In Silico Study for Acyclovir and Its Derivatives Against Mpro of nCoV: Temperature Dependent Molecular Dynamics Simulations

In the present work three molecules acyclovir (A) and ganciclovir (B) are reported to cure the infection from herpes virus and others. We have designed a derivative of hydroxymethyl derivative of ganciclovir (CH 2 OH of G, that is D) and investigated its potential against the Mpro of nCoV and compared with A & B. Density functional theory (DFT) calculations of A, G and D were performed using Gaussian on applying B3LYP under default condition to investigate the delocalization of electron density in their optimized geometry. Free energy of A, G and D were found to be -810.66964, -925.16575 and -1039.66047 Hartree per particle. It can be clearly seen that D have least free energy. Further, the molecular docking of the A, B and D against the Mpro of nCoV performed using iGemdock and the binding energy for A, B and D are found to be -105.068, -110.605 and -119.226 kcal/mol. It can be clearly seen the D showed effective binding, that is maximum inhibition. For better understanding for the inhibition of the Mpro of nCoV by A, B and D, molecular dynamics simulations were performed at different temperatures, (290, 300, 310 and 320 K). Various trajectories like RMSD, RMSF, Rg and hydrogen bond were extracted and analyzed. The results of molecular docking corroborate the MD simulations that is, a better inhibitor of Mpro of nCoV.


Introduction
Drug repurposing is the concept of utilising an FDA-approved drug for a new problem or illness other the one for which it was originally approved. These drugs save time and money, accelerated their admittance into experimental clinical trials. The process involved the activity based on experimental or computational approach to develop the new employ of drug [1][2][3].
COVID-19 can cause range of illness from common cold to severe respiratory syndrome. Coronaviruses (CoV) are a family of encapsulated viruses with single-strained RNA and pathogen. In compare to previously identi ed SARS-CoV (2002) and MERS-CoV (2013), SARS-CoV-2 is a more virulent variant [4][5][6][7]. New SARS-CoV has recently received attention worldwide and has been declared as a public health emergency of global concern. Acyclovir is a well-known antiviral drug mostly used for the treatment of the infections due to herpes group viruse and zoster virus. A clinical research report has been published, showing the acyclovir as promising drug against the infection due to SARS-CoV-2 [8]. Heidary et al. has done the study of on acyclovir as potent molecule in COVID-19 regimens [9]. Acyclo-GTP is more e cient inhibitor of viral DNA. Ganciclovir and acyclovir have good e cacy against cytomegalovirus infection [10]. A case report had showed that ganciclovir may act as an effective antiviral drug, even against SARS-CoV-2 [11].
Molecular dynamics (MD) simulations in drug development played an important role. The behaviour of proteins and other biomolecules is captured in atomic resolution in presence or absence of small molecules. A khan et al. has performed MD simulation of anti-HIV drugs as a potent inhibitor against SARS-CoV-2 main protease [12] The electronic structure of atoms, molecules and solid can be investigated using density-functional theory (DFT) calculations. The binding a nity between the protease and ligands can be determined using iGEMDOCK 2.1 tool but it provides a preliminary information. It is used to investigate the favorable site of receptor for an effective binding of ligand [13]. There is a possibility of vander Waals interaction, hydrogen bonding between the protease and ligand. Our research group has already provided preliminary information about the docking of acyclovir and its derivatives with Mpro of nCoV [14]. A research group has done the molecular docking of acyclovir and its derivative against the main protease of SARS-CoV-2 and their binding energy are -89.64 and -96.21 kcal/mol, respectively [15]. ADME is used to know the suitability of a molecule as potent drug. ADME is used to lter the compounds to get the leading molecule from high throughput screening (HTS) campaigns.
In this work, acyclovir (A), ganciclovir, (G) and hydroxymethyl derivative of ganciclovir (D) are taken for study to inhibit the activity of main protease (Mpro) of novel corona virus (CoV) using molecular docking. Further to make the information reliable and acceptable, molecular dynamics (MD) simulations of Mpro of nCoV with designed molecules at different temperature. Structures (acyclovir, ganciclovir and hydroxymethyl-ganciclovir) are drawn using chemdraw as in Figure 1.

Molecular Docking
The structure of Mpro of nCoV is downloaded from RCSB (PDB:6LU7). Before performing molecular docking, Mpro of nCoV is prepared using chimera. Structure of Mpro of nCoV was opened in chimera followed by removal of ligand and atoms were deleted and the structure was saved in PDB format [16][17][18]. All three ligands structures were optimized by applying MM2 force eld on chemdraw 3D before molecular docking. The docking was performed using iGEMDOCK 2.1 and gives binding energy in kcal/mol [19].

DFT Calculation
DFT calculation of A, G and D were performed by using Gaussian 16.0 and Gaussview 6 as an interface [20,21]. The parameter applied to perform calculation are optimization plus frequency with B3LYP and the basis set was 6-311G at 298 K.

Molecular dynamics (MD) simulations
Temperature dependent MD simulation of Mpro of nCoV with A, G and D were performed at 290, 300, 310 and 320 K by using online the web server WebGro (https://simlab.uams.edu/) and it uses GROMACS to perform the calculations [22][23][24][25][26]. Input parameter used are as ; force eld GROMACS9643a, box type triclinic, water model is SPC and salt type is NaCl. Equilibration and MD run parameter used pressure (1bar), number of frames per simulation 1000 and simulation time (100ns). The GlycoBioChem PRODRG2 is an online server was used (http://davapc1.bioch.dundee.ac.uk/cgi-bin/prodrg) used to create topology of small molecules [27].

Absorption, Distribution, Metabolism and Excretion (ADME) of A, G and D
A molecule may be a drug if it has the ability to reach its target location with su cient concentration and active in the body for the timespan of biological response. In this work, a free web tool swissADME (http://www.swissadme.ch) has been used to predict the different physiochemical and pharmacokinetics property of A, G and D.   [29]. Higher the energy of HOMO indicate more the ability to donate electron density. HUMO, LUMO and optimized geometry of A, G and D are given in Figure 3.

Result And
Further, different thermodynamics parameter such as zero-point Energy, thermal energy, Optimization energy and thermal enthalpy are calculated and no signi cant changes is found as in Table 3. Free energies are an important parameter for deciding the stability of compound. It is considered that lesser the free energy of molecule indicates higher stability. Among A, G and D, D has the least free energy -1039.7090 (Hartree per particle) and considered to more stable. Dipole moment is also an important parameter for predicting the solubility in aqueous medium. Table 3 shows that G has the highest dipole moment of 10.4241a.u. in all. Various physiochemical descriptors were also determined for above reported three molecules by DFT calculation as given  Table 4. Energy gap of HOMO and LUMO is a useful parameter for determining the reactivity of compound. The HOMO and LUMO energy are directly proportional to the ionization potential and electron a nity.

Molecular dynamics simulations
Molecular dynamics simulations is used to analyze the dynamic of macromolecular system. It uses the trajectories obtained from MD simulations and gives useful information to investigate the protein-ligand interactions [30,31]. MD simulations main protease of nCoV with of A, G and D were performed for 100 ns at different temperature. Root mean square deviation is the measure of the average of square root of deviation from the mean distance [32,33]. It provides the conformational stability of macromolecular system due to binding of ligand into binding cavity. RMSD values ranges between the 0.2 nm to 0.5 nm. It was also found that on increasing temperature the RMSD value increases. Lower temperature favors stabilization of protein-ligand complex. During simulations, no loop was seen which indicates less deviation due to binding of the ligands Figure 4.
Root mean square uctuation is used to study the uctuation values of the atomic coordinates of the atoms of amino acids [34]. Fluctuations values were used to analyze the con gurational changes in the presences of ligand at different temperature.
RMSF graphs for the protein with A, G and D were analyzed and given the Figure 5. Radius of gyration is the measure of the distance between center of mass and axis of rotation. It is used to study the stability of protein in presence of small molecules. Lesser the value of radius of gyration indicates higher the stability [35,36]. Radius of gyration values were analyzed for the main protease of nCoV in presence of A, G and D at different temperature (Figure 6). At low temperature, radius of gyration is less meaning more stability. But an increasing temperature value of Rg increases and thus the stability decreases. Results of molecular docking corroborate the results obtained by MD simulations.
Molecular dynamic simulation analysis used to examine their of Mpro of nCoV in presence of A, G and D over time span at various temperature. Conventional hydrogen bonding is more signi cant and formed between uorine, oxygen and nitrogen with hydrogen. The anchoring is tighter as the number of hydrogen bond increases and the length of hydrogen bond decreases [37,38]. The number of hydrogen bonds and their persistency were analyzed during simulation at different temperatures as given in Figure 7. Maximum number of hydrogen bonds were found 6 in case of acyclovir while in case of derivative and ganciclovir maximum hydrogen bonds are ve.
3.4 Absorption, Distribution, Metabolism and Excretion (ADME) of A, G and D ADME characteristics is as important in the drug development process [39]. ADME drug characteristics such as absorption, distribution, metabolism and elimination are critical to a drug candidate's clinical success. It is estimated that about 50% of the drugs fails due to insu cient effectiveness which includes low bioavailability due to inadequate intestinal absorption and poor metabolic stability [40][41][42][43]. Lipophilicity is one of the most important criteria to predict the molecule as a drug and it is a partition coe cient of n-octanol and water (log P O/W ). It determines the amount of solute dissolves in the water v/s organic solvent in a solution [44]. The partition coe cient is a signi cant indicator of a substance physical constitution and determines its behavior in various situation. For effective drug, the value of log P O/W is less than or equal to 5 [45]. Solubility is an important criterion for deciding a molecule to be drug, therefore, log S should be less than 6. According to Lipinski's rule of ve an orally active drug must have: (i) hydrogen bond donor less than or equal to ve (ii) hydrogen bond acceptor less than or equal to ten (iii) molecular mass less than 500g/mol (iv) log P values less than 5 [46-48]. Table 5 shows the physiochemical properties of A, G and D. All the studied molecules obey the Lipinski rule of 5. Physiochemical space for oral bioavailability Drug likeness is an important criterion for evaluating behavior of molecule during early stage of drug development. This factor may be regarded as a way to link physicochemical properties of a compound to its biopharmaceutical properties in the human body for oral delivery. Despite the fact that there are several methods for delivery of oral dosages is favored for patient comfort and compliance. At various stages of the discovery process, early assessment of oral bioavailability i.e. the percentage of the dosage that reaches the blood following oral administration, is a signi cant decision-making factor. Bioavailability is in uenced by a number of factors but gastrointestinal absorption is most important. The BBB may be regarded as a shield that protects the brain through a "physical" barrier and a "biochemical" barrier (e.g. P glycoprotein pumping out substrate from CNS tissues). Blood-brain barrier is a system that ensure the consistency of central nervous system environment by allowing brain tissues to interchange nutrient with the outside world. Effective BBB penetration is important for targeting CNS illness, but non-CNS drugs should have restricted BBB penetration due to side effect [49]. Despite the importance of active transport, passive diffusion is the primary route for drug to reach the brain from the blood. The method for assessing active e ux through biological membrane such as from the gastrointestinal wall to the lumen or from the brain, requires knowledge of compounds that are substrate or non-substrate of the permeability glycoprotein. P-gp has a number of functions, one of which is to protect the central nervous system from xenobiotics. TPSA is a key characteristic for drug likeness and its value must be smaller than 130 Å. TPSA is considered to be a useful indicator of a compounds ability to transport drug. In Table 6 bioactivity and drug likeness score of A, G and D are given. A shows high GI absorption, TPSA value 119.06 Å and following Lipinski rule with zero violation [50,51]. G and D have TPSA value more than 130 Å but they followed the Lipinski rule with zero violation. using Gaussian 16 to see the localization of electron density and stability. Result shows that D has minimum free energy of -1039.7090 Hartree per particle. Further, ADME properties of A, G and D were calculated using swissADME to evaluate solubility, bioavailability and potential of molecule to become effective drug. It has been found that A, G and D followed the Lipinski rule with no violation and falls in acceptable range for their bioavailability.

Declarations
Funding There is source of nancial support to perform the work.
Disclosure of potential con icts of interest and informed consent: I further con rm that the order of authors listed in the manuscript has been approved by all of us. The authors have relevant nancial and non-nancial interests to disclose.
Availability of data and material: "The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request."     Trajectory of RMSD t to backbone for the Mpro of nCoV with A, G and D at 290, 300, 310 and 320 K.  Trajectory of radius of gyration for the Mpro of nCoV with A, G and D at 290, 300, 310 and 320 K.