Molecular docking study on quercetin derivatives as inhibitors of PantothenateSynthetase (PanC) of Mycobacterium tuberculosis

The anti-TB drugs currently in the use are insuf(cid:977)icient to address these major health challenges. Hence, it is imperative to discover and develop new and ef(cid:977)icient drugs against TB. The enzyme pantothenate synthetase (PS or PanC), necessary for the production of pantothenate (vitamin B5), critical compo-nents of fatty acid synthesis, when inhibited will in turn affect the cell wall synthesis of bacilli. In the present study, an attempt will be made to (cid:977)ind the drug like molecules from quercetin derivatives prepared in silico to (cid:977)ind out possible inhibitors of PanCof M. tuberculosis . The 3D structure of PanC was obtained from RCSB database and quercetin from ZINC database. The derivatives of quercetin were prepared and were docked initially with iGEMDOCK docking tool. The (cid:977)inal docking was done in AutoDock vina software. The ADMET properties of the selected ligands were done in admetSAR online server tool. The present study revealed that four derivatives of quercetin has excellent binding with Pantothenate Synthetase (PanC) of M. tuberculosis. These derivatives can be taken for in vitro enzymatic assays for its inhibitory property in the search for new anti-TB drugs


INTRODUCTION
The tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the important bacterial infections that lead to millions of deaths all around the world (KJ et al., 2004). The TB is seen high in many developing countries where, with AIDS and immunocompromised diseases are common. The current treatment for TB has a prolonged course of antibiotics, which normally associated with poor patient compliance (Tomioka and Namba, 2006). This has now let to emergence of multi-drug resistant (MDR) and extensively drug resistant (XDR) strains of M. tuberculosis (WHO, 2014). In 2009, a new strain of "Totally drug resistant Tuberculosis" (TDR) emerged in some parts of the world (Velayati et al., 2009). These TDR strains showed invitro resistance to all irst-and second-line drugs. In 2012, the irst TDR strain was reported in India (Udwadia et al., 2012).
The anti-TB drugs currently in the use are insuficient to address these major health challenges. Hence, it is imperative to discover and develop new and ef icient drugs against TB (Zumla et al., 2013). The characteristics feature of M. tuberculosis is its lipid rich cell wall, which helps the bacteria of its intracellular survival and its pathogenicity. The cell wall synthesis pathway is a promising target for new anti-TB drug discovery (Jackson et al., 2013;Cole et al., 1998). The enzyme pantothenate synthetase (PS or PanC), is a key enzyme needed for the biosynthesis of coenzyme A (CoA). It produces pantothenate (vitamin B5), which is aprecursor for the biosynthesis of CoA. The CoA is a critical component of fatty acid synthesis (Abrahams et al., 2012). If this enzyme is inhibited, the fatty acid synthesis of M. tuberculosis will be affected which in turn will affect the cell wall synthesis (Sambandamurthy et al., 2002).
It is an established fact that the phytochemicals present in the plants are important source for the discovery and development of anti-microbial drugs (Zandi et al., 2009(Zandi et al., , 2011Chiu et al., 2012). Medicinal Plants contains naturally occurring phytochemicals (Calixto, 2000). Medicinal plants are rich in many types of phytochemicals like polyphenols and lavonoids proven to have many medical properties with drug like properties (Jassim and Naji, 2003). Quercetin, a lavonoid present in many medicinal plants, has been proved to have many medicinal properties and drug likeliness (Parasuraman et al., 2016).
Molecular docking is the insilico technique that looks for the best drug like molecules that can it in the binding site of the drug targets and thus helps in drug development for various diseases (Rajamani and Good, 2007). The irst in silico aided drug that came commercially is dorzolamide in 1996 (Kubinyi, 1999). Since then many drugs are developed successfully by this method and are being used as the treatment for various diseases including cancer.In the present study, an attempt will be made to ind the drug like molecules from quercetin derivatives prepared in silico to ind out possible inhibitors of Pantothenate Synthetase (PanC) of M. tuberculosis.

Target Protein preparation
The three-dimensional structure of Pantothenate Synthetase (PanC) (Figure 1) of M. tuberculosiswas retrieved from RCSB-PDB data base (www.rcsb.org )and was saved in the .pdb format. Its PBD code is 1N2H.

Active site prediction
As there is no three-dimensional structure of protein-ligand complex in the RCSB database for PanC, the 3DLIGANDSITE online server tool was used to determine the possible binding sites. The protein was uploaded in the server which gave various binding sites in the order of ranking (Wass et al., 2010). The binding site which was ranked irst was chosen for the study.

Generation and optimization of Ligand
The structure of quercetin was obtained from ZINC database ( Figure 2). Its zinc ID is ZINC-33980813. Its IUPAC NAME is (2S)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-2H-chromene-3,4-dione with a molecular weight of 302.2357 g/mol. The ACD chemsketch software tool was used to prepare the derivatives of quercetin. The quercetin was opened by the software and its side chains were modi ied to prepare the derivatives. The central ring structure was not changed during the preparation of the derivatives. The derivatives were saved in .mol format. Later, the derivatives are converted to .pdb format in OpenBabel software.    The rapid docking of the derivatives with the protein was performed in softwarei GEMDOCK v2.0 (Yang and Chen, 2004). Under docking option, the rough docking was selected with used a population size of 150 with 70 generation. After docking the binding energy and binding pose of each derivatives were analysed. The best derivatives in terms of binding energy and pose were further taken for docking in Autodock vina.

Protein-ligand docking
The docking of ligands was performed using AutoDockvina software using PyRx as GUI (Morris et al., 2009). First the protein was loaded in software and it was prepared for docking. During this process, the protein will be converted from .pdb format to .pdbqt format, a format needed for AutoDockvina for docking. Similarly, the derivatives (ligands) were also loaded and was converted to .pdbqt format. Before docking, the grid box was placed around the binding site so as enable the ligands binds only inside the grid box ie., in the binding site. After the grid box was set, the docking simulations were run for all the ligands. The AutoDock vina calculates the energy values using the Lamarckian Genetic Algorithm (LGA) algorithm. A total of eight binding con igurations was generated for each ligand and were arranged according to their root mean square deviation (RMSD) values. The binding con irmation ranked one was selected as the best in terms of binding energy and pose.

Visualization
The docking pose of Quercetin derivatives were visualized and analysed using software pymol software.

ADMET properties
The adsorption, distribution, metabolism, excretion and toxicity of the derivatives were analysed by admetSAR -2.0 server tool.
The docking poses of the derivatives with the PanC is shown in Figure 3. All selected ligands were shown to it into the binding pocket of the drug target.
The ADMET properties the ligands were analysed byadmetSAR server tool. All the selected four derivatives satis ied important properties (Table 3).
The treatment and management of drug sensitive TB is successful and brought down the TB cases under control. However, the continuous emergence of TDR and XDR TB poses threat and challenge in the management of TB caused by drug resistant M. tuber-  (Caminero, 2010). The discovery of streptomycin and subsequent use of it in the treatment of TB is not successful as the bacilli rapidly developed drug resistance (Council, 1948). This led to the strategy of developing combination therapy using at least two active drugs in the treatment of TB (Medical Research Council Investigation , 1952). Soon the multidrug-resistant (MDR) TB started to emerge wherein the TB bacilli showed resistant to isoniazid (INH) and rifampin (Zignol et al., 2006). Further, some MDR bacilli has been found to be resistant for any luoroquinolone and to at least one of three injectable agents (kanamycin, amikacin, or capreomycin) which are categorised as extensively drugresistant TB (XDR-TB).
After the drug resistance was identi ied, the underling mechanisms were explored and was found to be due to spontaneous mutations in the gene of drug targets and failure of binding of drug to the binding site of drug target (Miotto et al., 2015). Hence, vari-ous studies are being done to explore new drug targets and discovering potential drug that can bind to them (Abrahams et al., 2012). In the present study, the enzyme pantothenate synthetase has been used as the drug target to derive drug like molecules that can bind and inhibit the activity of the enzyme thereby can kill or inhibit the TB bacilli.
Few similar studies with other drug targets were done. In a study conducted by (Shilpi et al., 2015), it was found that ellagic acid derivatives from Ludwigiaadscendens and Trewianudi lora showed excellent binding ability with MabA of M. tuberculosis.

CONCLUSIONS
The molecular docking technique has paved way to discovery new drug like molecules for the treatment of TB. In the present study, the quercetin derivatives were prepared and subjected to the molecular docking technique in the quest to ind anti-TB drug like molecules. The present study revealed that four derivatives of quercetin has excellent binding with Pantothenate Synthetase (PanC) of M. tuberculosis. These derivatives can be further taken for in vitro enzymatic assays for its inhibitory property in the search for new anti-TB drugs.