key: cord-0803936-05ejg4ul
authors: Tabrez, Shams; Rahman, Fazlur; Ali, Rahat; Akand, Sajjadul Kadir; Alaidarous, Mohammed A.; Banawas, Saeed; Dukhyil, Abdul Aziz Bin; Rub, Abdur
title: Hesperidin Targets Leishmania donovani Sterol C-24 Reductase to Fight against Leishmaniasis
date: 2021-03-16
journal: ACS Omega
DOI: 10.1021/acsomega.0c05858
sha: d1d376d92756d8d46aba6dd4d8caee6263f15d59
doc_id: 803936
cord_uid: 05ejg4ul

[Image: see text] Hesperidin, a naturally occurring flavanoid, is present in citrus family of fruits. It was found effective against an array of pathogens including fungi, bacteria, viruses, and protozoa. Here, we evaluated its antileishmanial activity and possible mechanism of action through different in vitro and in silico experiments. It inhibited the growth and proliferation of the parasites significantly with a IC50 value of 1.019 ± 0.116 mM in vitro. It also reduced the growth of intra-macrophagic amastigotes with a IC50 value of 0.2858 ± 0.01398 mM. It induced the reactive oxygen species (ROS) in parasites in a dose-dependent manner. Through 2,7-dichloro dihydro fluorescein diacetate (H(2)DCFDA) staining, it was observed that around 96.9% parasites were ROS positive at 2.0 mM concentration of hesperidin. The ROS generated led to the apoptosis of parasites in a dose-dependent manner as observed by annexin/PI staining. Molecular docking with one of the very important and unique drug-targets of Leishmania donovani sterol C-24 reductase resulted in its significant inhibition. Here, we for the first time showed that hesperidin induced the antileishmanial response through the induction of apoptosis and inhibition of sterol C-24 reductase. Our study will be helpful in the development of a cost-effective antileishmanial lead with higher efficacy.

Leishmaniasis is a vector-borne disease caused by obligate intra-macrophage protozoa belonging to the genera Leishmania. The disease leishmaniasis is endemic in more than 89 different countries worldwide including Tropics, Subtropics, and Southern Europe. It is classified as a neglected tropical disease. 1 Worldwide, 0.7−1 million new cases of leishmaniasis are reported per year. 2 These days, paromomycin, amphotericin B, liposomal amphotericin, and miltefosine are the only available treatment of visceral leishmaniasis which have their own limitations due to high toxicity, resistance, and high cost. 2, 3 Leishmania exploits the host cholesterol biosynthetic machinery for its virulence, survival, and growth. 4−6 Leishmania requires ergosterol for its membrane organization and other biological functions. 7 Sterol C-24 reductase (LdSR), sterol alpha-14 demethylase, sterol 24-C-methyltranferase, and so forth are the major enzymes of the ergosterol biosynthetic pathway of Leishmania donovani which are still not studied in detail yet. 7 Out of these, LdSR converts ergostatetraenol to ergosterol. It is an important drug-target for the development of new molecules to fight leishmaniasis. 8 Hesperidin is a naturally occurring flavanone glycoside. 9 It is present in high quantity in Citrus aurantium, Citrus sinensis, and Citrus unshiu and other species of this genus. 10, 11 It showed profound antioxidant, anti-inflammatory, anti-proliferative, and anticancer activities. 12 The available literature reported that hesperidin inhibited the growth of different types of microbes including fungi, bacteria, virus, protozoa, and so forth. 13−15 Anti-fungal effects of hesperidin are well-documented on different fungal species including Aspergillus parasiticus, Aspergillus flavus, Fusarium semitectum, and Penicillium expansum. Hesperidin detoxifies the effects of patulin, a mycotoxin released from Aspergillus also. 16 Novel herbal micro-emulsion of hesperidin was found effective against bacterial species including Escherichia coli, Staphylococcus epidermidis, Bacillus cereus, Enterococcus faecalis, Salmonella typhimurium, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Hesperidin pre-treatment suppressed infection-induced endotoxic shock in mice and reduced bacterial colonies during infection. 17, 18 Besides the anti-fungal and anti-bacterial role, the anti-viral effects of hesperidin are well established against HIV and herpes simplex virus type 2. 19 Recently, hesperidin was shown to inhibit SARS-CoV-2 main protease and angiotensin-converting enzyme 2 receptor. 20, 21 In case of humans, hesperidin appeared to be safe with no adverse effects as reported in the treatment of hyperuricemia. 9 Keeping all these medicinal values of hesperidin in mind, we planned to evaluate its antileishmanial activity on L. donovani. We have also performed the molecular docking of hesperidin against an important drug-target LdSR to dissect the possible mechanism of action of it. The current work may open new possibilities for the development of effective treatment against leishmaniasis.

2.1. Antileishmanial Activity of Hesperidin. Logarithmic phase promastigote was treated with different concentrations of hesperidin. After 24 h of treatment, it inhibited the growth of parasite significantly. The IC 50 value was calculated as 1.019 ± 0.116 mM ( Figure 1A ) by the dose response curve.

The infective stage, promastigote that grows in the gut of sandfly, is transmitted to humans through the bite of sandflies. Macrophages phagocytose the promastigotes, where it gets transformed into an amastigote form. These amastigotes multiply within the macrophages and eventually propagate inside the body. Any drug-molecule must inhibit the growth of these intracellular amastigotes to be proven as an effective antileishmanial lead. Therefore, the differentiated macrophages were infected by L. donovani and treated with hesperidin. The IC 50 of the hesperidin on intra-macrophagic parasites was determined as 0.2858 ± 0.01398 mM ( Figure 1B) . A dosedependent inhibition of the intra-macrophagic parasite was observed in microscopic images ( Figure 1C ).

Reactive oxygen species (ROS) is the signal of stress and an important mediator of cell death and necrosis. We planned to check whether hesperidin-induced inhibition of parasite growth is related to the induction of apoptosis. To study this, hesperidintreated parasites were stained with H 2 DCFDA dye and analyzed through a flow cytometer. H 2 DCFDA fluoresces upon the generation of OH • radicals and H 2 O 2 . The fluorescence intensity is directly proportional to ROS generation, and the peak intensity was shifted to the right with increasing intensity. Hesperidin induced ROS generation in a dose-dependent manner in parasites ( Figure 2A ). It induced ROS in around 96.9% parasites at 2.0 mM concentration ( Figure 2A ).

2.3. Hesperidin Induced Apoptosis-Like Cell Death in L. donovani. Higher levels of ROS generally induce apoptosis in particular cells. Therefore, we further performed the annexin V/PI staining to check whether hesperidin-induced ROS ultimately induced the apoptosis-like cell death process in parasites. We observed a dose-dependent increase in a number of parasites undergoing apoptosis upon treatment of hesperidin. There were 96.9% parasites that have undergone apoptosis-like cell death at 2.0 mM concentration of hesperidin ( Figure 2B ). These results suggested that hesperidin induced ROS which ultimately led to the apoptosis-like cell death in parasites in vitro.

2.4. Homology Modelling and Molecular Docking of Hesperidin with LdSR. LdSR is one of the key enzymes of the ergosterol biosynthetic pathway which converts ergostatetraenol to ergosterol. It is an attractive drug-target for the identification of new drug against leishmaniasis. 8 We planned to perform the molecular docking of hesperidin against LdSR to investigate the possible mechanism of action. Hundred models of LdSR were generated and best one was selected based on the lowest dope score ( Figure Table 1 ). The binding pattern of hesperidin with LdSR depicts the loss of substrate accessibility and its inhibition ( Figure 4A ). Hesperidin interacts with Cys304(3), Thr307, and Arg464(2) binding site residues of LdSR by forming six intermolecular hydrogen bonds ( Figure  4B ,C). The bond length of hydrogen bonds was between 2.38 and 3.08 Å. One of the carbon ring of hesperidin formed πanion bond with Asp468 of LdSR to stabilize the interaction ( Figure 4B,C) . The π-anion is recognized as an important noncovalent interaction in the biological structure, driven by polarization and electrostatic effects. 22 Phe416 forms the π−π T-shaped as well as carbon hydrogen bond with the aromatic ring of hesperidin. This interaction plays an important role in the macromolecular structure and function. 23 The minimum binding energy and predicted inhibitory constant of hesperidin with respect to LdSR were calculated as −10.1 kcal/mol and 7.43 μM, respectively (Table 1 ). This value of inhibitory constant suggested the effective inhibition of LdSR and possible leishmanicidal activity of hesperidin. Overall, our study suggested the antileishmanial activity of hesperidin with ROS generation and apoptosis-like cell death induction in parasites along with the possible inhibition of a key ergosterol-biosynthetic enzyme, LdSR. In addition to this the generated results could not rule out the other possible targets of hesperidin in L. donovani that might be contributing in its antileishmanial response. . The logarithmic phase parasites were regularly passaged at 3−4 days at the density of 2 × 10 6 parasites/ml. THP-1 cell line (human leukemia cells) was cultured in RPMI-1640 media with 10% FBS and 1% penicillin−streptomycin antibiotic and maintained at 37°C and 5% CO 2 . The stock concentration of the compound was prepared in blank M199 media. THP-1 monocytic cells were stimulated with 20 ng/mL of phorbol myristate acetate for differentiation to macrophages.

3.2. Antileishmanial Activity of Hesperidin. For antileishmanial activity, logarithmic phase parasites were seeded at the density of 2 × 10 6 parasites/mL in complete M199 media; then, the compounds were added at different concentrations. After 24 h of incubation at 24°C, the parasites were fixed in 1% paraformaldehyde and enumerated using a hemocytometer. The percentage inhibition was calculated, by considering negative control as 100%. Dose−response curves were plotted using inhibitor concentration versus normalized response and IC 50 calculated automatically 24 3.3. Parasite Load Calculation in Macrophages. For amastigote assay, 2 × 10 5 THP-1 cells were seeded on the coverslip in a six-well plate, and the differentiated macrophages were infected with log-phase virulent promastigotes in the ratio of 1:10 (macrophage: Leishmania) for 24 h. The non- Figure 2 . Hesperidin-induced ROS and apoptosis in parasites (A) 2 × 10 6 parasites/mL were incubated with low and high doses of hesperidin for 24 h followed by staining with H 2 DCFDA dye for ROS analysis through a flow cytometer (B). Parasites were incubated with different concentrations of hesperidin for 24 h, followed by staining with annexin V-FITC/PI to assess the apoptosis through a flow cytometer as described in methods. phagocytosed parasites were washed, and infected macrophages were treated with different doses of hesperidin for 24 h post-infection. The coverslip was washed with 1× phosphatebuffered saline (PBS), fixed with chilled methanol, and stained using modified Giemsa stain for amastigote microscopic evaluation. About 100 macrophages were counted from different focus to evaluate the effect of the treatment on the intra-macrophagic parasite burden 25 3.4. Estimation of ROS Generation. To estimate the effects of hesperidin on ROS generation, 2,7-dichloro dihydro fluorescein diacetate (H 2 DCFDA) dye was used. The treated parasites were washed with PBS and incubated with 10 μM of H 2 DCFDA for 20 min in dark. The fluorescent signal of each sample was acquired using a BD FACS ARIA III flow cytometer, and the recorded data was represented in the form of histograms.

3.5. Apoptotic Assay. For the apoptotic assay, treated and untreated parasites were centrifuged at 3000g for 5 min and washed thrice with 1× PBS. Finally, the pellet was resuspended in 1× binding buffer (10 mM Hepes pH 7.4, 140 mM NaCl and 2.5 mM CaCl 2 ) containing 5 μL of annexin-FITC and 5 μL of propidium iodide for 25 min in dark at room temperature. The samples were analyzed through a BD FACSAria III Cell Sorter.

3.6. Molecular Docking, Homology Modelling, Receptor, and Ligand Preparations. PyRx was used for the virtual screening of the ligand with the target proteins of L. donovani. 26, 27 The detailed interactions and their types including hydrogen bonds, van der Waals interactions, alkyl, π-alkyl, halogen, and so forth were analyzed by BIOVIA Discovery Studio. 28 The most favorable binding poses of the compounds were analyzed by choosing the lowest free energy of binding (ΔG) and the lowest inhibition constant (K i ) which is calculated using the following formula

where ΔG is binding affinity (kcal/mol), R (gas constant) is 1.98 cal K −1 mol −1 , and T (room temperature) is 298.15 K. The protein sequence of LdSR (CBZ37214.1) enzyme of L. donovani was retrieved from NCBI. For the identification of similar templates, blastP 29 was performed against Protein Data Bank. The X-ray diffracted crystal structure 2.74 Å resolution (PDB id: 4QUV_A) was used as a template structure to model the 3D structure of sterol C-24 reductase. Modeller 9.24 30 was used for homology modelling, and the generated model was assessed by the PROCHECK program and PDB sum tool, and Ramachandran plots were also generated. 31, 32 The critical residues of the binding pocket was identified from the native catalytic pockets of the available crystal structure of proteins, CASTp, and Discovery studio. The 3D structure of hesperidin was retrieved from PubChem database. 33 The atomic coordinates of the hesperidin was changed to pdbqt set-up using Open Babel GUI, 34 and Universal Force Field (uff) was used for the energy minimization. 35 3.7. Statistical Analysis. All the experiments were performed in triplicate, and the results represented are the mean of the triplicate with SD. Statistical analysis was performed using Graph pad prism 7.0 software, and a Pvalue of less than 0.05 was considered significant. The statistical significance was calculated using one way ANOVA followed by Dunnett's multiple comparison test.

It is the first and preliminary study on the evaluation of antileishmanial potential of hesperidin. Further, our study will provide a platform for the future detailed in vitro and in vivo study in this context for the possible use of hesperidin in the treatment of visceral leishmaniasis.

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The authors would like to thank the Deanship of Scientific Research at Majmaah University, Al Majmaah, 11952, Saudi Arabia for supporting this work under the group project number RGP-2019-31.

The authors declare no competing financial interest.

The authors are thankful to the Central Instrumentation facility, Jamia Millia Islamia for support.