Assessing the efficacy of iso-mukaadial acetate and betulinic acid against selected Plasmodium falciparum glycolytic pathway proteins: in silico and in vitro studies

Abstract

Malaria is the extensive health concern in sub-Saharan Africa, with Plasmodium falciparum being the most lethal strain. The continued emergence of drug-resistant P. falciparum advocates for the development of new antimalarials. Our current study aimed to effectively explore the interaction capabilities of iso-mukaadial acetate (IMA) and betulinic acid (BA) against two essential P. falciparum glycolytic pathway proteins, PfLDH and PfHk. Recombinant PfLDH and PfHk were independently expressed in E. coli BL21 (DE3) cells and subsequently purified using affinity chromatography. Protein–ligand interaction studies probed in silico and in vitro approaches. Parasite inhibition studies confirmed potent antimalarial activity against the P. falciparum NF54 strains, with BA and IMA showing IC₅₀ values of 1.27 µg/ml and 1.03 µg/ml against the asexual stage of P. falciparum, respectively. FTIR experiments confirmed interactions between the compounds and the secondary structure of the proteins. Direct protein–ligand interaction studies analysis using microscale thermophoresis (MST) showed a K_D value of 0.1036 ± 0.6001 µM for the PfLDH-BA complex and 0.7473 ± 0.3554 µM K_D value for PfLDH-IMA. Meanwhile, PfHk-IMA had 0.39701 ± 0.16298 µM K_D value, while the PfHk-BA complex had no interaction detected. Molecular docking and molecular dynamics simulation studies were used to measure and confirm the interactive strength of complexes. Molecular docking reported a binding score of − 1.155 kcal/mol for the PfLDH-BA complex and a binding score of − 3.200 kcal/mol for PfLDH-IMA. The PfHk-BA complex had − 2.871 kcal/mol and PfHk-IMA complex had − 4.225 kcal/mol binding score. In conclusion, BA and IMA compounds had better interactions and remained bound within the binding sites of the glycolytic pathway proteins (PfLDH and PfHk).

Introduction

To date, malaria is still regarded a life-threatening disease, with nearly 247 million cases, and 619,000 deaths mostly caused by Plasmodium falciparum being reported annually [1]. Among the five known human-infecting strains, P. falciparum is the most lethal [2]. The continued development and spread of antimalarial drug resistant parasites have necessitated the development and exploration of new antimalarial drugs or treatments. Several medicinal plants with distinct biological activities have been exploited against diseases [3]. Recently, Iso-mukaadial acetate (IMA) and betulinic acid (BA) have gathered research interest due to their antimalarial activity potential [4]. It has been demonstrated that IMA selectively inhibits and impedes the downstream chaperone function of the selected malaria heat shock protein [4]. However, very little is known about the antiplasmodial activities of these compounds.

Recently, this has been a keen uptake of studies investigating how artemisinin derivatives may cause liver enzymes elevation and electrocardiogram abnormalities [5, 6]. Silva-Pinto and colleagues [7] demonstrated that artemisinin-based therapy might cause asymptomatic liver enzyme abnormalities and  that artemether-lumefantrine may result in the production of free radicals that can potentially lead multi-organ toxicity including the liver [7]. Additionally, artemisinin derivatives have also showed a delayed haemolysis in patients living in endemic regions. Furthermore, they showed haematological changes with pathological mechanisms that were not understood [5]. Thus, demonstrating the need for novel alternative non-toxic antiplasmodials [33]. Both IMA and BA have been shown to display antioxidant activities and liver protective activities, further justifying their exploration as potential antimalarials [8,9,10].

Within the human physiological environment of humans, the parasite invades hepatocytes and erythrocytes (asexual stage), and its existence leads to clinical symptoms of the malaria parasite [11]. The asexual stage, specifically the red blood cells (RBCs) infection has been linked with the rapid metabolization of glucose via the glycolytic pathway [12, 13]. Recent studies, showed that glucose consumption increased by at least a 100 fold in parasite infected RBCs compared to uninfected cells, thus highlighting the critical role of these parasite glycolytic enzymes [14]. In the current study we focused on two parasite glycolytic enzymes; P. falciparum hexokinase (PfHk) and P. falciparum lactate dehydrogenase (PfLDH). PfHk is a key enzyme involved in initiating the glycolysis pathway [15]. On the other hand, PfLDH, plays a central role in the production of NADH by converting pyruvate to lactate (PfLDH) [16]. Both PfHk and PfLDH are regarded as interesting drug targets due to their low (< 29%) sequence similarity identity that they share with their human homologues [15]. Thus, the current study, investigating the antimalarial and underlying mechanisms used by these compounds. We have recombinantly expressed, purified, and characterised PfLDH and PfHk. Furthermore, we aimed to understand the underlying mechanism of action in the molecular interaction between the purified glycolytic proteins and the compounds of interest (IMA and BA). Knowledge generated from this work has the potential to contribute to the advancements of parasitic diseases and the design of more effective and precise therapeutic strategies. Furthermore, data from this study may enhance the overall understanding and management of malaria parasite.

Methods and materials

ADMET prediction of selected compounds

The absorption, distribution, metabolism, excretion and toxicity (ADMET) screening of BA and IMA compound was conducted by subjecting these compounds to an in silico ADME analysis using SWISS tools (http://www.swissadme.ch). SMILES format was used to analyse lipophilicity, physicochemical properties, water solubility, pharmacokinetics, Bioavailability scores, drug-likeness properties, etc. Additionally, the pkCSM tool (https://biosig.lab.uq.edu.au/pkcsm/) was used to compare the results [17].

Inhibition studies of P. falciparum compounds

The malaria SYBR Green I-based fluorescence assay was used to measure the antimalarial activities of BA and IMA compounds by quantifying IC₅₀ values and investigating the compounds activities against the asexual stage of NF54 P. falciparum strain of malaria parasite. Human erythrocytes (O+) were used to maintain (37 °C, gaseous environment; 90% N₂, 5% O₂ and 5% CO₂) the P. falciparum parasite in the RPMI 1640 media which was supplemented with 25 mM HEPES, 20 mM d-glucose, 200 μM hypoxanthine, 0.2% sodium bicarbonate, 24 μg/ml Gentamycin, and 0.5% AlbuMAX II. The asexual ring stage of the parasite was separated by conducting culture synchronization using d-sorbitol (5% w/v). Equal volumes of the SYBR Green I lysis buffer and parasite culture were mixed after 96 h of growth followed by incubation at 37 °C for 1 h. Subsequently, the GloMax®-Multi + detection system with Instinct® software (Promega, excitation at 485 nm and emission at 538 nm) was used to measure the fluorescence. The experimental data obtained was analysed using GraphPad 8.0 and the experiments were conducted in triplicate with 3 times repetition (n = 3) [18].

Molecular docking

The in silico protein–ligand docking studies were conducted with Schrodinger software (https://newsite.schrodinger.com/) which was also used to further prepare both proteins and ligands. The proteins PfLDH (1CET) and PfHk (7ZZI) were retrieved from the protein data bank (https://www.rcsb.org/). The betulinic Acid compound was retrieved from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and Schrodinger (Maestro) was used to draw the IMA compound. The protein preparation wizard was used to preprocess proteins by adding missing hydrogens, optimizing them, removing water, and minimizing energy in the force field of OPLS4. The co-crystal ligand in each protein was selected to create a receptor grid. Furthermore, the ligand was prepared by Lig-prep for molecular docking. Thereafter, ligand docking was used; the generated receptor grid zip file and the ligand in the project table were selected. The generated complexes were viewed on ligand interaction [19].

Molecular dynamics of complex structures

Molecular dynamics (MD) simulation was performed for 100 ns using Desmond Schrodinger (https://www.schrodinger.com/ac). The system builder was used to prepare the desired framework, a predefined transferable intermolecular interaction potential three points (TIP3P) water model in an orthorhombic minimized volume of the boundary box was selected. Thereafter, the model was neutralization by adding counter 7 Cl⁻ ions where necessary and the natural physiological conditions were mimicked by adding 0.15 M NaCl. Furthermore, the experimental conditions were also mimicked by constant temperature and pressure (NPT ensemble). The models were relaxed before simulation and the trajectories were saved every 100 ns for analysis [20, 21].

Recombinant protein profiling

The pKK223 vector with the restriction enzymes BamH1 and HindIII and encoding PfLDH was transformed into E. coli BL21 (DE3) cells for protein expression. Cells were grown in Terrific Broth (TB) supplemented with a specific antibiotic (ampicillin) to prevent bacteria protein expression with shaking at 37 °C (180-rpm) until an optical density (OD) of 0.4 was reached. Culture cells were supplemented with 1 mM Isopropyl β-d-1-thiogalactopyranoside (IPTG) to induce protein expression at 180 rpm for 24 h, performing the protein expression time profile. To produce PfHk, the pET30(a) plasmid with restriction enzymes BamHI and XhoI and encoding PfHk was transfected into E. coli BL21-competent cells for the protein expression procedure. The starter culture was transferred into 500 ml Luria–Bertani (LB) media supplemented with 50 μg/ml of kanamycin (K₅₀) shaking at 25 °C until the OD₆₀₀ reached 0.4 to 0.6, followed by induction with 0.5 mM IPTG. The induced cell culture was left shaking for 24 h at 25 °C followed by recovery through centrifugation using a JA14 rotor (Beckman, Optima l-100XP) and resuspended in Bacterial Protein Extraction Reagent (B-PER). The recombinant protein expression was confirmed by running a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) [4, 22].

Purification of expressed proteins

Protein purification of the target protein was performed using the HisPur™ Ni–NTA Resin column (ThermoFisher Scientific, SA) following the manufacturer’s protocol. Briefly, the equilibration process was done by loading equal volumes of the crude fraction containing the His-tag protein and LEW buffer (supplemented with NaH₂PO₄, NaCl and 25 mM imidazole, pH7.5), followed by storage with vigorous shaking in ice cold to allow binding for 1 h. Thereafter, the unbound proteins were washed off with LEW buffer. The target protein was eluted using elution buffer (NaH₂PO₄, NaCl and 500 mM imidazole, pH7.5) and confirmed with SDS-PAGE (15%) and verified by western blot [4, 22].

Fourier transform infrared (FTIR) analysis

The purified proteins were each mixed with the compounds (1 mM BA and IMA), separately. The mixed samples were incubated at room temperature for 1 h to allow proper formation of the complexes. The sample reading of each complex was conducted at the frequencies between 400 cm⁻¹ to 4000 cm⁻¹ by depositing the samples on the diamond plate of the single reflection of a universal ATR Sampling Accessory. The results obtained were analysed with OriginPro 8.5 software [22, 23].

Microscale thermophoresis (MST) protein–ligand interaction

MST experiments for complexes were conducted by the Monolith NT.115 instrument (Nano Temper Technologies, Germany) at a temperature of 25 °C, 70% excitation, and 40% MST power. Malaria proteins (PfLDH and PfHk) were labelled with the Monolith His-Tag Labelling Kit RED-tris-NTA according to the manufacturer’s instructions. The phosphate-buffered saline (PBS) buffer (supplemented with 0.05% v/v tween 20; pH 7.4) was used to dilute the labelled protein to make a 50 nM solution and the binding affinity method previously reported by [24] was followed with modifications [24]. Briefly, 10 μl of the diluted labelled protein was added to 10 μl ligand of different concentration ranges and incubated at 37 °C for 30 min. Thereafter, the complexes were loaded into Standard Treated Capillaries (Nano Temper Technologies, Germany) and MO control software (Nano Temper) was used to measure the fluorescence signal. To calculate the dissociation constant (K_D), MO-Affinity analysis software, version 2.1.3 (Nano Temper Technologies, Germany), was used based on three independent measurements [18].

Results & discussion

ADMET analysis results

In this study, the SWISS ADMET and pkCSM tools were used to evaluate several parameters, including the physicochemical, pharmacokinetic, and drug-likeness of the BA and IMA compounds. Both compounds used in this study had molecular weights less than 500 Da, were less toxic and passed the Lipinski rule of five, which essentially shows the relative nature of bioavailability and physicochemical properties of the compounds, resolving the concept of drug-likeness. Furthermore, the BOILED-egg model was used to analyse parameters such as GI absorption and BBB penetration of compounds. The yellow region represents the BBB penetration and the white region represents the GI penetration probability. The results in Table 1 report that the compound BA has low GI absorption, 0.85 bioavailability score and no BBB permeant. Meanwhile, compound IMA has high GI absorption with a bioavailability score of 0.55 and without BBB permeant. Similarily, chloroquine (Table S1) has high GI absorption, 0.55 bioavailability score however with BBB permeant. Additionally, other parameters are shown in Table 1 and Table S1.

Table 1 ADMET prediction of target compounds (BA and IMA)

In vitro inhibition activities of BA and IMA against P. falciparum

The IMA and BA compounds used in this study were donated from our previous studies [8, 10]. The inhibition potency of the BA and IMA compound was quantified against the NF54 (drug-sensitive) strain of P. falciparum parasite (Table 2). BA and IMA had IC₅₀ values of 1.27 µg/ml and 1.03 µg/ml, respectively. Thus, both compounds had significant inhibition activity since they have IC₅₀ values less than 10 µg/ml. The obtained IC₅₀ values for the compounds significantly assisted in the determination of the concentrations that were utilized to narrowing down possible cause of parasite inhibition by targeting the parasite pathway (glycolytic pathway).

Table 2 IC₅₀ values of BA and IMA on P. falciparum parasite asexual stage

Molecular docking studies

Molecular docking studies were performed by the Schrödinger (maestro) software to investigate binding scores and interactions formed between compounds and protein crystal structures. The molecule with the lowest number for binding affinity (i.e., more negative) indicates the highest binding affinity to the protein [25]. The BA compound had the lowest binding score (− 1.155 kcal/mol) with PfLDH and IMA had the highest binding score of − 3.200 kcal/mol with the PfLDH protein. Meanwhile, the complex PfHk-IMA had − 4.225 kcal/mol and PfHk-BA had − 2.871 kcal/mol binding score. Therefore, IMA had better interaction with both proteins by having higher binding affinities and amino acid residues interacted are shown in Fig. 1. The interaction of PfLDH-chloroquine complex had a binding energy of − 3.211 kcal/mol and PfHk-chloroquine had a binding energy of − 4.155 kcal/mol as shown in the supplementary data (Figure S1).

Fig. 1

Three-dimensional (3D) and two- dimensional (2D) structural interactions of protein–ligand complexes and docking scores

The interaction of human lactate dehydrogenase and hexokinase were also evaluated with the IMA and BA compounds. The IMA compound showed possible interactions with human LDH (binding score − 4.277 kcal/mol) and docking interaction results are shown in the supplementary data (Figure S1). However, BA showed no possible interaction with the human LDH protein. Additionally, the interactions of human Hk with investigated compounds were not successful. The binding scores of formed complexes are usually not considered accurate. However, molecular dynamics simulation essentially gives an accurate understanding of the formed complex interactions and solvation effects depict the complex binding process in a more realistic way. Therefore, due to insufficient in silico data obtained for the human proteins’ interactions with the selected compounds in this study, the in vitro studies were not commenced [26].

Molecular dynamics simulation studies

The movement of the protein in the hydrated environment during simulation is important to check the structural stability of the ligand. This essentially contribute to the knowledge of its presence within the living host system and such information determines the efficiency and functionality of a ligand. After the molecular docking was performed, the compounds and proteins were further analysed using molecular dynamics studies to evaluate the formed complex interactions and the stability of each ligand within the active site of each protein.

Figure 2 demonstrates conformational PfLDH-BA complex analysis in which the protein reaches an equilibrium at an acceptable root mean square deviation (RMSD) value at approximately 3.1 Å. The ligand (BA) reached equilibrium after 30 nano seconds with the root mean square deviation (RMSD) value of approximately 2.3 Å within the protein until 70 nano seconds followed by a slight fluctuation and reasonably stable till the end of the remaining simulation. Several protein amino acids had different interactions and bond formation (Fig. 2B) with the ligand for 100 nano seconds. Additionally, (Fig. 2C) shows amino acids that interacted with the ligand during molecular simulation until the end of the molecular simulation process. The amino acid residue TYR85 had 46% interaction with a hydroxy group of BA, ASP53 had 31% interaction with a similar hydroxy group and LYS118 had 28% interaction with the carboxyl group. Figure S2 shows that chloroquine fluctuated lower than the protein however, towards the end of 100 ns chloroquine fluctuation increased. Additionally, at the end of simulation ASP53 amino acid residue interacted with the functional group of chloroquine. Therefore, ASP53 essentially played a vital role interacting each compound (BA or chloroquine) with the protein. These amino acids imply the specificity and selectivity of compound in engaging with the surrounding residues resulting in the formation of a hydrogen bonds and hydrophobic interactions at a distinct region of the compound.

Fig. 2

Conformational analyses of PfLDH and BA compound complex. A Root mean square deviation (RMSD) plot B interaction fraction of protein amino residues and C protein-ligands interaction after MD

IMA compound fluctuation within PfLDH protein is shown in Fig. 3A with an equilibrium obtained after 40 nano seconds at approximately 2.3 Å RMSD value. Figure 3B highlights the forces of interaction between the ligand and protein, including hydrogen bonds, hydrophobic interactions, and water-mediated bridges. The most significant interactions were observed between PHE-43 and LYS-113, with interaction fractions exceeding 0.6. Based on literature Hydrogen bonding plays a crucial role in maintaining the ligand’s specificity and binding stability [4]. Prominent water-mediated interactions were observed in residues ASP-53 and GLN-121. While amino acids residues such as LEU-112 and ILE-45 engage in moderate hydrophobic contacts. No significant ionic interactions were observed, suggesting that electrostatic forces do not significantly contribute to the ligand’s binding affinity in this system. Figure 3C shows an amino acid (SER245 with 37% interaction) that caused the compound to remain bound within the active site of the protein until molecular simulation was completed. The protein had one amino acid (SER245) with more than 30% interactions with IMA compound during the simulation time.

Fig. 3

Conformational analyses of PfLDH and IMA compound complex. A Root mean square deviation (RMSD) plot B interaction fraction of protein amino residues and C protein-ligands interaction after MD

BA compound had a lower RMSD than PfHk from 0 to 95 nano seconds followed by regular increased fluctuation within the protein (Fig. 4A). Also, the protein had the lowest 1.65 Å RMSD value followed by fluctuation at 95 nano seconds to 100 nano seconds however the RMSD value was still at the acceptable value. The lower RMSD value confirms less conformational changes. Furthermore, the protein had one amino acid ASP102 which had 72% interaction with hydroxy group of the compound that legitimately kept the compound bound within the protein's active site for 100 ns of simulation (Fig. 4C). Similarly, chloroquine also had amino acid residue ASP102 showing higher interaction of 67% with its functional group and other amino acids such as ASP470 and ILE265 (Figure S3).

Fig. 4

Conformational analyses of PfHk and BA compound complex. A Root mean square deviation (RMSD) plot B interaction fraction of protein amino residues and C protein-ligands interaction after MD

Both protein and ligand in Fig. 5 had lower RMSD value below 2.00 Å which is mostly acceptable. Therefore, the protein and ligand conformational changes were lower or less shift and ligand IMA remained bound within the active site of protein since IMA had lower RMSD than PfHk protein. Amino acid residue SER436 had 71% interaction with compound IMA and THR268 had 61% interaction with a similar compound functional group. Furthermore, ARG109 had 36% interaction and 58% interaction with IMA. The results show successful interaction and amino acids that bound the compound within the protein active site for 100 ns of MD simulation.

Fig. 5

Conformational analyses of PfHk and IMA compound complex. A Root mean square deviation (RMSD) plot B interaction fraction of protein amino residues and C protein-ligands interaction after MD

Expression and purification of the recombinant PfHk and PfLDH proteins

Using E. coli BL21 (DE3) cells, both proteins, PfHk and PfLDH, were expressed in high soluble and homogenous yields. The PfHk and PfLDH proteins expressed with an N-terminal His-tag which allowed high-affinity interaction with the Ni (₂₊)-nitrilotriacetic acid (Ni₂₊–NTA) to facilitate protein purification. Protonation of the side chains of interacting amino acids allowed the elution of target proteins from the resin, whereby a histidine precursor (such as imidazole) displaces the protein. The 12% SDS PAGE was used to analyse the purity of the target protein, and after washing away the non-specifically bound proteins, the target protein was eluted with the elution buffer containing a high concentration of imidazole (500 mM; pH7.5). The target proteins with a molecular weight of 55.26 kDa (PfHk) and PfLDH had a dimer at 100 kDa which is the active form of the protein and a monomer at 34.9 kDa are shown in the SDS PAGE in Fig. 6 (lane E1 to E3) and Figure S4 & S5. In this experiment both proteins were successfully expressed and purified for interaction studies.

Fig. 6

Successful production of recombinant PfLDH and PfHk. Images demonstrated the expression and purification of PfLDH (A) and PfHk (B). Lane M: molecular weight marker, lane NC: BL21(negative control), while lane 1HR-24HR shows time-expression profile studies. Lane E1-E3 shows the collected elution samples

Fourier transform infrared (FTIR) analysis

FTIR spectroscopy is well known for protein structure characterization particularly in the secondary structure [27]. Spectroscopy provides secondary structural information determining the empirical correlation frequency of amide I and amide II absorption [27]. The vibrations of C=O stretching result in the formation of the amide I mode readings between 1700 and 1600 cm⁻¹ with CN and CCN out of plane bending vibrations [4]. Substantially, the most important mode for protein characterization is amide I. The curve fitting on the amide I band is distinctively representing the fractions of the secondary structures in the protein. The NH (in-plane bending) and CN stretching contribute to the formation of amide II between frequencies of 1460 to 1590 cm⁻¹. Furthermore, amide III (1350 to 1229 cm⁻¹) is generally affected by protein side chains and varies with protein due to CC-stretching, NH, and C=O in-plane bending vibrations [4]. The lower frequencies from 750 to 400 cm⁻¹ represent the amide IV to amide VII of the protein structure [4, 28, 29].

The Fermi resonances between amide II and NH stretching vibrations generate amide A and amide B, usually located around 3000 and 3500 cm⁻¹, respectively [30]. The FTIR results of both proteins with selected compounds showed a smaller frequency shift, particularly in amide I, thus demonstrating structural rearrangement of both proteins in the secondary structures. Mostly, IMA reduced the transmittance percentage of PfHk at 1637 cm⁻¹ frequency compared to BA which had a lower effect on the protein (Fig. 7). The methylene C–H bend at 1457 cm⁻¹ had a reduced transmittance percentage for both compounds interacting with the PfHk protein. Furthermore, the frequency of 1097 cm⁻¹ representing skeletal C–C vibrations had shifted to a lower transmittance percentage. The higher the transmittance, the lower the number of bonds present absorbing the colour of light, and the lower transmittance, the higher the number of bonds present absorbing the light [31]. Therefore, the compounds decreased the quality of these proteins, particularly PfHk. The FTIR results showed better compounds’ interactions with PfHk protein than with the PfLDH protein in this study. PfLDH protein, less peak shifts were observed at 1635 cm⁻¹ for both compounds [31]. Meanwhile, the control compound used in this study (chloroquine) also had low effect on both proteins’ secondary structures as shown in Figures S6 and S7.

Fig. 7

FTIR spectra of PfLDH-compound interaction. The graphs show FTIR analysis for A PfLDH protein, PfLDH-IMA, and PfLDH-BA. B PfHk protein, PfHk-IMA, and PfHk-BA. (1 mM compound concentration)

Direct protein–ligand binding affinity interaction analysis

MST is an invaluable tool for investigating thermophoretic analysis interactions. The temperature gradient involved essentially alters sample components such as charge, size, and hydration shell influencing macromolecule mobilisation and interaction [32]. In this study MST was used to further evaluate protein–ligand binding affinity, high binding affinity is typically represented by a low K_D value. The PfLDH protein interacted successfully with both compounds (PfLDH-BA = 0.1036 ± 0.6001 µM and PfLDH-IMA = 0.7473 ± 0.3554 µM). The K_D value (Table 3) of PfLDH-BA showed better interaction than PfLDH-IMA complex and curve formation (Fig. 8A) demonstrates the interactions of compounds with PfLDH. The protein PfHk successfully interacted with the IMA compound with a K_D value of 0.039701 ± 0.016298 µM. The interacted molecules induced the fluorescence variation resulting to the curve formation. The compound BA had a higher K_D value than IMA, thus less interaction was observed (Fig. 8B). The newly formed cis- and trans-conformational changes increased protein binding with the ligands causing better interaction and peptide bond formation on complexes. Both compounds successfully induced conformational changes of the protein charge, surface area and hydration shell, altering mobility and yielding interpretable data.

Table 3 The K_D values of protein–ligand complexes

Fig. 8

Thermophoretic analysis interactions of A PfLDH-BA and PfLDH-IMA complex. B PfHk-IMA complex

Conclusion

Both compounds (BA and IMA) interacted with malaria target proteins and remained bound within active binding sites of both proteins. Additionally, BA and IMA directly interacted with the proteins mainly IMA demonstrated dual interaction and binding with both proteins. However, the BA compound did not show a strong interaction with the PfHk protein, possibly due to protein aggregation and low ligand concentration. Therefore, BA might require further evaluation by increasing its concentration. Possible interactions, cytotoxicity and antiplasmodial effects of the investigated compounds should also be assessed against human proteins (LDH and Hk). Furthermore, in vitro studies will essentially prove that there are no possible effects of these compounds towards humans.