Structure based virtual screening, molecular docking and molecular dynamics simulation of drug like small molecules against cell cycle regulator enzyme FtsZ of Mycobacterium tuberculosis


 Tuberculosis is a major public health problem in several developing and low income countries. It warranted 1.2 million deaths around the world in 2019. An active Tb patient can spread a large number of bacilli to the environment by coughing, spitting, speaking and sneezing which in turn help in development of new Tb cases. Further development of drug resistant strains of Mycobacterium tuberculosis to most powerful first line drugs viz., rifampicin and isoniazid put a hurdle in Tb elimination programme. Thus it is essential to explore new drug molecules to fight against Tb morbidity and mortality. In the present study an attempt has been made to screen new small drug like molecules using structure based molecular screening. 3D structure of cell cycle regulator enzyme FtsZ (PDB ID: 1RLU) was retrieved from the Protein Data Bank website and used it for structure-based virtual screening of drug like small molecules from PubChem database. The screened drugs were re-docked with FtsZ of M. tuberculosis to find out their affinity and antagonistic effect against M. tuberculosis. Molecular dynamics simulation studies were carried out for each of the docked complex to find out the stability and flexibility of the docked conformations. In silico ADME study was carried out to find out the drug likeliness of the selected molecules. A total of 1501 drug like small molecules were recovered through virtual screening of which three molecules, viz., Lig-24284406, Lig-49671233 and Lig-24791139 were taken for the docking study. It was observed that the selected drug molecules had a strong affinity towards the FtsZ. This was evident from the binding energy of the drug molecules towards FtsZ (-7.23, -9.67 and -5.51). The strong stability and flexibility of the docked structures were also evident from RMSD values i.e, < 2 Å and RMSF value i.e, > 1 Å for all the docking complexes. The present study suggested that three small drug like molecules could be taken for further clinical studies to find their efficacy against tuberculosis.


Introduction
Tuberculosis (Tb) is an infectious disease caused by Mycobacterium tuberculosis an acid fast bacterium of family Mycobacteriaceae. Despite of effective multi drug therapy Tb remains a major public health problem worldwide. Mainly the disease affects lungs and known as pulmonary Tb but it can affect other parts of the body like bone, brain, skin, intestine etc. and is known as extra-pulmonary Tb. According to global tuberculosis report 2019 an estimated 10 million people were suffered from tuberculosis and an estimated 1.2 million Tb deaths were reported among HIV negative patients in 2018 (WHO 2020). Two third of the total TB cases were reported from eight countries viz., India (27%), China (9%), Indonesia (8%), Philippines (6%), Pakistan (6%), Nigeria (4%), Bangladesh (4%) and South Africa (3%) (WHO 2020). Tb is spread from an active Tb patient to a healthy person through air. A large number of bacilli were spread in the air by coughing, spitting, speaking and sneezing of an active Tb patient.
People with latent Tb do not spread Tb. Treatment of Tb requires a long time period with multiple antibiotics. Although Tb is curable disease but development and spread of drug resistance Tb viz., MDR (Multi Drug Resistant) and XDR (Extensive Drug Resistant) strains are major hurdle to achieve the goal of Tb elimination (Fox et al. 2017). Multi drug therapy for Tb therapy implemented to avoid complications, increase the cure rate, decrease the mortality rate, preventing recurrences and also to restrict the dissemination of drug resistant strains of M.
tuberculosis (AlMatar et al. 2017). The development of resistance to most powerful Tb drug viz., rifampicin and isoniazid and horizontal transfer of MDR strains in the community (Anderson et al. 2014;Seung et al. 2015) put an obstacle in Tb elimination programme. Thus new antibiotics having pharmacological action against M. tuberculosis should be screened to treat both drug susceptible and resistant strains of M. tuberculosis to achieve the goal of elimination. Thus in this study FtsZ, a cell cycle regulator enzyme has been used as drug target to screen small molecule using structure based drug screening.
FtsZ is an essential gene for in vitro growth of M. tuberculosis (Griffin et al. 2011;DeJesus et al. 2017). FtsZ is involved in Z ring formation and the initiation of cell division (Bi, and Lutkenhaus 1991). FtsZ possesses many unique biochemical features, which include slow polymerisation kinetics, presence of charged amino acids in the C-terminal domain that interacts with a variety of other cell division proteins (Gupta et al. 2016). FtsZ is the most abundant cell division protein and plays an essential role in bacteria cytokinesis. The initiation of FtsZ polymerization requires GTP. The polymerization takes place in inner membrane at middle of the dividing cell and forwards bi directionally to form a highly dynamic helical structure known as the Z-ring that encircles the cell. Following recruitment of the other cell division proteins, Z-ring contracts to form septum (Huang et al. 2007).
Looking to the essential function of FtsZ protein in cell cycle regulation, an attempt has been made to screen small lead like molecules from PubChem small molecule database using 3D structure of FtsZ protein of M. tuberculosis and selected molecules were docked into the FtsZ and validated through molecular dynamics simulation analysis. The finding of this study might be able to provide a new paradigm for alternative regimen against MDR and XDR tuberculosis.

Retrieval of FtsZ and domain analysis
The holoenzyme of FtsZ was retrieved from the protein data bank (PDB) under the accession number 1RLU (Leung et al. 2004). The crystal structure of FtsZ consisted two identical subunits, A and B. The enzyme is composed of αβ-tubulin dimer (Nogales et al. 1999) and microtubules (Li et al. 2007). Structure editing was done using Discovery Studio Visualizer.
All the chains, hetero atoms and water were removed except chain 'A' containing the catalytic centre of the enzyme. The cleaned structure of FtsZ was saved in .pdb format for further processing. The protein family, superfamily and arrangement of domains were elucidated using InterProScan (Zdobnov and Apweiler 2001). Conserved domains of the FtsZ were investigated using the databases such as Pfam (Finn et al. 2014) andCDD (MarchlerBauer et al. 2009).

Preparation of protein, ligand, docking and visualization
3D conformers of ligands having lowest binding energy in the screening process were downloaded from PubChem using PubChem compound CID as sdf format. In this study three small molecules viz., N,N-diethyl-6-methyl-2-oxo-1,3-dihydrobenzimidazole-5-sulfonamide

Molecular dynamics (MD) simulation of rpoB-resistomycin complex
Molecular dynamics (MD) simulation is a principal tool for proofing the stability of proteinligand complex. MD simulation provides the detailed information on the fluctuations and conformational changes of proteins-ligand complex towards its stable conformation. Thus in the present study MD simulation was applied to understand the stability of FtsZ-ligand binding complexes. Files required for MD simulations were prepared using Visual Molecular Dynamics (VMD) v 1.9.3. Initially co-ordinates of FtsZ and ligands were written in .pdb format using save coordinates option of the VMD (Humphrey et al. 1996). The FtsZ was parameterized using automatic psf builder. For parameterization the topology parameter "top_all27_prot_lipid_na.inp was used". For parameterization of ligands ligand reader and modeller of CHARMM force filed was used (Jo et al. 2008) at Charmm Gui web site (http://www.charmm-gui.org/?doc=input). Then individual psf and pdb files of FtsZ and ligands were generated using TCL script of the VMD. The complexes were then solvated using TIP3P water box with 3.0 Å of water in each direction of the coordinate structure. Integrator parameters were included as 2 fs/step for all rigid bonds and nonbonded frequencies at 2.5 Å and electrostatic evaluations for 3 Å with ten steps for each cycle. Periodic boundary conditions and PME were adjusted using maxmin TCL script. Water constraints were eliminated by minimizing preliminary protein energy via 1000 steps of the Powel algorithm at a constant temperature of 310 K. Simulations were run for 200,000 runs for 2 ns Langevin dynamics to control the kinetic energy, temperature, and/or pressure of the system without a binding pocket, and the applying parameters do not change the protein conformation during the ligand unbinding (Feller et al. 1995;Pradeepkiran et al. 2015). MD simulation results were analyzed thoroughly for each of the FtsZ-ligand complexes and results were analyzed using VMD.
Root mean square deviation (RMSD) and root men square fluctuation (RMSF) were calculated to address the stability of atoms in the docked structures. The force field description of the interatomic forces reflects two important categories viz., the bonded terms and the non-bonded terms. The bonded terms are further regroup as simple covalent binding as well as the more complex hybridization and π-orbital effects, these are the bonds, angles, dihedrals and improper-dihedrals terms. Thus the bonding terms bonds, angles, dihedrals and improperdihedrals were plotted using NAMD plots.

Drug likeness of the small molecules
It is essential to predict ADME (Absorption, Distribution, Metabolism, and Excretion) properties of lead like small molecule to assess their drug likeliness. It reduces the probability of failure of a molecule as a drug candidate in the first and last stem of drug discovery (Kant et al. 2019). The selected small molecules as inhibitors of FtsZ of Mtb were analyzed for physicochemical and pharmacokinetics properties using the SwissADME web server (Daina et al. 2017).

Retrieval of rpoB and domain analysis
The unambiguous length of the FtsZ of M. tuberculosis is 379 amino acids long (Figure 1).

Docking and visualization
Molecule 24284406 Docking between FtsZ and 24284406 showed a strong binding pattern (Figure 2a Table 2). The backbones of protein-ligand structure were found to be stable in all the three MD trajectories. The average RMSF of residues contributing to the complex structure in each of the docked conformations were calculated as > 1.43 Å ( Figure 6).

Principal Component Analysis
Principal component analysis (

Drug likeness of the small molecules
SWISS-ADME was used to assess the drug likeness and pharmacokinetics properties of the molecules. In the SWISS-ADME analysis all the three molecules showed reasonable amount of drug likeliness and pharmacokinetics properties. Slight deviation in molecular weight was observed in case of compound 49671233 and 24791139. The results were shown in table 3.

Discussion
TB is a major health problem in developing and lower income countries worldwide. TB is one of the top 10 causes of death worldwide and the leading cause of death from a single infectious agent (WHO 2020). Nearly one-fourth of the world population is infected with TB. WHO set the goal to end TB by 2035 in a phased manner. In the first phase the target was set to reduce 35% of total TB cases by 2020, 75% reduction by 2025 in the second phase, 90% reduction by 2030 in the third phase, and 95% reduction by 2035 in the fourth phase (WHO, 2018).
Occurrence of drug resistant TB including MDR and XDR-TB are a major concern for the end TB strategy. Drug-resistant TB continues to be a public health threat and put obstacles in achieving eradication of the disease worldwide. In 2019 nearly half a million (465,000 in absolute number) people were infested with rifampicin resistant TB (RR-TB), of which 78% (362,700) had multidrug resistant TB (MDR-TB). In 2019, 3.3% of total new TB cases and 17.7% of previously treated cases had MDR/RR-TB (WHO, 2020). An effecting control of infectious disease needs either a good vaccine or regimens those can control the disease effectively. A number of new TB vaccines including genetically modified mycobacteria, mycobacterial antigens with or without adjuvant are in clinical trials phase. BCG is the only vaccine approved for TB and given just after the birth. According to some study BCG vaccine provides incomplete and variable protection against pulmonary TB (Dockrell and Smith 2017; Barreto et al. 2006;Arbelaez et al. 2000, Van-Dunem et al. 2015. Thus looking to the magnitude of the TB problem worldwide discovery of new cost effective, direct effect and having less side effect drugs is an important target for TB elimination programme. Thus in the present study an attempt has been made to screen lead like small molecules against FtsZ, a cell cycle regulation protein Mtb using structure based drug screening. In the present study, a 3D model of FtsZ protein of Mtb was downloaded from protein databank and had been used for virtual screening of potential inhibitors from diverse-lib implemented in MTiOpenScreen server (Labbe et al. 2018).
From the molecular interaction study, it was observed that the selected drug molecules viz., 24284406, 49671233 and 24791139 had strong affinities towards the FtsZ. This was evident from the free binding energy, bonding pattern and types of bonds involved in ligand and FtsZ interaction. The molecular interaction study showed a strong binding association exists between FtsZ and interacting drug molecules through hydrogen bonds, van der Waals interaction, Pi-Sigma bonds, alkyl bonds, Pi-alkyl interactions, halogen bond and Pi-lone pair interactions. Hydrogen bonds are major player for negative free energy change and contribute to the stability of protein-ligand binding interface complex (Fu et al. 2018).
Hydrogen bonds are also known to enhance the protein ligand interaction at the binding interface (Chen et al. 2016). In this study we observed 7 conventional hydrogen bonds and 2 carbon hydrogen bonds for FtsZ-24284406; 5 conventional hydrogen bonds and 2 carbon hydrogen bonds for FtsZ-49671233 and four conventional hydrogen bonds for FtsZ-24791139 docking complexes. These interactions showed a strong association and stabilization of FtsZligand interface.
Though van der Waals interactions are weaker than covalent and electrostatic bonding but this bond in large is responsible for free energy change and made the major contribution to the total binding energies (Sood et al. 2018;Mohanty et al. 2018;Keretsu et al. 2019). These interactions are known to stabilize the binding interface structures through intermolecular energy minimization Arthur and Uzairu 2019). In this study we observed a large number of van der Waals interactions stabilizing the FtsZ and interacted small molecule docking complex. We observed 9, 12 and 14 van der Waals interactions between FtsZ and 24284406, 49671233 and 24791139 docked structures respectively. Thus we summarised that these van der Waals interactions were largely contributed to the negative free energy change and stabilize the FtsZ and each lead like molecule docked interface.
Other FtsZ-49671233 and FtsZ-24791139 respectively. These findings were also supported by the cosine content value of each PCA coordinates, i.e., less than 6 for all three docking complexes (Maisuradze et al. 2007).
It is an essential to predict absorption, distribution, metabolism, excretion and toxicity of any lead like molecule to understand the action, behaviour and bioavailability of the molecules before entering into a clinical experimentation. In this study SWISS-ADME was employed to all the proposed molecules to assess the toxicity and bioavailability of the lead like molecules.
We found that all the three molecules passed the ADME test could be used for clinical experimentation against M.tb.
The molecular interaction study showed that a strong binding association exists between modelled protein FtsZ and interacting lead molecules. The molecular interaction patterns between the drug molecules and one of the binding site residues (Gly-18, Gly-19, Asn-22, Gly-105 and Thr-106) of FtsZ might interrupt protein function.