|Year : 2017 | Volume
| Issue : 1 | Page : 49-54
Inhibition of mycobacterial CYP125 enzyme by sesamin and β-sitosterol: An in silico and in vitro study
Gauri Wankhade1, Sarika Kamble2, Shraddha Deshmukh1, Lingaraja Jena1, Pranita Waghmare3, Bhaskar C Harinath2
1 Bioinformatics Centre, Mahatma Gandhi Institute of Medical Sciences, Wardha, Maharashtra, India
2 JB Tropical Disease Research Centre, Mahatma Gandhi Institute of Medical Sciences, Wardha, Maharashtra, India
3 Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Wardha, Maharashtra, India
|Date of Web Publication||24-Jul-2017|
Bhaskar C Harinath
JB Tropical Disease Research Centre, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha - 442 102, Maharashtra
Source of Support: None, Conflict of Interest: None
Background: Cholesterol degradation pathway is one of the important pathways in survival of Mycobacterium tuberculosis (Mtb) bacilli, and steroid C26-monooxygenase (CYP125) enzyme of Mtb associated with this pathway is reported to be novel drug target. This study aims to find out novel, safe, and effective inhibitors against CYP125 from natural phytochemicals with reported anti-tubercular activity. Methods: Bioinformatics approach such as homology modeling, virtual screening, and molecular dynamics (MD) simulation was applied to identify best hits among all the shortlisted 148 compounds. The Mtb H37Ra bacilli growth was measured at optical density at 600 nm in minimal media supplemented with cholesterol and monitored for 10 days. Two promising compounds, namely, sesamin and β-sitosterol, were studied to determine their effective minimum inhibitory concentrations (MICs) in Mtb H37Ra bacilli culture. Results: In virtual screening, 15 compounds showed comparatively better binding affinity than natural substrate (choletst-4-en-3-one). In MD simulation study, the protein structure was observed to be stable in alls the interaction complexes, i.e., with choletst-4-en-3-one, sesamin, and β-sitosterol. The MICs of sesamin and β-sitosterol were observed to be 2 μg/ml, inhibiting the growth of the Mtb bacilli by 51% and 53%, respectively. Conclusions: From the above experimental findings, sesamin and β-sitosterol may be proposed as safe and potential inhibitors of CYP125 resulting in diminished growth of Mtb bacilli.
Keywords: Minimum inhibitory concentration, molecular dynamics simulation, Mycobacterium tuberculosis, phytochemicals, virtual screening
|How to cite this article:|
Wankhade G, Kamble S, Deshmukh S, Jena L, Waghmare P, Harinath BC. Inhibition of mycobacterial CYP125 enzyme by sesamin and β-sitosterol: An in silico and in vitro study. Biomed Biotechnol Res J 2017;1:49-54
|How to cite this URL:|
Wankhade G, Kamble S, Deshmukh S, Jena L, Waghmare P, Harinath BC. Inhibition of mycobacterial CYP125 enzyme by sesamin and β-sitosterol: An in silico and in vitro study. Biomed Biotechnol Res J [serial online] 2017 [cited 2021 Jun 13];1:49-54. Available from: https://www.bmbtrj.org/text.asp?2017/1/1/49/211404
| Introduction|| |
Increasing incidence of multidrug-resistant cases of tuberculosis (TB) and difficulty in treating these cases requires urgent need to find safer and effective anti-TB drug. Globally, 33% of the population is considered to be infected with Mycobacterium tuberculosis (Mtb) infection, with 10.4 million new patients.
Anti-TB drugs were introduced till 1980s, reporting 98% chance of cure, which include streptomycin and other highly specific drugs such as isoniazid, rifampicin, and ethambutol. These drugs have major serious side effects such as psychosis, convulsive seizures, mental confusion, coma, vasculitis, peripheral neuropathy, and clinical hepatitis. Recent rise in HIV and TB coinfection has caused more trouble with drug-resistant and multidrug-resistant TB. Therefore, more effective drugs with minimal side effects are the need of the day for effective TB control.
In spite of host defense system against microbes, Mtb resides in a modified phagosomal compartment of macrophage to acquire nutrients from the human host cells., When Mtb is in the active phase of infection, it utilizes cholesterol as whole carbon and energy source of the bacilli. In Mtb, cholesterol is degraded by a specialized pathway, i.e., cholesterol degradation pathway and steroid C26-monooxygenase, an important enzyme of this pathway is reported to be a good drug target.,, It converts cholest-4-en-3-one to 26-hydroxycholest-4-en-3-one. Accumulation of this intermediate product (cholest-4-en-3-one) is toxic to Mtb bacilli. This enzyme is present in three isoforms, i.e., CYP125 (Rv3545c), CYP142 (Rv3518c), and CYP124 (Rv2266) collectively named as cytochrome P450 isoforms. Among these isoforms, CYP125 is highly efficient. The present study is proposed to find out a drug that can inhibit CYP125 which may lead to accumulation of cholest-4-en-3-one to kill the pathogen.
| Methods|| |
Hardware and software
Dell Workstation with Linux Operating System, having a hard disc of 2 TB and 32 GB RAM, was used for in silico study. Further, computational analysis was performed using bioinformatics tools, such as AutoDock Vina version 1.1.2 (AutoDock Vina is an open-source program for doing molecular docking. It was designed and implemented by Dr. Oleg Trott in the Molecular Graphics Lab at The Scripps Research Institute), GROMACS version 5.0.4 (GROMACS was first developed in Herman Berendsen's group, department of Biophysical Chemistry of Groningen University, and now lead in Stockholm from the Science for Life Laboratory), and other online resources.
One hundred and forty-eight phytochemicals with reported antitubercular activity were identified from online resources (Dr. Duke's Phytochemical and Ethnobotanical Databases) and literature survey.,,,,
Mycobacterium tuberculosis CYP125 enzyme
The amino acid sequence of CYP125 (Rv3545c), i.e., steroid C26-monooxygenase enzyme, was retrieved from Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The experimentally determined structure of this protein (protein data bank [PDB] Identifier (ID): 2×5W) obtained through the X-ray diffraction experiment was retrieved from the PDB.
Structure prediction and validation of CYP125 enzyme
As there were some missing residues in PDB structure of CYP125 (PDB ID: 2×5 W), its three-dimensional (3D) structure was further modeled using Modeller 9 version 14 (MODELLER is maintained by Ben Webb at the Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry, and California Institute for Quantitative Biomedical Research, Mission Bay Byers Hall, University of California San Francisco, San Francisco, CA 94143, USA) by taking the same PDB structure as template. The predicted protein structure was validated and the reliability of refined model was assessed through protein structure analysis (ProSA), protein quality predictor (ProQ), and Research Collaboration for Structural Bioinformatics (RCSB) validation server.
The chemical structures of selected 148 phytochemicals were retrieved from the National Center for Biotechnology Information PubChem database in sdf format. These structures were then converted to pdbqt format using Open Babel version 2.3.2 (Open Babel is a chemical toolbox designed to speak the many languages of chemical data. It's an open, collaborative project allowing anyone to search, convert, analyze, or store data from molecular modeling, chemistry, solid-state materials, biochemistry, or related areas. It is available at http://openbabel.org/.).
Virtual screening prototype is now being widely used for filtering large dataset of compounds for drug discovery. All the 148 compounds were subjected to virtual screening along with the natural substrate of CYP125, i.e., cholest-4-en-3-one using AutoDock Vina version 1.1.2 virtual screening program. Molecular docking of all the ligands with target protein was performed on the binding site of substrate. A grid of 20, 20, and 20 points in X, Y, and Z directions was centered on the known active site residues of proteins (PHE100, ILE104, ASP108, GLN112, VAL115, LEU117, MET200, PRO213, LYS214, SER217, VAL263, VAL267, ALA268, THR272, and TRP414). Ligands showing binding energy less or equal to that of cholest-4-en-3-one were selected for further studies.
Molecular dynamics simulation
GROMACS version 5.0.4 (GROMACS was first developed in Herman Berendsen's group, department of Biophysical Chemistry of Groningen University, and now lead in Stockholm from the Science for Life Laboratory) package along with GROMOS96 54a7 force field was used for performing molecular dynamics (MD) simulation of CYP125. The standard procedures used by Jena et al. were elaborately followed for performing MD simulation in this study. Finally, 100 ns MD simulations of each system were performed. Further, topology of ligands was generated by the PRODRG server and the docking complexes of ligands (cholest-4-en-3-one, sesamin, and β-sitosterol) with CYP125 were subjected to MD simulation for 100 ns using the same protocol followed by Jena et al. in their study. All the important analyses were performed using GROMACS analysis programs. Microsoft Excel was used for preparation of the graph.
Role of cholesterol and inhibitors on the survival and growth of Mycobacterium tuberculosis H37 Ra
The role of cholesterol in the growth of Mtb H37 Ra was demonstrated by the method explained by Chang et al. with required modifications. Previously, bacilli were grown in Middlebrook 7H9 medium supplemented with 0.05% Tween 80 and albumin-dextrose-catalase at 37°C with hygromycin (50 μg/ml). The growth was transferred to minimal media (MM) containing per liter, 0.5 g asparagine, 0.1 mg ZnSO4, 1.5 g Na2 HPO4, 10 mg MgSO4.7H2 O, 1 g KH2 PO4, 0.5 mg CaCl2, 50 mg ferric ammonium citrate, and 1 ml 1:1 (vol/vol) tyloxapol-ethanol, pH 7.2. A 100 mM stock of cholesterol was prepared by heating the mixture of cholesterol in tyloxapol and ethanol for 30 min at 65°C. Log-phase bacteria were washed with MM and diluted to an optical density at 600 nm (OD600) of 0.1. Finally, media (MM with cholesterol [MM + C]) containing 0.2 mM cholesterol were prepared. Equal volumes of diluted bacteria and 0.2 mM cholesterol medium were mixed to obtain a final concentration of 0.1 mM cholesterol at an OD600 of 0.05. All the procedure is performed in aseptic conditions.
The growth of Mtb H37 Ra in the MM without any carbon source, with cholesterol as sole carbon source along with MM (MM + C) and in the presence of two best hits at different concentrations (10, 5, 2, 0.2, and 0.1 μg/ml), i.e., sesamin (scivation) and β-sitosterol (source naturals) along with MM (MM + C), was monitored at 1 day interval for 10 days at OD600 simultaneously to calculate percentage inhibition and to determine the minimum inhibitory concentration (MIC) of both compounds.
Ethical approval was not required for this study.
| Results|| |
Protein structure modeling and validation
The stereochemistry of the newly modeled CYP125 [Figure 1]a (procheck analysis) revealed that 93.3% of residues were situated in the most favorable region and 6.2% were in allowed regions, whereas only 0.3% (one residue) fell in the disallowed region of the Ramachandran plot [Figure 1]b. ProSA-web evaluation revealed a compatible Z-score value of −9.36 [Figure 1]c. The 3D model of CYP125 showed Log (LG) score of around 3.422 by the ProQ tool, implying the high accuracy level of the predicted structure.
|Figure 1: (a) Three-dimensional structure of CYP125. (b) Ramachandran plot of CYP125, (c) Z plot of CYP125|
Click here to view
From virtual screening, it was observed that the CYP125 enzyme binds with its natural substrate, i.e., cholest-4-en-3-one with binding energy of −10.9 kcal/mol. Further, among 148 compounds screened, 15 compounds, i.e., amentoflavone, tiliacorinine, 2-nortiliacorinine, α-amyrinone, α-amyrin, taraxerol, friedelin, diospyrin, ergosterol-5, 8-endoperoxide, ursolic acid, lupeol, rottlerin, artonin F, sesamin, and β-sitosterol, showed binding energy lower than substrate.
In this study, two compounds (sesamin and β-sitosterol) were selected for further studies because these two are already marketed as nutraceuticals. From docking study, it was found that the binding energy levels of CYP125 with sesamin and β-sitosterol were −11 kcal/mol and −10.9 kcal/mol, respectively. Both these compounds were observed to be interacting with CYP125 at the enzyme active site like the natural substrate [Figure 2]. These two compounds were further considered for MD simulation.
|Figure 2: Docking interaction of CYP125 with (a) natural substrate choletst-4-en-3-one, (b) sesamin, (c) β-sitosterol in the enzyme active site|
Click here to view
Molecular dynamics simulation
In this study, MD simulations were performed to determine the stability of CYP125 in the interaction complex with the natural substrate (cholest-4-en-3-one) as well as with the virtual screened hits (sesamin and β-sitosterol).
To observe the stability of protein structure, root mean square deviation (RMSD) values were determined for CYP125. It was shown that RMSD graph of CYP125 enzyme fluctuate from 0.1 ns to 0.50 ns, but from 50 ns it was observed to be stable throughout simulation. For determining dynamic behavior of residues, Root Mean Square Fluctuation (RMSF) values of CYP125 enzyme were calculated. RMSF value of native residues fluctuates from a range 0.06 to 2.1 nm in the entire simulation period. The radius of gyration (Rg) provides an observation into global dimension of protein. Rg graph for alpha-carbon atoms of protein versus time at 300 K was depicted. A ligand binding interaction always influences the stability of the receptor protein. It was shown in [Figure 3]a that the RMSD graph of CYP125 with sesamin and β-sitosterol are almost similar to RMSD graph of CYP125 with substrate during most of the time scale. It was shown that the RMSD graph of CYP125 with substrate and ligands deviated after 0.1 ns till 100 ns. At 44.99 ns, CYP125 protein with substrate attained a maximum deviation of about 0.36 nm whereas, and sesamin and β-sitosterol attained a maximum deviation of about 0.38 nm and 0.40 nm, respectively.
|Figure 3: (a) Root mean square deviation plot, (b) Root Mean Square Fluctuation plot, (c) radius of gyration plot of CYP125 in unbound state as well as in complex with natural substrate (choletst-4-en-3-one), sesamin, and β-sitosterol obtained from molecular dynamics simulation analysis|
Click here to view
The RMSF value of CYP125 substrate fluctuates from a range 0.05 to 0.53 nm in the entire simulation period. Moreover, CYP125 with sesamin and β-sitosterol exhibited flexibility of ~0.41 nm and ~0.45 nm, respectively [Figure 3]b. In Rg plot [Figure 3]c, we observed a major fluctuation of CYP125 in unbound state than in bound state with substrate and ligands between time periods of 0 and 100 ns.
Inhibition of Mycobacterium tuberculosis H37 Ra bacilli by sesamin and β-sitosterol
At the concentration of 2 μg/ml, sesamin and β-sitosterol showed inhibition of 51% and 53%, respectively. Therefore, the MIC of both the compounds is 2 μg/ml in the presence of cholesterol [Table 1] and [Figure 4].
|Table 1: Percentage inhibition of Mycobacterium tuberculosis growth by sesamin and β-sitosterol at various concentrations to determine the minimum inhibitory concentrations|
Click here to view
|Figure 4: Growth of Mycobacterium tuberculosis H37Ra in cholesterol supplemented media and effect of phytochemicals on their growth. (a) Effect of sesamin, (b) effect of β-sitosterol. MM - Minimal media, MM + C - Minimal media with cholesterol|
Click here to view
| Discussion|| |
Cholesterol has importance in various stages of pathogenicity of Mtb including mycobacterial uptake and persistence during chronic infection. Different genes of Mtb associated with cholesterol modification were reported to cause virulence., Further, igr locus contains different genes which are involved in the metabolism of cholesterol.
The major hurdle in the current TB treatment is increasing multidrug-resistant and extensively drug-resistant cases. To provide a new, more effective, and less toxic drug, an effort has been made by analyzing potential phytochemicals against CYP125 (a new drug target). CYP125 (steroid C26-monooxygenase) is proved to be a key enzyme in a specialized pathway which degrades cholesterol as a carbon and energy source. To come up with a new drug which is from natural source, 148 anti-TB phytochemicals were screened against CYP125 by virtual screening using AutoDock Vina version 1.1.2. The best hits were selected on the basis of binding energy less than that of substrate, i.e., cholest 4-en 3-one (−10.9 kcla/mol). We have found fifteen compounds, of which few compounds are already marketed for various uses. We have studied effect of sesamin and β-sitosterol which are commercially available food supplements. Sesamin is commonly present in Piper species, and sesame seeds (Sesamum indicum) have been found with anti-hair loss and anti-canitie uses. Sesamin also helps promote fat oxidation, prevents fat storage, increases insulin sensitivity, prevents free radical damage, promotes anti-inflammatory effects, reduces cholesterol, and acts as a potent antioxidant. β-sitosterol is useful in treating heart disease, high cholesterol, colon cancer, gallstones, the common cold and flu (influenza), HIV/AIDS, rheumatoid arthritis, TB, allergies, fibromyalgia, asthma, hair loss, bronchitis, chronic fatigue syndrome, and for symptoms of menopause. It is also used by marathon runner to reduce pain and swelling after a run.
Overall stability of the protein structure was observed to be stable in all the interaction complexes, i.e., with choletst-4-en-3-one, sesamin, and β-sitosterol. These results suggest that these two phytochemicals are capable to compete with choletst-4-en-3-one while interacting with CYP125 and thus may be used as possible CYP125 inhibitors. Demonstration of effect of inhibition of sesamin and β-sitosterol at various concentrations revealed their MIC as 2 μg/ml. As shown in [Figure 4], it is clear that the growth of Mtb H37 Ra bacilli is significantly enhanced in the presence of cholesterol as compared to only MM. The Mtb bacilli growth is significantly inhibited by sesamin and β-sitosterol in the presence of cholesterol [Figure 4]. Form these results, we can hypothesis that addition of sesamin and β-sitosterol inhibits the growth of Mtb bacilli leading to accumulation of toxic intermediate product, i.e., choletst-4-en-3-one which cannot be degraded further due to inhibition of CYP125 by sesamin and β-sitosterol.
| Conclusion|| |
This study shows the usefulness of computational approach for screening effective inhibitors from large dataset of phytochemicals against CYP125 enzyme, a novel drug target of Mtb. Further,in vitro experiments employed in this study confirmed sesamin and β-sitosterol as safe and significant inhibitors for Mtb growth when subjected to grow on cholesterol. These results suggest that these phytochemicals can be used as anti-TB drug supplements.
This study was partially supported by Tropical Disease Research grant from Kasturba Health Society, Sevagram, Wardha, Maharashtra, India. The authors would like to thank the Department of Biotechnology, Ministry of Science and Technology, Government of India for financial support to Bioinformatics Centre wherein the in silico study has been carried out. The authors convey thanks to Shri Dhiru S. Mehta, President, KHS, for his keen interest and encouragement.
Financial support and sponsorship
This study was partially supported by Tropical Disease Research grant from Kasturba Health Society, Sevagram, Wardha, Maharashtra, India. The authors would like to thank the Department of Biotechnology, Ministry of Science and Technology, Government of India for financial support to Bioinformatics Centre wherein the in silico study has been carried out.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]