Anti-malarial Drugs as Potential Inhibitors of Leishmania Glycolytic Enzymes: Development of New Anti-leishmanial Agents

Leishmaniasis is one of the most important endemic diseases in Sudan. The glycolytic pathway is one of the essential pathways in the survival and pathogenicity of the leishmania parasite. This study aimed to evaluate the antileishmanial activities of three antimalarial drugs through targeting the glycolytic pathway of the parasite. Anti-leishmanial activities of artesunate, quinine and mefloquine were evaluated using an in vitro anti-promastigote assay. Then, in silico molecular docking was conducted using Autodock 4.0 software to study the molecular interactions of antimalarial drugs to different key glycolytic enzymes. The results of the current study, Artesunate, quinine, and mefloquine showed effective inhibitory activities against L. donovani with IC50 values of 58.85, 40.24, and 20.06 μg/ml, respectively. Molecular docking analysis revealed interesting interactions between different antimalarial drugs and various glycolytic enzymes (Glucose-6-phosphate isomerase, Triosephosphate isomerase, Glycerol-3-phosphate dehydrogenase, Glyceraldehyde-3-phosphate dehydrogenase and Pyruvate kinase). Moreover, these drugs interact with different amino acid residues of the proteins with satisfactory binding energies, particularly with artesunate. According to binding energies, Glycerol-3-phosphate dehydrogenase was represented the most potential target for three tested drugs. Collectively, our results showed promising antileishmanial activities of different antimalarial that may mediated through inhibition of glycolysis process in leishmania donovani promastigote.


Introduction
A leishmaniasis is a group of parasitic diseases caused by Leishmania species, which is transmitted by the bite of a Lutzomyia or Phlebotomus sand fly. 1

,2 Leishmaniasis
One of the most important endemic diseases in Sudan is visceral leishmaniasis (VL). Sudan represents one of the main VL foci globally, as 90% of VL incidence appears in 5 countries (India, Bangladesh, Brazil, Nepal, and Sudan). 3,5,6 VL was spread over a wide belt along the Sudanese-Ethiopian border from the Atbara River in the northeast and extending west across the White Nile to the Sobat River in South Sudan. It was focused in the Kapoeta area, the Nuba Mountains and Nassir, and Malakal and scattered areas in the Darfur region. 5,7,8 The glycolytic pathway is one of the essential pathways in the survival and pathogenicity of the Leishmania parasite. 9 Due to the lack of the functional Krebs cycle, the parasite utilizes the host carbons as a source of energy through the glycolytic pathway, representing the only source for Adenosine triphosphate (ATP) generation inside the parasite. 10,11 Therefore, inhibition of the glycolytic pathway by various agents could be considered as an excellent strategy to target leishmaniasis, as the glycolysis is the only source of energy. 12 The leishmania glycolytic pathway consists of multiple enzymes that play an essential role in the cascade of the glycolysis process, such as pyruvate kinase 13 , glucose-6-phosphate isomerase 14 , UDP-glucose pyrophosphorylase 15 , glyceraldehyde-3phosphate dehydrogenase 16 , glycerol-3phosphate dehydrogenase 17 , triosephosphate isomerase 18 , thiol-dependent reductase 1 19 , and phosphomannomutase. 20 Most anti-leishmanial drugs are toxic and less effective, and most of these medications are used parenterally for prolonged periods, especially for visceral leishmaniasis; also, it has several limitations drug-resistance, length of treatment, and cost lines. 21,22 So, there is an urgent need to search for new antileishmanial compounds of advantage that can outweigh the problem of the available anti-leishmanial drugs. In Sudan, malaria is co-endemic with visceral leishmaniasis, and co-infections with both diseases are common. 23 Previous clinical observation from Ecuador showed that significant efficacy of anti-malarial drugs, mefloquine and artesunate against leishmania. 24 In this study, the in vitro and in silico evaluation of the efficacy of different antimalarial drugs, including artesunate, quinine and mefloquine for their potential antileishmanial activity was assessed.

Methods
Parasite culture L. donovani promastigotes used in this study were kindly provided by the Department of Microbiology, Medicinal, Aromatic Plants and Traditional Medicine Research Institute (MAPTRI), Khartoum, Sudan. Promastigotes were cultured at 26°C in culture flasks containing RPMI 1640 medium (Sigma, St. Louis, MO) and supplemented with 5% fetal bovine serum (FBS), 2 mM glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin (Sigma, St. Louis, MO). Promastigotes were harvested after four or five days of incubation, and the growth of it was observed daily by using an inverted microscope (CK40; Olympus, Tokyo, Japan).

In vitro Evaluation Against Leishmania Donovani Promastigotes
The in vitro anti-promastigote assay was performed as previously described with trivial modifications 25. Briefly, the harvested promastigotes of L. donovania were suspended in a concentration of (2×106 parasites/ml) in culture medium and then incubated in 96-well microtiter plate at 26 ºC for 72 hr in fresh medium, in the absence and the presence of antimalarial drugs (artesunate, quinine, and mefloquine) at several concentrations. 40μl of the maximum concentrations of the drugs solutions artesunate (500 μg /ml), quinine (600 μg /ml), and mefloquine (100 μg /ml) were added in the first column wells (C-1). 20μl of complete RPMI 1640 medium (HyClone China Ltd., China) was added to the other wells (C-2 and C-6). Serial dilutions of the drugs were performed by taking 20μl from the previous wells and added to next wells. Then, 80μl of culture medium that contained the parasite was added to each well. Pentamidine was used as positive control whereas untreated cells were used as a negative control (culture medium plus parasites). For checking the activity, trypan blue were mixed with the samples, and then counted using by using a hemocytometer with a 20× objective under standard light microscopy. The results were represented as the mortality rate % and the mean inhibitory concentration (IC 50 ) after 72 h of incubation period.

In silico Molecular Docking Study
The 3-D structure of five enzymes involved in glycolysis; glucose-6-phosphate isomerase (PDB ID: 1T10) 14 , triosephosphate isomerase (PDB ID: 2Y63) 18, glycerol-3-phosphate dehydrogenase (PDB ID: 1M67) 17, glyceraldehyde-3-phosphate dehydrogenase (PDB ID: 1I33) 16, and pyruvate kinase (PDB ID: 3PP7) 13, were retrieved from the RCSB Protein Data Bank in .pdb format at models quality of 2.35 Å, 1.97 Å, 2.50 Å, 3.00 Å, and 2.35 Å resolution, respectively. The surface features and functional regions of the proteins were determined using the Computed Atlas for Surface Topography of Proteins (CASTp 3.0) web server http://sts. bioe.uic.edu/castp/. 26 Three anti-malarial compounds; artesunate, quinine, and mefloquine were selected as potential candidates for drug repositioning for leishmaniasis. The anti-malarial compounds and the positive control pentamidine ( Figure   1) were docked into the selected enzymes of glycolytic pathway. Molecular docking was carried out using Autodock 4.0 software 27 , based on Lamarckian Genetic Algorithm. 28 The protein structure was prepared by removal of water molecules and hetero groups. Then, we were added the polar hydrogen atoms to the protein targets, and computed the Kollman united atomic charges. The default docking algorithms were set following standard docking protocol. Ten independent docking runs were carried out for each ligand and results were retrieved as binding energies. Poses that showed lowest binding energies were visualized using BIOVIA Discovery Studio Visualizer (DVS) software. 29 Target proteins were re-docked with their cocrystallized ligand for validation of docking protocol.

Statistical analysis
All tests were repeated three times. The results were expressed as the mean ± SD. A two tailed Student's test was used to evaluate the significance of the results. Data were analyzed using GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA, USA) and P value < 0.05 was considered statistically significant.

Results and discussion
A reduction of the number of viable promastigotes was noticed after incubation for 72 h with different concentrations of various antimalarial drugs, and the reduction was significantly increased with the increasement of concentrations of these drugs ( Figure 2). The IC 50 value of the positive control anti-leishmania drug, pentamidine was 1.3±0.37 µg/mL, while the other tested anti-malarial compounds; quinine, artesunate and mefloquine showed less IC 50 values of 40.24±8.24 µg/mL, 58.85± 9.73 µg/mL, 20.06±4.1 µg/mL, respectively.
The results in hand suggest fair antileishmanial activities of these compounds as compared to the positive control pentamidine. The potential anti-leishmanial activity for quinine, artesunate and mefloquine had been reported in different studies. Herein, a previous study had reported the antileishmanial activity for quinine against L. donovani. 30 . Also, a recent finding reported the effective anti-leishmanial activity of quinine formulated as quinine sulphate microparticles 31 , and this could plausibly explain the poor activity for quinine in our finding. Similarly, mefloquine was also reported to be effective as anti-leishmanial agent and exerts a noticeable toxic effect against promastigotes in axenic cultures among other tested antimalarial drugs. 32 Artesunate reported a poor anti-leishmanial activity as compared to other tested drugs. 33,34 It was reported previously the divergence of artesunate activity as anti-leishmanial agent and pertained to the difference in genotypes between Leishmania species. 35  Molecular docking has been used efficiently for drug repurposing predictions. 36 Thus, we have attempted to study the possible interactions that would occur between the compounds and enzymes involve in glycolysis pathway. Table 1 and Figure 3-6 showed the interactions between the antimalarial drugs and the positive control pentamidine with several key proteins in the parasite glycolysis pathway. Glycolysis is a vital and sole metabolic pathway for ATP supply in the parasite. 14 . The initial enzymes involved in glycolysis are sequestered in the glycosome, a specialized organelle, and thus inhibition of these enzymes block the growth of the parasite. 37 . So far, the molecular  Artesunate and mefloquine formed interesting hydrogen and halogen bonding interactions with GLN-91, respectively, as this residue was required to be conserved in the design of the inhibitor N6 substituents. 16 Mefloquine and quinine showed hydrogen bonding and π-sulphur interactions with MET-39 located in the hydrophobic cleft; a region exploit for the design of potent selective inhibitors. 16 Moreover, they have shown hydrogen bonding with the conserved residue ASP-38. The hydrophobic substituents that pack against LEU-113 gain affinity as well as selectivity. 16 Herein, the docked compounds showed π-alkyl interactions with LEU-113.
Finally, the enzyme pyruvate kinase (PYK) catalyzes the last step reaction of glycolysis in which P-enolpyruvate and ADP are converted into pyruvate and ATP. 32 Pentamidine showed π-π stack and hydrogen interactions with the hydrophobic residues PRO-29 and TYR-59, respectively. Other conventional hydrogen bondings were observed with the residues HIS-54, ASN-51, THR-26, and ILE-27. The binding energy for pentamidine was reported ˗6.22 Kcal/mol. Similarly, docking results of the compounds showed stacking interactions with vital residues in the active pocket as TYR-59, HIS-54 and PRO-29 13 . The two hydrophobic residues PRO-29 and TYR-59 are essential and provide stability via helping to hold the molecule in the ATP/ADP binding site of the enzyme PKY. 13 Artesunate and mefloquine formed conventional hydrogen bonding with some of these residues, while quinine has formed van der Waals and π˗π stacked interactions. Table 1 indicates that artesunate and quinine showed the lowest binding energies (-9.31 and -7.43 kcal/mol, respectively) when docked with glycerol-3-phosphate dehydrogenase (PDB ID: 1M67). While mefloquine showed lowest binding energy (-6.91 kcal/mol) when

Conclusions
The preliminary results of this work showed relatively promising activities of artesunate, quinine and mefloquine against leishmania donovani promastigote. The in silico analysis suggested that anti-leishmanial activities mediated through inhibition of glycolysis pro1qacess in the parasite. Artesunate and quinine showed better interactions, in terms of binding energies, when docked with glycerol-3-phosphate dehydrogenase, while mefloquine showed lowest binding energy when docked with glyceraldehyde-3-phosphate dehydrogenase. Further future studies should focus on their detailed mechanism of action on in vivo models for the development of potential therapeutic agents.