Characterization of PfTrxR inhibitors using antimalarial assays and in silicotechniques
- Ranjith Munigunti†1,
- Symon Gathiaka†2,
- Orlando Acevedo2,
- Rajnish Sahu3,
- Babu Tekwani3 and
- Angela I Calderón1Email author
© Munigunti et al.; licensee Chemistry Central Ltd. 2013
Received: 31 July 2013
Accepted: 5 November 2013
Published: 10 November 2013
The compounds 1,4-napthoquinone (1,4-NQ), bis-(2,4-dinitrophenyl)sulfide (2,4-DNPS), 4-nitrobenzothiadiazole (4-NBT), 3-dimethylaminopropiophenone (3-DAP) and menadione (MD) were tested for antimalarial activity against both chloroquine (CQ)-sensitive (D6) and chloroquine (CQ)-resistant (W2) strains of Plasmodium falciparum through an in vitro assay and also for analysis of non-covalent interactions with P. falciparum thioredoxin reductase (PfTrxR) through in silico docking studies.
The inhibitors of PfTrxR namely, 1,4-NQ, 4-NBT and MD displayed significant antimalarial activity with IC50 values of < 20 μM and toxicity against 3T3 cell line. 2,4-DNPS was only moderately active. In silico docking analysis of these compounds with PfTrxR revealed that 2,4-DNPS, 4-NBT and MD interact non-covalently with the intersubunit region of the enzyme.
In this study, tools for the identification of PfTrxR inhibitors using phenotyphic screening and docking studies have been validated for their potential use for antimalarial drug discovery project.
Malaria, a tropical parasitic disease, continues to be the dominant cause of death in low-income countries especially in Africa and is considered to be one of the top three killers among communicable diseases . Malaria caused by Plasmodium falciparum is considered to be the most deadly and also the one with highest rate of drug resistance . Research investment in new and improved interventions will improve malaria cure, control, increase the cost-effectiveness of interventions and support efforts to eliminate malaria .
P. falciparum requires efficient antioxidant and redox systems to prevent damage caused by reactive oxygen species. In recent years, it has been shown that P. falciparum (Pf) possesses a functional low molecular weight thiol thioredoxin (Trx) system . Thioredoxin reductase (TrxR) is an important enzyme of this redox system that helps the parasite to maintain an adequate intracellular redox environment during intraerythrocytic development and proliferation. This antioxidant enzyme (PfTrxR) is essential for the survival of Plasmodium parasites for combating intraerythrocytic oxidative stress. Disruption of this enzyme is a feasible way to interfere with intraerythrocytic development and proliferation of the malaria parasites . The current chemotherapy for malaria as recommended by WHO focuses on artemisinin-based combination therapies (ACTs) as the front line of treatment for malaria disease. The main drawbacks of combination therapies are high cost, adverse drug reactions and a high degree of pharmacokinetic mismatch between components leading to prolonged exposure of parasites to low doses of partner drug and its active metabolites which may facilitate development of resistant parasites .
Development of parasites’ resistance to the known antimalarials remains a major challenge for the effective management of malaria. Intensive drug discovery programs have aimed at developing new antimalarials or modifying current antimalarials to improve their efficacy and reduce evidence of resistance.
In silico molecular modeling methods, such as docking can aid in the drug discovery process by ascertaining the binding affinities of existing and hypothetical compounds towards PfTrxR and the human isoform of this enzyme. Ideally, the simulations can also elucidate the origin behind the observed inhibition, as crystalline enzyme/inhibitor complexes of the thioredoxin protein for x-ray structure determination have not been reported. A comparison with other disulfide reductases including glutathione reductases reveals the most common inhibitor binding sites are at the active site and at the crystallographic 2-fold axis in the large cavity at the dimer interface. These sites can be exploited for structure-based inhibitor development. The dimer interface shows non-competitive or uncompetitive behavior and their interaction with the protein is purely non-covalent [7–10]. Docking calculations are well suited for exploration of this interface; however, the simulations are unable to reproduce covalent inhibitors that bind irreversibly at the active site. A combined experimental and computational effort may counterbalance this deficiency and provide an enhanced avenue for inhibitor development.
A comparison between the hTrxR and PfTrxR structures shows that they have 46% sequence identity and overlay with an RMSD of 0.91 Å between the 374 monomer atom pairs. The most important difference that can be exploited for selective inhibition between the two enzymes is at the dimer interface. The interface in PfTrxR is narrower than in hTrxR due to the presence of Tyr101 and His104 and can therefore host smaller molecules. Their counterparts in the human isoform are Gln72 and Leu75 and this difference can determine the chemical nature of suitable inhibitors . The molecular surfaces of the parasite and the human enzymes also indicate that the charges on the cavity walls are different, with the hTrxR’s being more negatively charged compared to the PfTrxR’s .
The current study is aimed to employ the combined approach of in silico molecular docking for identification of key interactions of PfTrxR inhibitors to improve selectivity and phenotypic antimalarial assays for identification of activity against susceptible and drug-resistant P. falciparum blood stage cultures to assure the identification of specific PfTrxR inhibitors as scaffolds for lead optimization.
Results and discussion
Pf TrxR inhibitory and antiplasmodial activities of tested compounds
Pf(D6) CQ sensitive IC50(μM)**
Pf(W2) CQ resistance IC50(μM)**
8.9 ± 2.3
16.7 ± 3.7
38.5 ± 0.76
91.2 ± 11.3
72.3 ± 11.3
79 ± 3.51
8.3 ± 2.1
9.8 ± 1.9
80 ± 1.15
18.5 ± 1.9
28.3 ± 5.6
70.5 ± 3.69
0.055 ± 0.006
0.440 ± 0.045
Predicted physico-chemical properties of the compounds
log P octanol/water (M)
log S aqueous solubility (M)
Polar surface area (PSA)
Apparent caco-2 permeability (nm/sec)
Apparent MDCK permeability (nm/sec)
Range 95% of drugs
(−2.0 / 6.5)
(−6.5 / 0.5)
(7.0 / 330.0)
(<25 poor, >500 great)
(<25 poor, >500 great)
The lack of antiplasmodial activity of 3-DAP, a Mannich base, may be due to (i) non-specific alkylation of cellular thiol groups, and also (ii) due to the absence of active transport to red blood cells and parasites. The correlation between inhibition of PfTrxR in the enzyme inhibition assays and antiplasmodial activity in cell culture allows for a better evaluation of biological activities of inhibitor compounds. The active compounds namely, 1,4-NQ, 2,4-DNPS, 4-NBT and MD showed more toxicity than 3-DAP against the 3T3 cell line. The 3T3 cells are epithelial cells that reflect toxicity against proliferating mammalian cells.
The 1,4-NQ chemical features and the ability to generate ·OH suggest the proficiency in altering intracellular redox status . The antimalarial naphthoquinones (1,4-NQ and MD) are believed to perturb the major redox equilibria of the targeted P. falciparum infected red blood cells, which might be removed by macrophages. This perturbation results in development arrest and death of the malaria parasite at the trophozoite stage .
Comparison between computed binding affinities at the dimer interface in Pf TrxR and experimental IC 50 values
Computed binding affinity (kcal/mol)
Exptl. PfTrxR IC50(μM)
Comparison between computed binding affinities at the dimer interface and experimental IC 50 values in Pf TrxR and h TrxR
Calc. binding affinity (kcal/mol)
Calc. binding affinity (kcal/mol)
In this study, tools for the identification of PfTrxR inhibitors using phenotypic screening and docking studies have been validated for their potential use for antimalarial drug discovery project.
Chemicals and enzymes
Deionized water generated by a Milli-Q water system (Millipore, MA) was used in the experiments. All reagents were purchased from Sigma–Aldrich.
Briefly, antimalarial activity of the compounds were determined in vitro on chloroquine sensitive (D6, Sierra Leone) and resistant (W2, IndoChina) strains of P. falciparum. The 96-well microplate assay is based on the effect of the compounds on growth of asynchronous cultures of P. falciparum, as determined by the fluorometric SYBR green assay .
Cytotoxicity in terms of cell viability was evaluated using 3T3 cells by AlamarBlue assay . This assay was conducted on compounds designated as active in the PfTrxR functional assay and the antimalarial phenotypic screening.
Accelerated generation and accumulation of reactive oxygen intermediates (superoxide radical, hydroxyl radical and hydrogen peroxide) are mainly responsible for oxidative stress . The intraerythrocytic formation of ROS was monitored in real-time with 2′7′-dichlorofluorescein diacetate (DCFDA), a fluorescent ROS probe . Human erythrocytes collected in citrate phosphate anticoagulant were used. The erythrocytes were washed twice with 0.9% saline and suspended in PBSG at a hematocrit of 10%. A 60 mM stock of DCFDA was prepared in DMSO and added to the erythrocytes suspension in PBSG (10% hematocrit) to obtain the final concentration of 600 μM. Erythrocytes suspension containing 600 μM of DCFDA was incubated at 37°C for 20 min and centrifuged at 1000 g for 5 min. The pellet of DCFDA loaded erythrocytes was suspended in PBSG to 50% hematocrit and used for kinetic ROS formation assay. The assay was directly set up in a clear flat-bottom 96 well microplate. The reaction mixture contained 40 μl of DCFDA loaded erythrocytes, the test compounds (50 μM)) and potassium phosphate buffer (100 mM, pH 7.4), to make up the final volume to 200 μl. The controls without drug were also set up simultaneously. Each assay was set up at least in duplicate. The plate was immediately placed in a microplate reader programmed to kinetic measurement of fluorescence (excitation 488 nm and emission 535 nm) for 2 hours with 5 min time intervals.
AutoDock Vina  was used to dock inhibitors to the respective targets. Initial Cartesian coordinates for the protein-ligand structures were derived from reported crystal structures of hTrxR (PDB ID: 3QFA)  and PfTrxR (PDB ID: 4B1B) . The protein targets were prepared for molecular docking simulation by removing water molecules and bound ligands. AutoDockTools (ADT)  was used to prepare the docking simulations whereas Chimera was used to analyze the docking poses. All ligands were constructed using PyMol  with subsequent geometry optimizations carried out using the semi-empirical method PDDG/PM3 [20, 26, 27]. Polar hydrogens were added. ADME properties logP, logS, polar surface area, and apparent Caco-2 permeability for each ligand were computed using QikProp [28, 29]. Conjugate gradient minimizations of the systems were performed using GROMACS . A grid was centered on the catalytic active site region and included all amino acid residues within a box size set at x = y = z = 20 Å.
AutoDock Vina details
Standard flexible protocols of AutoDock Vina using the Iterated Local Search global optimizer  algorithm were used to evaluate the binding affinities of the molecules and interactions with the receptors. All ligands and docking site residues, as defined by the box size used for the receptors, were set to be rotatable. Calculations were carried out with the exhaustiveness of the global search set to 100, number of generated binding modes set to 20 and maximum energy difference between the best and the worst binding modes set to 5. Following completion of the docking search, the final compound pose was located by evaluation of AutoDock Vina’s empirical scoring function where the conformation with the lowest docked energy value was chosen as the best.
We are deeply indebted to Dr. Katja Becker from the Justus-Liebig University, Germany for supplying the enzyme PfTrxR. NCNPR is partially supported by USDA-ARS cooperative scientific agreement.
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