Molecular Dynamic Simulations and Binding Free Energy Evaluations of Thiazolo-[2,3-b] Quinazolinone Derivatives With wtEGFR-TKD and "TMLR" Mutant EGFR-TKD

Cancer causes innumerable deaths every year globally. Breast cancer and non-small cell lung carcinoma (NSCLC) are the most prevalent worldwide. The epidermal growth factor receptor tyrosine kinases play a pivotal role in manifestations of cellular signals in carcinoma cells and thus are endorsed as therapeutic targets in cancer management. EGFR-TKD is a good option for the treatment of cancer, but the resistance shown by rst-generation TKIs leads to hyperphosphorylation, overexpression, and mutations of EGFR-TKD. The new molecular scaffolds of thiazolo-[2,3-b] quinazolinones were evaluated against EGFR-TKD via molecular docking simulations and thereby linked with molecular dynamic simulations to identify the ligand stability with EGFR-TKD’s. The binding energy of thiazolo-[2,3-b] quinazolinones is approximately similar to that of reference molecules found against both wild type wtEGFR-TKD and EGFR-TKD mutant "TMLR" (T790M/L858R) in this study. According to ADMET analysis, thiazolo-[2,3-b] quinazolinone derivatives (5ab, 5aq, and 5bq) are safe. The stability was investigated at an atomistic level by molecular dynamic simulations, and the strength of the bindings was calculated by the molecular mechanics Poisson-Boltzmann surface areas continuum solvation MM-PBSA method. The RMSD, radius of gyration, and SASA trajectories were studied in detail. The ΔGbind generated by heterocyclic 5aq thiazolo-[2,3-b] quinazolinone was found to be -63.723 ± 0.419 kJ/mol against the EGFR-TKD mutant "TMLR" and it was -51.551 ± 0.409 kJ/mol against wtEGFR-TKD. This study concludes that the top ranked complexes 5ab, 5aq, and 5bq with both wild and mutated types of EGFR-TKD corroborate and z =-24.769. All the amino acids present in the binding site are covered in a grid box and form a binding site for molecular docking. The Autogrid module of AutoDock4 is used to build maps of atoms present in a ligand. The parameter search genetic algorithm is selected. 10 docking poses are opted for every ligand against both proteins (wtEGFR-TKD and EGFR-TKD mutant "TMLR"). The Lamarckian GA module is used to generate nal docking poses by Autodock4. Each docking conformation is analysed by free energy binding scores. These scores are calculated on the basis of the sum of the distances between atom pairs. of cancer cells. The stability and binding energy of thiazolo-[2,3-b] quinazolinone derivatives towards both wtEGFR-TKD and EGFR-TKD mutant "TMLR" are calculated by molecular dynamics simulation and MMPBSA (g-mmpbsa) methods. According to MD simulations, the thiazolo-[2,3-b] quinazolinone 5ab, 5aq, and 5bq derivatives are stable in the binding cavity of wtEGFR-TKD and EGFR-TKD mutant "TMLR". The free energy binding of 5ab and 5aq was much lower than that of 5bq against wtEGFR-TKD, whereas 5aq showed the lowest negative free energy binding against the EGFR-TKD mutant "TMLR", which was investigated by the MM-PBSA method. According to predictions, thiazolo-[2,3-b] quinazolinone has ADMET properties and is non-toxic, but both reference ligands AQ44 and 4ZQ are hepatotoxic and are inhibitors of the human Ether-go-to-go gene (HerG-II). Furthermore, we conclude that the selected lead candidates of thiazolo-[2,3-b] quinazolinone derivatives may have EGFR-TKD inhibitory potency. This study will help to design the most potent inhibitor against wtEGFR-TKD and EGFR-TKD mutant "TMLR". Also, these molecular scaffolds will be taken to In vitro and In vivo levels for future study of interest.

towards tyrosine kinase inhibitors (TKIs). This resistance blocks the kinase inhibitor's ability to bind with EGFR-TKD 13 . Overall, mutations by nonspeci c drugs towards EGFR-TKD are a major concern in the present scenario. The resistance by marketed drugs, as well as their lethality, are major concerns in conventional therapeutics 14,15 .
Therefore, the urge for novel effective therapeutic anticancer drugs is a remarkable challenge worthy of attention in cutting-edge technology 13,16 . Previous research has demonstrated that quinazolinones are EGFR-TKD inhibitors, and it has been proposed that the development of new heterocyclic thiazolo- [2,3-b] quinazolinones could be used as an EGFR-TKD inhibitor In this study we aimed to explore the molecular interactions via molecular docking simulations, molecular dynamic simulations and free energy binding (FEB) of newly synthesised heterocyclic thiazolo- [2,3-b] quinazolinones library against wild type EGFR-TKD (wtEGFR) and EGFR-TKD mutant "TMLR".

Material And Methods
System preparation and protein-ligand docking The synthesised congeners 5aa-5aq, 5ba-5bq and reference ligands of wtEGFR-TKD (AQ44) and EGFR-TKD mutant "TMLR" (4ZQ) were sketched by using Avogadro software 17  www.rscb.org 20, 11 . The resolution of wtEGFR-TKD 1M17 is 2.60 Å and total weight is 38.27 kDa and EGFR-TKD mutant "TMLR" 5CAS is 2.10 Å and total weight of protein is 38.13 kDa. Both protein structures were minimized by using Gromacs 5.1.1 for 10ps and dumped in PDB format after minimizations. The binding site of both proteins is de ned by using Argus Lab software 21 . The reference ligand (AQ44) of 1M17 was selected and made into a ligand group. The ligand group is selected and module "make a binding group" is used to identify the amino acids present in the binding site of the EGFR-TKD (1M17). A similar procedure was applied to the EGFR-TKD mutant "TMLR" and the amino acids present in the binding site were identi ed. These amino acids will be selected in a grid box for molecular docking simulations. AutoDock4 22 is used for estimation of the inhibition constant, ligand e ciency, and binding energy. The proteins were prepared by deleting water molecules, incorporating polar hydrogens, adding a Kollam charge. The library of thiazolo- [2,3-b] quinazolinones was prepared by selecting a torsion tree, adjusting the number of torsions, and saving them in pdbqt format. The grid dimensions of 1M17 at centre are 62 × 46 × 58 (x, y, z) and grid spacing resolutions are adjusted at 0.358, Å, grid box centre x = 25.517, y = 0.052 and z = 54.719.
The 5CAS grid dimensions at the centre are 54 × 42× 70 (x, y, z), and the grid spacing resolution is set to 0.358, with grid box centre x =-51.385, y = 1.059, and z =-24.769. All the amino acids present in the binding site are covered in a grid box and form a binding site for molecular docking. The Autogrid module of AutoDock4 is used to build maps of atoms present in a ligand. The parameter search genetic algorithm is selected. 10 docking poses are opted for every ligand against both proteins (wtEGFR-TKD and EGFR-TKD mutant "TMLR"). The Lamarckian GA module is used to generate nal docking poses by Autodock4. Each docking conformation is analysed by free energy binding scores. These scores are calculated on the basis of the sum of the distances between atom pairs.
where, epair(d)=w1*Gauss1(d)+w2*Gauss2(d)+w3*Repulsion(d)+w4*Hydrophobic(d)+w5*HBond(d) Molecular dynamic simulations: The molecular dynamics simulations were carried out using Gromacs 5.1.1 23,24 . Ligand topologies are generated by using https://cgenff.umaryland.edu [25][26][27] . The charmm36 force eld and recommended water model TIP 3-point are used 28,29 . Both Ligand and protein topologies les are incorporated in a single gromacs le. Gradually, this complex le is placed in the simulation box, which is 1 nm apart from the walls of the periodic boundary and solvates the box 30 . The prepared system is neutralized by using Na + and Cl. The Steepest decent method is used for energy minimization. The temperature was kept at 300K and the pressure at 1 bar. Berendsen coupling of pressure and V-rescale coupling temperature is used. The Particle Mesh Ewald (PME) algorithm is used. The shortrange distance cut-off and coulomb cut-off are kept at 1.2 nm. The LINICS algorithm is used to constrain the bond length 31 for 2 fs. Both NVT and NPT methods are used to equilibrate the system for 100 ps 32 . 8 production runs of 20ns simulations are carried out by applying a time step of 2 fs.

Free Energy Calculations:
Free energy binding is calculated by using g_mmpbsa. Three input trajectory les (trr or xtc), a topology parameter le (tpr), an index le (ndx), and an mdp python script for mmpbsa calculations are obtained from https://github.com/RashmiKumari/g_mmpbsa. The above-mentioned input les are obtained after completion of molecular dynamic simulation 33 .
The binding free energy of wtEGFR-TKD and EGFR-TKD mutant "TMLR" is calculated by the given equation.
ΔGbinding = G complex -(G protein + G ligand ) (II). G complex denotes the total free energy of the protein-ligand complex, whereas the isolated total free energy of protein and ligand are represented as G protein and G ligand in the above given equation.
The free energy of an individual entity is summed up by the equation given below: The protein ligand complex is denoted by X, and E MM denotes an average molecular mechanics potential energy in vacuum, as calculated by equation (IV). G solvation denotes the free energy of solvation, and TS denotes the entropic free energy contribution in vacuum, while T denotes temperature and S entropy. The pkCSM 34 Molsoft L.L.C 35 and SwissADME 36 servers are used to calculate toxicity, druglikeness and pharmacokinetic properties of thiazolo- [2,3-b] quinazolinones and reference molecules of wild type EGFR-TKD (AQ44) and EGFR-TKD mutant "TMLR" (4ZQ). Overall, these servers predict the toxic fragments of molecular structure in a very robust manner. The scrutiny of the molecular scaffolds is calculated on the basis of a weighed drug index and pharmacokinetic properties that are very useful in drug discovery. First, universal SMILES are obtained from Open Babel 37 then these SMILES are used as inputs to obtain druglikeness, toxicity, and pharmacokinetics.

Results And Discussions:
Generally, many kinase inhibitors bind directly with the EGFR-TKD and inhibit the function of p-glycoprotein in malignant cells 38 . The synthesized quinazolinone based rhodamines are potent anti-cancerous agents 39 . Imidazole's and benzimidazoles are the functional precursors of polyazaheterocycles that are synthesized with the aid of microwave-mediated multi domino reactions MDRs 40,41 . New thiazolo [2,3-b] quinazolinone derivatives 5ab, 5aq and 5bq ( Figure 1) are synthesized by microwave-assisted organic synthesis (MAOS) and multi-domino reaction (MDR). The thiazolo [2,3-b] quinazolinones have ADMET properties. Both the MDR and MAOS methods are cost effective, e cient and have remarkable structural attributes for anticancer activity. These molecular structures contain pyrimidine, cyclohexanone, and thiazole, which have a broader range of pharmaceutical properties. Quinazolinone has been synthesized by the substitution of phenyl and methyl groups over the benzene ring, which shows cytotoxic activity 39 . The thiazolo [2,3-b] quinazolinone derivatives share structural identity with the reference ligands of wtEGFR-TKD and EGFR-TKD mutant "TMLR", which contain a substituted pyrimidine moiety showing anticancer activities.

Molecular docking simulations:
The wild type EGFR-TKD and EGFR-TKD mutant "TMLR" are selected for in silico investigations. The PDB ID of wild type is noted as 1M17 and EGFR-TKD mutant "TMLR" as 5CAS. The thiazolo-[2,3-b] quinazolinone library is synthesized, and ADMET studies show that the derivatives 5ab, 5aq, and 5bq are not toxic as per predictions (Table   SI-SIII). Furthermore, the comparative in silico investigations of three selected thiazolo-[2,3-b] quinazolinone derivatives (5ab, 5aq, 5bq) and reference ligands (AQ44 and 4ZQ) of wild type and EGFR-TKD mutant "TMLR" were  Also, amino acid Lys745 interacts with thiazolo [2,3-b] quinazolinone derivatives (5ab, 5aq, and 5bq) and reference ligand (4ZQ) via an aryl substituted functional group with a binding cavity. Among these three derivatives, 5ab exhibits interactions with the N atom of LYS745 and ASP 855 ( Figure 3). Similar interactions are found in 5ad and 5bq and the interactions are showed with ASP855 by the OH group anked by cyclohexanone and pyrimidine rings at position 9a. The reference (4ZQ) ligand of the EGFR-TKD mutant "TMLR" showed interactions with the N atoms MET793. Besides these interactions, THR854 also interacts with the N atom of the pyrimidine ring during molecular docking simulations. The free binding energy, ligand e ciency, and inhibition constant of thiazolo [2,3-b] quinazolinone derivatives against wtEGFR-TKD are given in (Table I) and for EGFR-TKD mutant "TMLR" in (Table II). The mean energy of binding is obtained in triplicates. The highest negative binding energy is found in 5aq =-11.97 kcal/mol. towards the EGFR-TKD mutant "TMLR". The results of molecular docking revealed that the selected thiazolo [2,3-b] quinazolinone derivatives may be the inhibitors of both wtEGFR-TKD and EGFR-TKD mutant "TMLR". Further molecular docking is linked with molecular dynamic simulations and MM-PBSA calculations for conformations of ligand movement, root mean square uctuations, and free energy binding at an atomistic level against both wild type and EGFR-TKD mutant "TMLR". After completion of molecular docking simulations, protein-ligand complexes were introduced to obtain interactions exploited by thiazolo-[2,3-b] quinazolinone and reference ligands (AQ44, 4ZQ) with the catalytic site of wild and EGFR-TKD mutant "TMLR" by using MOE v2009 [18][19] . All the molecular interactions exploited are presented in Figure   2  The ligand conformations When 5ab, 5aq, 5bq, and reference ligand (AQ44) are superimposed into the respective ligand, the RMSD is calculated (Figure 4b). The RMSD of 5ab is 0.04 -0.13 nm, 5aq is 0.03 -0.12 nm, and 5bq is 0.03 -0.1 nm during simulations, and the RMSD of reference ligand (AQ44) is between 0.05 to 0.2 nm during simulation period. The 1M17 receptor Cα RMSD is analysed when simulated with 5ab, 5aq, 5bq and AQ44 The exibility of amino acids is determined by root mean square uctuations and is corroborated that the exibility of amino acids is higher in the EGFR-TKD mutant "TMLR" than in wtEGFR-TKD 45,46 . The RMSF of wtEGFR-TKD is is 0.07 -1nm at the N terminus, 0.07 -0.4 nm at the C terminus, and the remaining amino acid uctuations are 0.07 -2.2 nm. The present ndings support previous results 45,46 . The exibility of various amino acids in the EGFR-TKD mutant "TMLR" is higher than in wtEGFR-TKD.
The Rg and SASA values re ected during simulations of EGFR-TKD mutant "TMLR" (5CAS) are found in the range of 2 -2.08 nm and the SASA value of the protein lies within 157 -175 nm2 when simulated with the 5ab derivative.
When simulated with 5aq, the Rg of the main chain is found to be 2-2.12 nm, and the SASA of the protein is 155- The radius of gyration analysis shows EGFR-TKD mutant "TMLR" has more variations in protein stability in comparison to wtEGFR-TKD. These ndings agree with previously reported protein stability of wtEGFR-TKD and mutated EGFR-TKD 46 .

Hydrogen bond analysis:
The hydrogen bond module of gromacs is used to obtain the number of hydrogen bonds formed between protein and ligand. The hydrogen bond analysis among thiazolo [2,3-b] quinazolinone derivatives and reference ligands of wtEGFR-TKD and EGFR-TKD mutant "TMLR" during MD simulations has been determined. The hydrogen bond formed by thiazolo [2,3b] quinazolinone derivative 5ab with wtEGFR-TKD is 1, 5aq, 5bq derivative and reference ligand AQ44 formed 2 hydrogen bonds each (Figure 6a-d). EGFR-TKD mutant "TMLR" 5ab and 5aq derivative formed 4 hydrogen bonds, 5bq derivative and reference ligand (4ZQ) formed 2 hydrogen bonds, which is higher than the wtEGFR-TKD complexes (Figure 7a Figure 8), but 5bq also has a high negative free binding energy and they are parallel with AQ44 reference ligand.   All thiazolo-[2,3-b] quinazolinone derivatives (5ab, 5aq, and 5bq), AQ44, and 4ZQ against wtEGFR-TKD and EGFR-TKD mutant "TMLR" are selected for the contribution of free energy decomposition, which is calculated by using the MmPbSaDecomp.py python script. The decomposition of contribution energy of each amino acid during free energy calculations is determined. Residues of wtEGFR-TKD and EGFR-TKD mutant "TMLR" contribute to free energy decomposition. The decomposition free energy contribution of the wtEGFR-TKD-5ab complex is found in amino acids, Leu694 = -2.1811, Val720 = -5.7595, Ile720 = -2.0354 and Leu820 = -4.3941. In the 5aq-wtEGFR-TKD complex, free energy contributions are found in amino acids. Lys693 = -4.8364, Val720 = -4.5307 and Leu768 = -2.5720 amino acids. 5bq-wtEGFR-TKD complex free energy contributions are found in Leu694 = -6.0328, Val702 = Contribution free energy decomposition leads to the conclusion that the 5aq derivative of thiazolo- [2,3-b] quinazolinone has many similarities with the reference ligand (4ZQ). Average negative free energy binding calculations help to nd the most potent molecule against wtEGFR-TKD and TMLR-EGFR-TKD therapeutic targets.
Despite the free energy contribution energy by amino acids of wild type and mutated type tyrosine kinases during calculations, the selected ligands also contribute towards free energy decomposition. The reference ligand AQ44 shows lower negative free energy contributions than 5ab, 5aq, and 5bq, but 5aq derivative shows a slight difference of-4 kJ/mol than AQ44. In the case of the EGFR-TKD mutant "TMLR", the 5aq derivative shows higher negative decomposition than the reference ligand 4ZQ. These conclusive results suggest that 5bq, a derivative of thiazolo- [2,3-b] quinazolinone, may also be a potent anti-cancer agent and may inhibit the functioning of both wild and EGFR-TKD mutant "TMLR".

ADMET predictions:
The pkCSM, SWISSadme, and Molsoft L.L.C. were used to investigate the toxicity, druglikeness of thiazolo [2,3-b] quinazolinones and reference ligands of both wtEGFR-TKD and MT EGFR-TKD. The only three 5ab,5aq and 5bq are found safe but reference ligands AQ44 and 4ZQ are inhibitors of HerG-II and show hepatotoxicity as per predictions. All the molecules taken in this study did not show AMES toxicity. Furthermore, none of the molecules violated the Lipski rule of 5. The druglikeness index of 5ab = 0.66, 5aq = 0.37, 5bq = 0.62, 4ZQ = 0.79 and AQ44 = 0.90. Higher druglikeness was reported in AQ44 and 4ZQ because they hold toxic fragments as per predictions.
Thereafter, the ADMET analysis suggests toxic scaffolds have higher druglikeness than non-toxic drug candidates (Table SI-SIII). Experimental ndings supported predicted results that erlotinib (AQ44) is a hepatoxic molecule 46 . We present non-toxic thiazolo-[2,3-b] quinazolinone derivatives synthesised using Insilico methods, which will be further evaluated by in vivo methods.

Conclusion
The goal of this research is to nd the best inhibitor from thiazolo-[2,3-b] quinazolinone derivatives against wtEGFR-TKD and EGFR-TKD mutants "TMLR". Preliminary research and deep machine learning data suggest that thiazolo-[2,3-b] quinazolinone derivatives could be the next generation anti-cancer drugs. Widely designed EGFR-TKD inhibitors were studied, and some of them are available on the market. These inhibitors have certain complications, and their speci city towards a target is still not known. We therefore introduced two chemotherapeutic targets (wtEGFR-TKD and EGFR-TKD mutant "TMLR") as they are present in a wide range of cancer cells. The stability and binding energy of thiazolo-[2,3-b] quinazolinone derivatives towards both wtEGFR-TKD and EGFR-TKD mutant "TMLR" are calculated by molecular dynamics simulation and MMPBSA (g-mmpbsa) methods. According to MD simulations, the thiazolo-[2,3-b] quinazolinone 5ab, 5aq, and 5bq derivatives are stable in the binding cavity of wtEGFR-TKD and EGFR-TKD mutant "TMLR". The free energy binding of 5ab and 5aq was much lower than that of 5bq against wtEGFR-TKD, whereas 5aq showed the lowest negative free energy binding against the EGFR-TKD mutant "TMLR", which was investigated by the MM-PBSA method. According to predictions, thiazolo-[2,3-b] quinazolinone has ADMET properties and is non-toxic, but both reference ligands AQ44 and 4ZQ are hepatotoxic and are inhibitors of the human Ether-go-to-go gene (HerG-II). Furthermore, we conclude that the selected lead candidates of thiazolo-[2,3-b] quinazolinone derivatives may have EGFR-TKD inhibitory potency. This study will help to design the most potent inhibitor against wtEGFR-TKD and EGFR-TKD mutant "TMLR". Also, these molecular scaffolds will be taken to In vitro and In vivo levels for future study of interest.

Data And Software Availability:
The data mentioned in this article will be available upon the author's request.    Molecular interactions exploited by thiazolo [2,3-b] quinazolinones (5ab, 5aq and 5bq) and reference ligand 4ZQ with catalytic site of EGFR-TKD mutant "TMLR".    The binding energy of thiazolo-[2,3-b] quinazolinone derivatives with wtEGFR-TKD complexes is mentioned in upper along with reference ligand complex and binding energy of thiazolo-[2,3-b] quinazolinone derivatives with EGFR-TKD mutant "TMLR" complexes were given in below panel, the color representations were given as 5ab black, 5aq red, 5bq green and reference ligands blue. The contribution by amino acids in free energy decomposition were plotted, left row represents wtEGFR-TKD and right row EGFR-TKD mutant "TMLR"