98:5648\u0026ndash;5652. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1063/1.462066\u003c/span\u003e\u003cspan address=\"10.1063/1.462066\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eW. J. Hehre, R. Ditchfield and J. A. Pople (1972) Self\u0026ndash;Consistent Molecular Orbital Methods. XII. Further Extensions of Gaussian\u0026ndash;Type Basis Sets for Use in Molecular Orbital Studies of Organic Molecules. J. Chem. Phys. 56, 2257. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1063/1.1677527\u003c/span\u003e\u003cspan address=\"10.1063/1.1677527\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLanfranchi DA, Belorgey D, M\u0026uuml;ller T, et al (2012) Exploring the trifluoromenadione core as a template to design antimalarial redox-active agents interacting with glutathione reductase. Org Biomol Chem 10:4795. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/c2ob25229e\u003c/span\u003e\u003cspan address=\"10.1039/c2ob25229e\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBirth D, Kao W-C, Hunte C (2014) Structural analysis of atovaquone-inhibited cytochrome bc1 complex reveals the molecular basis of antimalarial drug action. Nat Commun 5:4029. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ncomms5029\u003c/span\u003e\u003cspan address=\"10.1038/ncomms5029\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-modeling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmmo","sideBox":"Learn more about [Journal of Molecular Modeling](https://www.springer.com/journal/894)","snPcode":"894","submissionUrl":"https://submission.nature.com/new-submission/894/3","title":"Journal of Molecular Modeling","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Malaria, Plasmodium falciparum, antimalarials, Glutathione Reductase, PfGR, Molecular Docking","lastPublishedDoi":"10.21203/rs.3.rs-3952252/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3952252/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMalaria remains a significant global health challenge, with emerging resistance to current treatments necessitating the development of novel therapeutic strategies. \u003cem\u003eP. falciparum\u003c/em\u003e Glutathione Reductase (PfGR) plays a critical role in the defense mechanisms of malaria parasites against oxidative stress. In this study, we investigate the potential of targeting PfGR with conventional antimalarial drugs and dual drugs combining aminoquinoline derivatives with GR inhibitors using molecular docking and molecular dynamics simulations. Our findings reveal promising interactions between PfGR and antimalarial drugs, with the naphthoquinone Atovaquone (ATV) demonstrating particularly high affinity and potential dual-mode binding with the enzyme active site and cavity. Furthermore, dual drugs exhibit enhanced binding affinity compared to reference inhibitors, suggesting their efficacy in inhibiting PfGR. Insights into their interaction mechanisms and structural dynamics are described. Overall, this research provides valuable insights into the potential of targeting PfGR and encourages further exploration of its role in the mechanisms of action of antimalarial drugs, including dual drugs, to enhance antiparasitic efficacy.\u003c/p\u003e","manuscriptTitle":"Analysis of the interaction of antimalarial agents with Plasmodium falciparum Glutathione Reductase through molecular mechanical calculations","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-15 07:04:28","doi":"10.21203/rs.3.rs-3952252/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-02-14T12:58:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-14T12:58:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-13T13:30:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Molecular Modeling","date":"2024-02-13T00:09:08+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-molecular-modeling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmmo","sideBox":"Learn more about [Journal of Molecular Modeling](https://www.springer.com/journal/894)","snPcode":"894","submissionUrl":"https://submission.nature.com/new-submission/894/3","title":"Journal of Molecular Modeling","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f6dc4061-a7f4-4bb2-bec0-5c74d0b5e1c7","owner":[],"postedDate":"February 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-06-06T09:08:53+00:00","versionOfRecord":{"articleIdentity":"rs-3952252","link":"https://doi.org/10.1007/s00894-024-05968-3","journal":{"identity":"journal-of-molecular-modeling","isVorOnly":false,"title":"Journal of Molecular Modeling"},"publishedOn":"2024-05-23 09:08:53","publishedOnDateReadable":"May 23rd, 2024"},"versionCreatedAt":"2024-02-15 07:04:28","video":"","vorDoi":"10.1007/s00894-024-05968-3","vorDoiUrl":"https://doi.org/10.1007/s00894-024-05968-3","workflowStages":[]},"version":"v1","identity":"rs-3952252","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3952252","identity":"rs-3952252","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}