Uridine-Derived 4-Aminophenyl 1-Thioglucosides: DFT Optimized FMO, ADME, and Antiviral Activities Study

The biological roles of carbohydrates, especially their synthetic glycoconjugates, are at the forefront of research, and their applications and research are in the development of an impressive area of carbohydrate-derived vaccines. Considering this, several thioglucosides 2-6 conjugated with paraaminophenyl and uridine moieties are investigated for their possible application as hepatitis antiviral applications. Geometry optimization of these uridine thioglucosides by density functional theory (DFT) indicated that their glucopyranose rings are in C1 chair conformation with β-glucosidic linkage. Frontier molecular orbital (FMO), molecular electrostatic potential (MEP), lower hardness, and softness calculations indicated their higher electrophilic nature. Solubility and ADMET studies indicated that the uridine thioglucosides might be safer drugs to use. With these encouraging results, molecular docking of 2-6 was conducted with hepatitis C virus (HCV, 3su4) and hepatitis B virus (HBV, 5e0i) proteases where the binding affinities for 2-6 were found higher than the standard anti-hepatitis drug sofosbuvir. Overall, the thioglucosides showed a better binding affinity with HBV protease (5e0i) than the HCV protease (3su4), indicating their prospect against these viruses.


Introduction
Changes in glycosylation can alter inflammatory responses, promote cancer cell metastasis and enable viral immune escape, including damage to kidney function that affects health problems and diseases. In this respect, glycoconjugation of selected monosaccharides/carbohydrates with chosen proteins and other biomolecules improve the ability to fine-tune immunological responses and boost immune responses to cancer. A sufficient fundamental understanding of glycoconjugates' structural insights and functions may enrich glycomedicine. Most glycoconjugate relies on a covalent link between carbohydrates and chosen molecule or macromolecule (protein) [1]. The biosynthesis of highly glycosylated glycoproteins found on the surface of several viruses is prepared by glycosyltransferases (GTs) Hepatitis B (5e0i) virus. Uridine-based thioglucosides are structurally related to the standard HCV drug sofosbuvir. Correlations have been conducted for acyl groups among uridine, glucose, and both uridine-glucose units. In addition to molecular docking, drug-likeness properties ADME of these compounds have been discussed in this study.

Materials.
In the present study, uridine (1), its 4-aminothiophenylglucoside 2, and acetate 3-6 are chosen ( Figure 2). For the rigorous comparison, acetyl groups in uridine moiety (3)(4), in glucoside moiety (5), and both moieties (6) are considered. Several of these compounds and related glucosides were synthesized by various researchers [4,5,37]. Structurally thioglucosides 2-6 contain an amido functional group, a pyrimidine ring, and pyranose and furanose rings. Dglucopyranose and uridine are linked together via para-aminothiophenol unit ( Figure 2).  The basic geometry of glucopyranoside and uridine (1) was initially taken from the online structure database known as ChemSpider. Having these structures, the necessary molecules 2-6 with appropriate geometry were drawn in the GaussView (5.0) program [46]. All the thioglucosides and uridine compounds were then optimized with Gaussian 09 program at DFT (B3LYP) computing method [47] with a 3-21G* basis set [47][48][49]. It should be noted that due to the larger molecular sizes, higher basis sets couldn't be used. For optimization, dry conditions at 298 K and 1 atm were maintained. The DFT optimized structures were used for the FMO (frontier molecular orbital) energy calculations like HOMO (highest occupied molecular orbital), LUMO (lowest unoccupied molecular orbital), and HOMO-LUMO gap, etc.
All the chemical reactivity descriptors are compared with the standard medication named sofosbuvir (brand name Sovaldi) used to treat the hepatitis C virus. DOS plots were obtained from GaussSum 3.0. Molecular electrostatic potential (MEP) was also calculated at the same level of DFT, and MEP calculation was conducted by an online WebMO demo server [44,50].

Calculation of ADMET and Lipinski rule.
Knowing structural features and chemical descriptors, these glucoconjugates were run on SwissADME online database (http://www.swissadme.ch) [51] for evaluating their Lipinski rule satisfaction. In addition, prediction of pharmacokinetic (PK) properties is generally conducted ahead of in vivo tests which tremendously reduce time and cost for drug discovery procedure. The ADMET properties were completed by the online database amdetSAR (http://biosig.unimelb.edu.au), the most acceptable database for predicting the AMDET parameters [52]. The optimized structures of 1-6 in InChIKey and SMILES formats were used for their ADMET calculation. For rigorous comparison, a standard hepatitis C drug, sofosbuvir was used.

Method for molecular docking.
Common antiviral drugs (telaprevir, danoprevir, vaniprevir) inhibit the HCV viral NS3/4A protease (PDB ID: 3su4). However, the sustainable applications of the common drug class are challenged by the quick emergence of resistance. Hence, potential strategies for developing robust therapies against this rapidly developing virus are essential. In addition, hepatitis B virus (HBV, a double-stranded DNA virus) inhibitors bound to HBV's core protein capsid Y132A (PDB ID: 5e0i) and found an important target for the antiviral drugs by inhibiting its replication. Based on the structural and mechanistic understanding, the selected uridine glycoconjugates are docked with 3su4 and 5e0i followed by comparing docking score with a standard antiviral drug (Sofosbuvir; approved for the treatment of chronic HCV infected patients).
Ligand preparation: For the ligand preparation, the DFT optimized structures (Section 2.2.1) were executed for the molecular optimization by utilizing vibrational frequency from the DMol3 code of Material Studio 08 [53]. The functional B3LYP and Basis set DNP+ were used from DMol3 code. Finally, the optimized ligands were exported as the PDB file for molecular docking study.
Protein preparation: The original three-dimensional (3D) crystal structure of Hepatitis C (3su4; Resolution: 2.25 Å) and Hepatitis B (5e0i; Resolution: 1.95 Å) viruses were collected from the RSCB Protein Data Bank (PDB) [54]. After taking the proteins from PDB, these were viewed by the PyMOL software version using PyMOL V2.3 (https://pymol.org/2/) [55]. The crystal structure of the protease was adjusted and verified based on their lowest energy eliminated water molecules and was saved as PDB files. In the next step, the pdb file was energetically minimized in another software called Swisspdb.
Molecular docking: Molecular docking is mainly investigated to calculate the binding affinity with protein, bonding interaction, bonding mode in their mechanism for bonding between ligand and protein [56], which are the key factors to study biologically active compounds against pathogenic protein strains. The docking procedure was performed with the help of AutoDock Vina incorporated from PyRx Virtual Screening Tools. After loading ligands (compounds) and proteins, their energy is minimized and saved as pdbqt file format. Both the files are forwarded for docking. The grid center points were set in X = -6.0679 Å, Y =2.8496 Å, Z = 20.8678 Å, and the dimension in X = 74.8931 Å, Y = 57.6001Å, Z = 92.1773Å. The grid box dimensions were selected and set up to wrap the protein's substrate-binding region. The BIOVIA Discovery Studio Visualizer 2017 was used to analyze the non-covalent interaction between the ligands and the pathogenic protein [57].

Optimized structures of thioglucoside 2-6.
Molecular structure, conformation, and shape greatly influence their interactions with receptor proteins and their biological functionality [44]. Thus, initially, the molecular structures of these uridine-glycoconjugates are determined. As shown in Figure 3, the glucoconjugates 2-6 glucopyranose rings exist in 4 C1 chair conformation in β-thioglucosidic linkage.
In compounds 2-6, the ribofuranose part of uridine is attached to the pyrimidine ring with β-linkage. Interestingly, the primary hydroxyl group or its acetyl group of glucopyranose unit and CO/NH of pyrimidine unit of 2-6 are found closer/cis to each other ( Figure 3). Thioglucosidic bond angle (∠C1-S-C) was found 101.3-101.6° and ribose to amide (∠C4′-C5′-CO) 110.6-111.9° for compound 2-4. Whereas, attachment of C-6 acetyl group at glucopyranose unit, as in 5-6, reduced both the bond angles (98.2-98.7° and 109-109.6°, respectively). This is probably due to the attractive force between the acetyl carbonyl oxygen and the nitrogen atom of the pyrimidine ring.

Molecular orbitals and chemical reactivity descriptors.
The chemical descriptors generally carry a special significance for any organic compound or biologically active molecule. The magnitude of ԑLUMO, ԑHOMO, and energy gap (Δԑ), ionization potential (I), electron affinity (A), chemical potential (µ), electronegativity (χ), hardness (ղ), electrophilicity (ω) and softness (S) of the six molecules 1-6 are presented in Table 1. These data were calculated by the DFT function. Table 1. Frontier molecular orbitals and reactivity descriptor analysis of 1-6. It is evident from Table 1 that both uridine and sofosbuvir possess a similar HOMO-LUMO gap (Δԑ). Whereas, all the glucoconjugates 2-6 had lower Δԑ values than the uridine (1), which indicated their better chemical reactivities. It should be noted that the smaller the energy gap Δԑ, the greater the reactivity for a molecule [46,48,58]. The higher electrophilicity index ω values for 1-6 (3.7-4.2 eV) than sofosbuvir (2.5 eV) indicated them as stronger electrophiles than the standard drug. The addition of acetyl group in the uridine thioglycosides, as in 3-6, gradually decreased their hardness and increased softness compared to uridine (1) and sofosbuvir, indicating their more reactive nature (according to the maximum hardness principle). DOS plot indicating HOMO-LUMO gap (Δԑ) of 5 and 6 are shown in Figure 4.

Frontier molecular orbital: HOMO and LUMO.
The DFT method has been used to determine the HOMO and LUMO orbital diagrams. HOMO generally refers to the maximum amount of electron density in the part of the molecule where an electrophile can easily attack. From the following pictures ( Figure 5), it can be seen that the LUMO parts are present in the alkyl chains and pyrimidine moieties of the uridine portion. In comparison, the HOMO part extends on the sugar (ribofuranose, glucopyranose, and hydroxyl group) part. Also, electronegative atoms, i.e., mainly oxygen atoms of those regions, are more likely to have HOMO orbitals. After analyzing their optimized structures, this study clearly showed this conceptual knowledge about HOMO from the frontier molecular orbital. On the other hand, the term LUMO refers to the lack of electrons where an electron-withdrawing group or nucleophilic group can easily be added. From Figure 5, it is seen that the LUMO of these molecules is associated with the alkyl group/part(s).

Molecular electrostatic potential (MEP).
Molecular electrostatic potential (MEP) correlates various physicochemical properties of compounds such as dipole moment, electronegativity, and partial charges especially MEP forecast the reactive electrophilic and nucleophilic sites of molecules. MEP is created in the space around a molecule by its nuclei and electrons (formed as static distributions of charge) It is a useful property for explaining and predicting its reactive behavior. Computationally predicted MEP of 1-6 and sofosbuvir are shown in Figure 6. The molecular MEP of 1-6 are predicted from their DFT optimized structures using the WebMo server [17]. other hand, the highest electrostatic potential energy value is represented by the blue zone in MEP, which indicates the maximum positive area and favorable site for nucleophilic attack. Again, the green color indicates zero potential areas. It is evident from Figure 6 that uridine (1), uridine thioglucosides 2-6, and sofosbuvir (7) possess almost similar red and blue zones indicating that these compounds are almost equally favorable for electrophilic reaction than nucleophilic attack. However, a critical observation showed their higher red zones compared to blue zones, informing their better electrophilic reaction.

Pharmacokinetics: drug-likeness study.
Optimal drug-likeness in compounds should be targeted in the early phases of drug research since it contributes to individual ADMET (absorption, distribution, metabolism, elimination, and toxicology), blood-brain barrier (BBB) penetration, and clearance. According to Lipinski rules (rule of five), drug-like molecules need to possess the five parameters such as drug molecule should have-(i) less than 5 hydrogen bond donors (HBD), (ii) less than 10 hydrogen bond acceptors (HBA), (iii) more than three number of rotatable bonds (NBR), (iv) molecular mass (MW) less than 500 Daltons, and (v) octanol-water partition coefficient (log Po/w) is not greater than 5 [44]. The HBD, HBA, NBR, MW, log Po/w, log S, and Lipinski rule violations of the glucofuranose compounds are presented in Table 2. It is found that almost all uridine thioglucosides molecules 2-6 partially obeyed the Lipinski rule. Also, these compounds possess topological polar surface areas above 140 Å² like standard hepatitis virus drugs. The n-octanol/water partition coefficient (log Po/w) is a key physicochemical parameter for drug discovery, design, and development. It is indicated as the ratio of the concentration of the non-ionized compound at equilibrium between organic and aqueous phases. All the target compounds 2-6 have lower lipophilicity and higher water solubility than uridine (1) and standard drug sofosbuvir (7).

Pharmacokinetics: ADMET studies.
Pharmacokinetic (PK) drug testing is needed in preclinical efficacy species (typically mice) at the early stage of drug discovery to ensure whether a potential drug has the necessary exposure to achieve efficacy following in vivo dosing [59]. It mainly consists of ADMET (absorption, distribution, metabolism, excretion, and toxicity) and indicates what the body does to the drug. A very similar term PD (pharmacodynamics), outlines what the drug does to the body. Nowadays, both PK and PD can be predicted using any software. In the present study, pkCSM protocol (http://biosig.unimelb.edu.au) [52] was applied to predict ADMET of 1-6 and presented in Table 3. Initially, absorption is predicted as Caco-2 permeability (C2P), human intestinal absorption (HIA), and P-glycoprotein inhibitor (P-gpI). Like sofosbuvir (7), all the compounds are found non-P-gpI. Their C2P and HIA are lower than the standard drug 7. The blood-brain barrier (BBB) and central nervous system (CNS) permeability represent the distribution of 1-7. BBB and CNS are found to be in good agreement with standard sofosbuvir. In terms of metabolism, all the compounds are not metabolic substrates of CYP3A4.
The uridine thioglucosides have slightly lower renal excretion as compared to 7. Interestingly, all the compounds 1-6 have lower toxicity in terms of human ether-a-go-go-gene (hERG) and oral rat acute toxicity (LD50) ( Table 3). As these compounds are predicted as noninhibitor of hERG, they should have fewer side effects (less or non-toxic). Thus, the uridine thioglucosides might be a safer drug to use.

Molecular docking: anti-hepatitis activities.
Molecular docking studies were conducted to validate the obtained pharmacological data [60] and provide evidence for the binding affinity of drug compounds 2-6 with proteins of Hepatitis C virus (HCV; 3su4) and Hepatitis B virus (HBV; 5e0i). Sofosbuvir is deliberately chosen as a standard drug due to its encouraging use in chronic HCV treatment and structural resemblance with the target compounds under investigation. Sofosbuvir (Figure 7) prevents HCV viral replication by binding with HCV polymerase's active site and preventing further replication of HCV genetic material. Several direct-acting antiviral agents (DAAs) for HCV target the essential 3su4 protease (NS3 protease, NS4A protein, or simply NS3/4A serine protease) and are approved by the FDA. Hence, at first, molecular docking of the uridine thioglucosides was conducted with 3su4 protease (HCV). The binding affinity and interactions, as shown in Table 4 and Table  5, all the compounds exhibited better binding affinities as compared to the uridine (1) and sofosbuvir (7, Figure 7). Compound 5 with two acetyl groups at the ribofuranose part showed the highest binding affinity (-8.1 kcal/mol), which was found higher than that of standard antiviral drug 7 (-6.8 kcal/mol). -6.9 5 5 Absent 10 7 -6.8 3 6 Absent 9 * Standard docking score value is ≤-6.00 kcal/mol Table 7 indicated that all the uridine-based drug compounds formed different hydrophilic and hydrophobic interactions with the 3su4 protein chain (Figure 8). It was reported that DAA-like telaprevir showed four hydrogen bonds with the amino acid residues present in the active site of the HCV NS3/4A protease [61]. These thioglucosides also showed several hydrogen bonds and non-bond interaction with this protease (Table 5).   Again, the HBV core protein is the major target for developing new therapies to treat HBV-infected patients. Hence, molecular docking was conducted with 5e0i, and the binding affinities of 1-7 are shown in Table 6. In both the cases (3su4 and 5e0i), the binding affinities for 2-6 were calculated higher than the standard anti-hepatitis drug sofosbuvir. Overall, the thioglucosides showed a better binding affinity with HBV protease (5e0i) than HCV (3su4). It should be noted that the target protein 5e0i with a 6-chain structure is the core protein of the HBV and is essential for HBV replication. So, it is an important target for antiviral drug discovery. The uridine thioglucosides 2-6 (-7.9 to -8.7 kcal/mol) showed better binding affinities than the uridine (1, -6.0 kcal/mol). The addition of the acetyl group at ribofuranose, glucopyranose, or both the parts did not change significant binding affinities with 5e0i. The best binding affinity was found for 2 and 3 (-8.7 kcal/mol), higher than antiviral sofosbuvir (-8.0 kcal/mol). The results were also supported by their good hydrogen bond and hydrophobic interaction with different amino acid residues of the protein chain of 5e0i (HBV; Table 7; Figure 8). Thus, further studies may establish these uridine-based thioglucosides as potential antiviral drugs, especially against HBV and HCV.

Conclusions
The available HCV and HBV antiviral therapeutics boceprevir, telaprevir, etc., cause severe side effects and long-term application of these drugs create drug resistance, which is a global health concern.  Absent Note: Short names of AA residues are mentioned in Table 5.
To develop more potent next-generation HCV and HBV protease inhibitors, several uridine-structured 4-aminophenyl 1-thioglucosides were docked against related proteins 3su4 and 5e0i, respectively. The thioglucosides showed a better binding affinity with HBV protease (5e0i) than HCV (3su4). In addition, all the thioglucosides 2-6 have a higher electrophilicity index, which indicates their stronger electrophilic nature than the standard drug sofosbuvir. The present drug-likeness, FMO, MEP, and molecular docking results and related study will help design uridine-based potential strategies to develop robust therapies against these deadly HCV and HBV soon.

Funding
This research was funded by Research and Publication Cell (University of Chittagong), COVID-19 Special 2021, 124/5.