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Long-range replica exchange molecular dynamics guided drug repurposing against tyrosine kinase PtkA of Mycobacterium tuberculosis

Tuberculosis (TB) is a leading cause of death worldwide and its impact has intensified due to the emergence of multi drug-resistant (MDR) and extensively drug-resistant (XDR) TB strains. Protein phosphorylation plays a vital role in the virulence of Mycobacterium tuberculosis (M.tb) mediated by protein kinases. Protein tyrosine phosphatase A (MptpA) undergoes phosphorylation by a unique tyrosine-specific kinase, protein tyrosine kinase A (PtkA), identified in the M.tb genome. PtkA phosphorylates PtpA on the tyrosine residues at positions 128 and 129, thereby increasing PtpA activity and promoting pathogenicity of MptpA. In the present study, we performed an extensive investigation of the conformational behavior of the intrinsically disordered domain (IDD) of PtkA using replica exchange molecular dynamics simulations. Long-term molecular dynamics (MD) simulations were performed to elucidate the role of IDD on the catalytic activity of kinase core domain (KCD) of PtkA. This was followed by identification of the probable inhibitors of PtkA using drug repurposing to block the PtpA-PtkA interaction. The inhibitory role of IDD on KCD has already been established; however, various analyses conducted in the present study showed that IDDPtkA had a greater inhibitory effect on the catalytic activity of KCDPtkA in the presence of the drugs esculin and inosine pranobex. The binding of drugs to PtkA resulted in formation of stable complexes, indicating that these two drugs are potentially useful as inhibitors of M.tb.

engulfed by alveolar macrophages into phagosomes. In principle, fusion of lysosomes and phagosomes leads to the elimination of foreign particles; however, M.tb has evolved mechanisms that prevent the fusion of phagosomes and lysosomes, thereby promoting its survival inside the alveolar macrophages 3 . M.tb possesses various signal transduction mechanisms that include eleven two-component systems, eleven eukaryotic-like Ser/Thr protein kinases (PknA-PknL) 4 , two protein tyrosine (Tyr) phosphatases (PtpA and PtpB) and a recently discovered protein tyrosine kinase (PtkA) 5,6 . These signaling molecules block the host defense pathway and allow the bacteria to adapt and survive within the host macrophages 7 . M.tb PtpA binds to the H subunit of the vacuolar H + -ATPase (V-ATPase) and dephosphorylates the host macrophage protein VPS33B, which prevents phagosome-lysosome fusion and phagosome acidification 8,9 . PtkA, which is encoded within the same operon as PtpA, mediates the phosphorylation of PtpA on Tyr128 and Tyr129 and enhances the activity of PtpA, which suggests a central role for PtkA in M.tb virulence 8,10 .
Structural studies of PtkA (30.6KDa; 291 amino acids (AA)) have shown that it contains two domains, an intrinsically disordered domain (IDD) and a kinase core domain (KCD). The IDD is the N-terminal domain, IDD PtkA (80AA) 11 and the KCD is the C-terminal domain KCD PtkA (216 AA). The KCD PtkA contains a catalytic loop (D 85 LD motif), lysine residues (Lys184, Lys217, and Lys270) for ATP binding, and an autophosphorylation site at Tyr262. The catalytic site of PtkA resides on the KCD, and its activity depends upon its interaction with the IDD. The IDD inhibits the activity of PtkA, as it masks the auto phosphorylation site on KCD PtkA . PtkA shows two conformational states-an open state conformation in which IDD is away from Tyr262 (auto phosphorylation site), making it accessible for phosphorylation and thereby increasing the activity of PtkA; and a closed state, in which IDD masks the Tyr262, thereby decreasing the activity of PtkA. Phosphorylation of IDD by Ser/Thr kinases (PknA) induces conformational changes in IDD, which move IDD away from the autophosphorylation site and promotes an open state of PtkA 12 .
The catalytic activity of KCD PtkA alone is greater than the catalytic activity of KCD PtkA measured along with IDD PtkA , which suggests an inhibitory role of IDD PtkA for the catalytic activity of PtkA 12 . Here, we present a rigorous study in which we have performed Replica Exchange Molecular Dynamics (REMD) to investigate the structural conformers of the disordered domain IDD PtkA. Further we performed protein-protein docking of IDD PtkA -KCD PtkA to analyse the impact of IDD on the catalytic activity of PtkA via study of IDD PtkA -KCD PtkA interactions and through modification of the KCD region involved in catalysis. In the subsequent steps, molecular docking and simulations were performed with FDA-approved drugs against PtkA (IDD PtkA -KCD PtkA ) to identify potential M.tb PtkA inhibitors.

Results and Discussion
iDD ptkA modelling and validation. The structure of IDD of PtkA was modelled using the I-TASSER server 13 . The highly significant structural templates used in the modeling of the IDD protein domain through I-TASSER are listed in Table 1. As is evident from the good coverage and high Z score (>1 stands for a good alignment) in case of most of the templates, the generated threading alignment pointed towards a good and confident model. The quality of the modelled structures was validated through Protein Server Validation Suite (PSVS) 14 and the highest quality model was selected on the basis of Ramachandran analysis of ψ/ϕ angle from PROCHECK. The plot showed a coverage of 61.7% of the residues in the most favored region, 26.7% of the residues in additionally allowed regions, and with a stretch of about 3.3% residues in disallowed regions. This indicated that, overall, 94% of the residues were in allowed regions, showing that the quality of the protein model was suitable for further study.
conformational analysis of iDD ptkA . The binding of IDD to KCD in PtkA has been shown to play an inhibitory role and to decrease the catalytic activity of KCD 12 . In a recent study, the conformation of IDD PtkA has been characterized under varying biophysical conditions and phosphorylation using NMR-spectroscopy and it was confirmed that N-terminal domain of PtkA exists as an unstructured state 12 . REMD simulations was conducted on IDD PtkA to examine the conformational behavior of the peptide at different temperatures. A total of 28 replicas were generated in the temperature range of 300-500 K, and REMD analysis was conducted for a replica at 300 K. Conformational analysis of IDD provided insight into the unstable and disordered structure of IDD. Since IDD is an intrinsically disordered protein, the root mean square deviation (RMSD) plot exhibited large www.nature.com/scientificreports www.nature.com/scientificreports/ fluctuations. However the fluctuations lied within a range of 0.7-1.3 nm throughout the simulations, thus indicating the characteristic flexible nature of IDD as well as convergence of the simulations. Since the protein was stable throughout the simulations period; the protein was considered for further analysis. Figure 1 depicts the plot of (A) RMSD and (B) secondary structure elements in IDD PtkA. The secondary structure analysis of IDD PtkA revealed that throughout the entire simulations period (50 ns), IDD acquired multiple conformations; these were mostly random coils, along with short stretches of unstable α-Helices/β-Sheets. Snapshots of IDD taken every 2.5 ns (Fig. 2) during the 50 ns MD simulations evidently speak about this conclusion that the protein tyrosine kinase A (PtkA) has an 80-residue intrinsically disordered N-terminal region. The coil percentage in IDD was 54%, whereas the α-Helices and β-Sheets were present at a mere 3% and 1%, respectively. This indicated that no persistent secondary structure elements were present in IDD PtkA .
inhibitory activity of iDD ptkA on KcD ptkA . Since the inhibitory effect of IDD on KCD has already been verified 12 , protein-protein docking and MD simulations of IDD PtkA -KCD PtkA were conducted to explore the  www.nature.com/scientificreports www.nature.com/scientificreports/ hydrophobic and hydrogen bond interaction patterns between the two domains. The top-scoring cluster having a HADDOCK score of −58.6+/− 9.0 kcal/mol and Z-score equivalent to −1.2 was considered for MD simulations. The top cluster is considered to represent the most reliable cluster on the basis of Z-score which indicates the magnitude of standard deviations between the given cluster and the average of all other clusters (the more negative the better). RMSD, root mean square fluctuation (RMSF), radius of gyration (Rg), and solvent accessible surface area (SASA) analysis of PtkA in comparison to PtkA-drug complexes during MD simulations is described later in the manuscript. The residues His23, Gln24, Ser26, Arg27, Ser38, Gly39, Arg43, and Thr54 of IDD formed hydrogen bonds with the KCD residues Asp116, Thr119, Asp240, Trp260, Gly261, Tyr262, Asp266, Ile268, and Ser272. Among these were residues that have been reported to form interactions between IDD and KCD that include Glu114 and Trp260; the other active site and surrounding residues formed hydrophobic interactions. This pointed out that IDD is important for regulating the catalytic activity KCD and thus reducing the efficiency of PtkA. Figure 3 illustrates the hydrogen bonding pattern and the hydrophobic interactions between IDD and KCD.
Identification of lead compounds against PtkA. In order to identify drugs that can be repurposed as agents against M.tb PtkA, we performed molecular docking studies using the FDA-approved drugs as ligands. Initially, high throughput virtual screening (HTVS) docking was performed, followed by extra precision (XP) docking using the top scorers of HTVS (having docking score <−5.0). The two top scoring drugs, esculin (DrugBank ID: 13155) and inosine pranobex (DrugBank ID: 13156), had Glide scores equivalent to −8.44 kcal/ mol and −8.82 kcal/mol, respectively. Esculin had a Glide energy of −70.99 kcal/mol and inosine pranobex had a Glide energy of −67.97 kcal/mol. Esculin formed one hydrogen bond each with Val122 and Ser209, while inosine pranobex formed four hydrogen bonds with residues Ile21, Lys184, and Ser209.
We also conducted docking of both drugs with KCD PtkA without IDD PtkA to clarify the role of IDD PtkA in preventing the catalytic activity of PtkA. The docking score of KCD PtkA -inosine pranobex was −6.11 kcal/mol and that of KCD PtkA -esculin was −4.59 kcal/mol. The lower docking scores in the case of KCD without IDD suggested a strong binding of drugs in the presence of IDD, indicating a significant role of IDD in the structure of KCD. The docking scores, hydrogen bonding, and hydrophobic interactions of PtkA (IDD-KCD) with esculin and inosine pranobex and with PtkA (KCD) are provided in Table 2.
elucidation of the mechanism of binding of drugs to ptkA. In order to gain insights into the thermodynamic behavior and to further examine the mechanism of drug binding and its impact on PtkA, the docked protein-ligand complexes were subjected to 50 ns MD simulations. The RMSD plots of PtkA, PtkA-esculin, and PtkA-inosine pranobex showed that the protein and protein-drug complexes were stable during the simulations and were therefore suitable for further analysis (Fig. 4(A)). The Rg graph showed a decrease in the overall Rg value of the protein structure docked with drugs, indicating that PtkA in complex with drugs was in a compactly packed state and had stable folding (Fig. 4(B)). The SASA calculation suggested that the exposure of the protein surface to the solvent and the changes in SASA could lead to conformational changes.   Table 3. The values indicate that inosine pranobex binding had an overall more stabilizing effect on PtkA when compared to esculin.      (Fig. 6).
The flexibility of the protein was analyzed to a larger extent by calculating the trace value for diagonalized covariance matrix, which is a matrix of Eigen vectors and diagonal Eigen values. Figure 7 shows the diagonalized covariance matrix of PtkA, PtkA-esculin, and PtkA-inosine pranobex. The trace values, which are the sums of the Eigen values, were 74.10, 66.96, and 47.43 nm 2 for PtkA, PtkA-esculin, and PtkA-inosine pranobex, respectively. Therefore, unbound PtkA appeared to cover a larger conformational space due to its greater flexibility when compared with the protein-drug complexes. free energy analysis. Gibbs free energy landscape (FEL) plot for unbound protein (PtkA) and protein-drug complexes (PtkA-esculin and PtkA-inosine pranobex) was generated using PC1 and PC2 coordinates. The ΔG values for PtkA, PtkA-inosine pranobex and PtkA-esculin were 12.5, 13.8 and 14 kJ/mol respectively. The blue, cyan and green regions in the plot signify low energy state with highly stable protein conformation while the red region denotes high energy conformation. A smaller and more concentrated blue minimal energy area in PtkA-inosine pranobex and PtkA-esculin indicates a highly stable complex as compared to PtkA alone (Fig. 8).

Secondary structure analysis. The secondary structure elements included rigid (α-Helices and β-Sheets)
conformations and flexible (coils and turns) conformations. The unbound PtkA was conquered by coil (28%) which decreased to 26% and 27% in case of PtkA-inosine pranobex and PtkA-esculin respectively (Table 4). An increase in β-Sheet and turn was observed in case of PtkA-inosine pranobex complex favouring stability in the presence of inosine pranobex. Furthermore Table 5 provides the secondary structure elements adopted by   www.nature.com/scientificreports www.nature.com/scientificreports/ the active site residues of unbound PtkA and PtkA-drug complexes. An increase in rigid structural elements at active site residues was observed in case of PtkA-inosine pranobex. Most of the active site residues retained their conformation in case of PtkA-esculin except Lys270 which changed its conformation from coil to turn favouring a stable complex.    www.nature.com/scientificreports www.nature.com/scientificreports/ conclusion Protein phosphorylation and dephosphorylation at tyrosine residues have a significant role in the survival of M.tb. PtpA and PtkA are the two protein tyrosine phosphatases secreted by M.tb. PtpA is an essential protein for M.tb pathogenesis, as it prevents phagosome acidification and plays a significant role in virulence. The structure of PtpA shows a high similarity to that of human protein phosphatase; thus, it cannot be considered a suitable target for drug development. The phosphatase activity of M.tb PtpA is regulated by PtkA, which is composed of a disordered IDD domain and a rigid catalytic KCD domain. PtkA phosphorylates Tyr128 and Tyr129 of PtpA, thereby enhancing its activity, and it has also recently been reported to increase the growth of M.tb in macrophages. Thus, M.tb PtkA can be a suitable potential target for drug development against TB. The structure analysis of PtkA showed that the disordered domain, IDD, modulates the arrangement of the helices involved in the catalytic activity of KCD, thereby showing a significant role for IDD. We examined the molecular mechanism behind the role of PtkA in regulation of phosphorylation by M.tb. PtpA by performing an extensive computational study on PtkA that included modelling, REMD, MD, and molecular docking. Since the structure for IDD was not available, it was modelled and validated for quality assessment and subjected to REMD. Notably, IDD was largely composed of random coils that transiently bind to the substrate binding site of KCD. The docking and MD of IDD and KCD domain, performed to look at the inhibitory effect of IDD on KCD, revealed strong bonding between IDD and KCD.

PtkAesculin
Potential inhibitors against PtkA (IDD-KCD) were identified using molecular docking and resulted in two top scoring drugs, esculin and inosine pranobex. Various analyses, RMSD, RMSF, Rg, SASA, and PCA, done using MD trajectories indicated that IDD, in the presence of the drugs, had a greater inhibitory effect on the catalytic activity of KCD. The results also revealed that PtkA was highly stable in the drug-bound form, when compared to unbounded PtkA. This study proposes the repurposing of two FDA approved drugs, esculin and inosine pranobex, for use as agents against PtkA and consequent inhibition of the growth of M.tb.

Methodology
Homology modeling of IDD ptkA and quality assessment. The sequence of IDD PtkA (N-terminus) of M.tb was retrieved from UniProt-KB database [UniProt ID: P9WPI9] 15 . Since the crystal structure of IDD was not available, the structure was modeled on the basis of sequence homology through I-TASSER server 13 . I-TASSER has regularly been the top ranked method in CASP (Critical Assessment of Protein Structure Prediction) method (Moult et al. 2014). I-TASSER initiates modelling by identifying template proteins from PDB library. The models are then constructed by continuous assembling of the aligned templates and initiating ab initio folding for the unaligned regions based on replica exchange Monte Carlo simulations. These simulations generate an ensemble of conformations which are further clustered on the basis of free energy. Further lowest energy structures are subjected to refinement resulting in final structural model 16 . Structure quality assessment was performed by PSVS 14 through quality scores and constraint analysis programs. The modelled protein structure was subjected to 50 ns REMD simulations and the stable conformation was used for further analysis.

Replica exchange molecular dynamics (ReMD) simulations. REMD is a molecular simulations
method that enhances the sampling efficiency. A set of replicas in a canonical ensemble are simulated at different temperatures, and at particular intervals, the replicas are swapped with a transition probability such that each independent system maintains a canonical ensemble. These exchanges allow the molecule to jump out of the minima and sample efficiently on the conformational space 17 . It has been reported that the 80-residue N-terminal domain of PtkA protein is an intrinsically disordered region which lacks a stable structure and goes through conformational exchange on changing the environmental settings such as temperature, chemical denaturant and phosphorylation 12 . In the present study, to explore the conformations of IDD PtkA , REMD was performed at varying temperatures ranging from 300-500 K.
REMD simulations of IDD were performed using GROMACS version 5.0 18 with GROMOS 43a1 force field. The peptide was provided with periodic boundary conditions (PBC) and placed in a cubic box filled with a single point charge (SPC) 19 water model containing 710 water molecules. Na and Cl ions were added to neutralize the overall charge on the system. A total of 28 replicas were swapped in a temperature range of 300K-500K (300.00, 306. 23 Docking and molecular dynamics simulations study of iDD ptkA -KcD ptkA . The X-ray crystal structure of KCD was obtained from a protein data bank (PDB ID: 6F2X). The IDD and KCD of PtkA were docked using the HADDOCK 21 web server. The server performs data-driven flexible docking, in which it integrates the experimental data (X-ray and NMR) as restraints and uses these to implement docking, in addition to a combination of energetics and shape complementarity. An AIR (ambiguous interaction restraints) file which consist of active and passive residues at the interaction interface for each molecule based on NMR data, was supplied to the server. Active residues are the residues that are experimentally determined to be involved in the interaction between the two molecules. For the current study the active residues involved in the interaction between IDD and KCD has been obtained from experimental data 12 . The active site of IDD included residues Ala10, Ser41, and Cys61, and the KCD active site consisted the residues Glu114, Gly134, Asp162, Thr188, Ile192, Ile205, Gly212, Leu231, Met237, Trp260, and Ile282. The docked complex (IDD PtkA -KCD PtkA ) was simulated under the dynamic behavior of molecular systems as a function of time using GROMACS version 5.0 with the GROMOS 43a1 force field. The complex was provided with PBC and placed in a cubic box filled with the SPC water model. The energy of the system was minimized until the system attained the maximum force <1000.0 kJ/mol/nm to ensure no steric clashes on the system. The system was first equilibrated under an isothermal-isochoric ensemble (NVT, constant Number of particles, volume, and temperature) using a Berendsen thermostat (a v-rescale) applied for temperature coupling for a total of 50000 steps. This was followed by equilibration under an isothermal-isobaric ensemble (NPT, constant number of particles, pressure, and temperature) using Parrinello-Rahman applied for pressure coupling, again for 50000 steps. The two phases of equilibration stabilized the system at 300 K and 1 bar pressure, and the system was subjected to MD simulations for 50 ns. The LINCS constraints algorithm was applied to fix the lengths of the peptide bonds and angles 22 . The MD trajectory files were analyzed using various inbuilt scripts of GROMACS.
Molecular docking of ptkA (iDD-KcD) and MD simulations. The complete protein structure of M.tb PtkA was then used for the molecular docking study to identify potential inhibitors of PtkA. The docking studies were conducted using the Schrodinger suite. Prior to the docking, the target protein was pre-processed by adding missing hydrogen atoms, correcting bond orders, and capping protein termini using the Protein Preparation Wizard 23 . A library of 2355 FDA approved drugs was obtained from the DrugBank 24 database and LigPrep 23 was used to prepare the chemical structures of the drugs. This resulted in energy-minimized, chemically broad, and structurally diverse 3D structures of the drug molecules. The Receptor grid generation panel was used to generate a grid around the active residues (Asp85, Leu86, Asp87, Tyr146, Tyr150, Lys184, Lys217, Tyr262 and Lys270) of KCD. The docking was performed in two steps: HTVS docking and XP, using the Glide module of Schrodinger 25 . The large chemical library was first screened using the HTVS approach. A rapid screening of the ligands was executed, followed by the XP method, which implements a rigorous screening of the top-scoring HTVS ligands and results in compounds having the best binding mode to the receptor. The top-scoring protein-ligand complexes were subjected to 50 ns MD simulations using GROMACS 5.0. The webserver PROGRG 26 was used to generate GROMACS topologies for the ligands.
Molecular dynamics simulations analysis. The MD simulations runs performed in the present study were analyzed using various GROMACS tools. RMSD was calculated using g_rmsd to study the convergence of the simulations. Rg of the protein, which is a measure of the protein folding and compactness, was calculated using g_ gyrate 27 . RMSF for deviation in the position of atoms was calculated using g_rmsf. The SASA for the protein, which gives an idea of the area of the residues exposed to the surface, was calculated using g_sasa. We also performed essential dynamics or PCA, which gives a view of the large-scale combined motion of atoms, thereby revealing the behavior of the protein structure underlying the atomic variations. The initial step in PCA is to construct a covariance matrix (g_covar), which captures the linear relationship of atomic fluctuations for individual atomic pairs. This is followed by diagonalization of this matrix, which results in a matrix of eigenvectors and eigenvalues. Eigenvectors determine the movement of atoms and eigenvalues yielded by diagonalization represent the extent that an atom participates in motion. The Eigen vectors were analyzed using the g_anaeig tool. Further FEL was calculated using g_sham module and the secondary structures of PtkA and PtkA-drug complexes were analysed using gmx_dssp. LigPlot 28 was used to explore the hydrogen and hydrophobic interactions, and Chimera 29 was used to create the images depicting the interactions.