Selectivity profile comparison for certain γ-butyrolactone and oxazolidinone-based ligands on a sigma 2 receptor over sigma 1: a molecular docking approach

Sigma receptors (σ1 R and σ2 R) are pharmacologically characterized membrane-bound receptors that bind a wide range of chemical compounds. Alzheimer's disease, traumatic brain injury, schizophrenia, and neuropathic pain have all been associated with abnormal σ2 activity. The σ2 receptor has recently been identified as a potential therapeutic target for inhibiting the formation of amyloid plaques. Numerous laboratories are now investigating the potential of σ2 ligands. Small molecule discovery is the focus of current research, with the goal of using target-based action to treat a variety of illnesses and ailments. Functionalized γ-butyrolactone and oxazolidinone-based ligands, in particular, are pharmacologically important scaffolds in drug discovery research and have been thoroughly examined for σ2 receptor efficacy. The purpose of this study was to evaluate the pharmacophoric features of different σ2 receptor ligands using in silico techniques. This study used a library of 58 compounds having a γ-butyrolactone and oxazolidinone core. To investigate the binding characteristics of the ligands with the σ2 receptor, a 3D homology model was developed. To understand the binding pattern of the γ-butyrolactone and oxazolidinone based ligands, molecular docking studies were performed on both σ1 and σ2 receptors. Furthermore, MM/GBSA binding energy calculations were used to confirm the binding of ligands on the σ2 over σ1 receptor. These in silico findings will aid in the discovery of selective σ2 ligands with good pharmacophoric properties and potency in the future.


Introduction
In 1976, W. R. Martin et al. published the results of a study on the impact of morphine, ketocyclazocine, and (AE)-SKF-100047 in dogs with chronic spinal disease. They found that these three compounds elicited distinctly different responses and hypothesized that each was interacting with a different pharmacological target. They labeled these previously unidentied targets as the m-opioid receptor (morphine type, MOR), the kopioid receptor (ketocyclazocine type, KOR), and the s-opioid receptor (SKF-100047 like). 1 Follow-up studies with the individual enantiomers of SKF-100047 revealed that each isomer interacted with a different biochemical target. (À)-SKF-100047 interacts with MOR and KOR to produce an opioid type response, but (+)-SKF-100047 produces a non-opioid response through the sigma receptor (sR). 2 Eventually, it was determined that there are two subtypes of this receptor, which have been designated as sigma-1 (s 1 ) and sigma-2 (s 2 ). 3 An X-ray structure of human s 1 was reported in 2016, 4 but to date there are no known ligands for this receptor.
The true nature of s 2 , however, remained a mystery for nearly 40 years. In 2017, A. C. Kruse et al. demonstrated that this receptor is identical to the Transmembrane Protein 97 (TMEM97, also known as MAC30 (Meningioma-associated protein)), 5 and an X-ray structure was published in 2021. 6 Similar to s 1 , there are no known natural functional s 2 ligands. It has been demonstrated that this protein is present in the endoplasmic reticulum (ER) and lysosomes where it binds to cholesterol, but the pharmacological role of s 2 has not been determined. 7 Numerous disease states such as schizophrenia, 8 Alzheimer's disease, 9 neuropathic pain, 10 traumatic brain injury, 11 Niemann-Pick disease, 12 and cancer 13 have been linked to s 2 and this has prompted many research teams to investigate the potential therapeutic utility of s 2 ligands. Numerous in vivo active s 2 ligands have been identied and some have reached human clinical trials. UKH-1114 (1, s 2 K i ¼ 46 nM) is efficacious in animal models of pain, 14 while Siramesine (2, s 2 K i ¼ 0.12 nM) is efficacious in animal models of depression and anxiety. 15 The radioligand [ 18 F]ISO-1 (3, s 2 K i ¼ 6.9 nM) has been studied as a possible PET ligand in the treatment of breast cancer, 16 while CT1812 (4, s 2 K i ¼ 8.5 nM) has been the subject of clinical trials as a potential Alzheimer's disease therapy (Fig. 1). 17 Recently, we reported our discovery of two novel classes of s 2 ligands, oxazolidin-2-ones (5) and functionalized g-butyrolactones (6). [18][19][20] The synthetic protocol for the synthesis of these two classes of compounds is outlined in the ESI † (Schemes 1 and 2). These compounds demonstrated a range of selectivity for s 2 over s 1 . As part of an effort to develop a better understanding of the structural features that drive selectivity, we have studied generated in silico models of both receptors. In addition, we have conducted docking studies and calculated binding energies of the exemplary compounds from our previously disclosed s 2 ligands.

Homology modeling
Homology modeling is a technique for creating and predicting an atomic resolution model of a target protein based on the experimentally established 3D structure of a homologous protein called the template protein. The four fundamental stages in homology modeling are (1) locating the template structure sequence, (2) matching the query sequence with the template structure sequence, (3) creating the query's model structure based on the template structure information, and (4) assessing the projected model. As a result, homology modeling may be used to predict the structure of unknown proteins, such as the human sigma 2 receptor.
The model architectures of the human sigma 2 receptor were predicted using a homology modeling approach. Maestro was used for all computational and molecular modeling of the human sigma 2 receptor (Schrödinger, LLC, 2019-1). 21 The human sigma 2 amino acid sequence, which consists of 176 residues, was retrieved from the Uniprot database (UniProt ID: Q5BJF2). 22 PSI-BLAST was used to scan the nonredundant PDB database for template identication. The X-ray crystalline structure of the Bos taurus sigma-2 receptor bound to Z1241145220 (PDB ID: 7M95) 23 exhibited measurable similarities to the query sequence and was therefore utilized to generate the model. Protein Data Bank was used to acquire the coordinates. The alignment of the template and the target was the initial stage. To choose the best alignment, the alignments were ordered by identities, score, positives, expectation value, and gaps, and statistically analyzed.
The Prime Module was used to create the homology models. In the model, the co-crystallized ligand Z1241145220 was retained. Using Prime functionality, loop renement and numerous loop conformations were produced. Side-chain predictions and all-atom minimizations were used to score these conformations. The model was further optimized and minimized when the model construction calculations were completed.

Molecular docking studies
Schrödinger's Glide docking tool (Schrödinger, LLC, New York, 2019) was utilized to investigate ligand-receptor interactions. Glide is a novel method for nding favourable interactions between proteins and many ligands. As a result, Glide docking allows you to compare the binding mechanism and affinity of different ligands to the protein. In exible docking, ligand posture refers to the location and orientation of a ligand with regard to the protein, as well as its shape. A number of hierarchical lters are used to analyze the ligand postures obtained by Glide docking. The ChemScore empirical scoring function is used by Glide. This algorithm detects the protein's and ligand's favourable hydrophobic and hydrogen-bonding interactions. Through many evaluation trials, Glide has been regarded as one of the best docking tools presently available.

Ligand preparation.
Glide takes the ligand structure in 3D format as an input le for ligand preparation. Maestro Molecule Builder was used to create the 3D structures of all ligands. The ligand molecule is prepared for Glide docking using the Schrodinger ligand preparation product LigPrep. LigPrep (LigPrep, 2019) is a technique that renes the ligand structure via a series of processes. LigPrep was used to remove unnecessary structures, add hydrogens, and optimize and minimize ligand structures. Because of its enhanced parameterization and coverage, the ligand structures were reduced using the OPLS-2005 force eld. Furthermore, among the force elds, the OPLS-2005 force eld was selected for grid generation due to its metal handling capabilities (LigPrep, 2019). As a consequence, a single low-energy 3D structure was created with the right chiralities. LigPrep is a tool for creating highquality 3D structures by changing chemical structures like as stereochemistry and protonation state (LigPrep, 2019). As a result, LigPrep was used to establish the protonation states of all the ligands. The protonation states of the ligand may be derived from the structure of the ligand if structural data is available. The structural data for all of the ligands is available, and the protein active site was chosen before molecular docking. As a result, the proper protonation states were assigned to the ligand binding mode.
2.2.2 Protein preparation. The RCSB Protein Data Bank was used to get the protein crystal structure of human sigma-1 receptor bound to PD144418 (PDB ID: 5HK1). Using Maestro's Protein Preparation Wizard, the human sigma-1 protein crystal structure and the modelled sigma-2 receptor were rened further. The protein's multimeric complex structure is simpli-ed because a small number of atoms in the complex structure is preferred for computer efficiency. Except for those that have bridging connections between the protein and the ligand, all crystallographic water molecules are eliminated from the receptor molecules' 3D structure.
Customizing the protein, metal ions, and cofactors is simple using the Protein Preparation Wizard. The wizard lls in any missing residues around the protein's active site. The formal charges and ligand bond ordering were changed. Following these stages, the Impact Renement module was used to perform restrained minimization (Impref, 2019). The revised ligand/protein/water structures were examined to guarantee the right formal charges, bond ordering, protonation states, and nal changes were made to the protein structures. The receptor grid les were created using the prepared protein structures.
2.2.3 Receptor grid generation. Several distinct sets of elds were used to depict the shape and features of the grid on the receptor, providing increasingly more precise scoring of the ligand poses. One of the input les for ligand docking is the receptor grid. A prepared structure (an all-atom structure with proper bond ordering and formal charges) is required for receptor grid production, which is obtained during the protein preparation stage. A receptor grid generation panel was used to set up and produce the receptor grid for all of the proteins. The receptor structures were dened using the choices from this panel. Co-crystallized ligands were eliminated during this stage, and the location and size of the active site, which would be indicated by receptor grids, were identied. The active site of the receptor was determined using the ligands in the crystal structures. To properly address metals and a broad variety of atom types described, the force eld OPLS 2005 was employed.
2.2.4 Docking studies. Glide ligand docking needed previously produced receptor grids and ligand structures built using LigPrep. By choosing XP mode on the Glide ligand docking screen, the docking was completed. The ligand docking panel's exible docking option with default settings was chosen because it guides the docking process to build conformations internally. In terms of Glide score and docking score, Glide calculated the ligand-receptor binding affinity.

Prime MM/GBSA binding energy calculations
One of the most oen used computational approaches for estimating relative binding affinities of target protein-ligand complexes is molecular mechanics with generalised born surface area (MM/GBSA). Through employing the MM/GBSA technology tools available in the Prime module of Schrodinger 2019-1, ligand-binding energies were calculated based on docking complex. The MM/GBSA calculations were performed using the protein-ligand complexes produced from the docking investigations. DG bind , the relative binding free energy, was calculated using the following equation: where DG bind is the computed relative free energy that takes into account both ligand and receptor strain energy. The MM/GBSA energy of the minimized complex is E complex (minimized), and E ligand (unbound, minimized) is the MM/GBSA energy of the ligand aer it has been removed from the complex and allowed to relax. Aer separating the protein from the ligand, the MM/ GBSA energy of the E receptor (unbound, minimized) is calculated.

Homology modeling and binding site analysis
To the best of our knowledge, the crystal structure for sigma-2 receptor of Homo sapiens has not been reported yet. As a result, we used a comparative modeling approach to create a three-dimensional (3D) model for the sigma-2 receptor of H. sapiens. The homology model of the H. sapiens sigma-2 receptor was created in Schrödinger Suite 2019-1 (Schrödinger, LLC, New York, NY) using Meastro, Prime. The H. sapiens sigma intracellular receptor 2 query sequence was obtained from the universal protein resource (Uniprot, Entry Id: Q5BJF2). The template was chosen from the crystallographic structure of the Bos taurus sigma-2 receptor (PDB ID: 7M95, 2.41 resolution) with 78.4 percent sequence identity with the target. Prime homology modeling methods were used to create a 3D model of the human sigma-2 receptor. The Ramchandran plot and protein reports were used to analyze the predicted 3D structure. The sequence alignment of the sigma-2 receptor of H. sapiens (UniProt ID:Q5BJF2) and Bos Taurus (PDB ID: 7M95) is shown in Fig. 2. The homology-modeled structure of the human sigma-2 receptor and its Ramchandran plot are shown in Fig. 3 and 4. The Ramchandran plot of the human sigma-2 receptor structure revealed that >95 percent of residues are favored and allowed 4, c backbone conformational regions.

Molecular docking
All of the compounds were subjected to molecular docking studies in order to discover signicant binding mechanisms responsible for their action on the sigma 1 and 2 receptors. Molecular docking was used to conrm the binding pose and conformation of these analogues. All of the compounds were docked into the active site of a homology-modeled sigma 2 receptor as well as the crystal structure of the sigma 1 receptor. The results of molecular docking, hydrogen bonding, and arene-arene interactions of compounds with sigma 1 and 2 receptors are shown in Tables 1 and S1. † The initial set of compounds (LACT1-LACT41) comprised of a series of g-butyrolactone analogues with a heterocyclic primary core and varied electron donating, electron withdrawing, hydrophobic, H-bond acceptor, and donor groups. When compared to the 1-diethyl lactone derivative (LACT1) of the g-butyrolactone family, the 1,1-dimethyl lactone derivative (LACT2) demonstrates a restricted number of interactions with the active site of sigma 2 receptor. The active site residues (Asp29, Glu73) of s 2 R formed two hydrogen bonds with LACT1. The protonated N-atom of phenyl piperazine serves as an Hbond donor, forming an H-bond with Asp29 (d ¼ 2.57 A). Butyrolactone's carbonyl oxygen atom functions as an H-bond acceptor, forming an H-bond with Glu73 (d ¼ 2.49 A). Furthermore, piperazine's N-atom forms a Pi-cation interaction with Tyr147. LACT1 has also exhibited a variety of hydrophobic interactions with active site residues. The phenyl ring of LACT1 forming a Pi-Pi (arene-arene) contact with Tyr50. Furthermore, LACT1 formed only one Pi-Pi interaction with the s 1 R residue Tyr103 and made no H-bond interactions with it. There are fewer interactions with both s 1 and s 2 R for the 1,1-dimethyl lactone derivative (LACT2).
LACT3 with carbonyl group on its ethyl linker chain has lower docking scores towards both s 1 & s 2 R. LACT4 with piperazin-2-one ring has average docking scores with fewer interactions on s 1 & s 2 R. LACT5 with piperadine ring has shown good docking scores with s 1 & s 2 R. The N-atom of piperadine scaffold has made two strong Pi-cation interactions with the active site residues Tyr147 (d ¼ 5.59 A) and Tyr150 (d ¼ 4.55 A) of s 2 R. Interestingly, the N-atom of piperadine also made a salt bridge with Asp29 (d ¼ 3.07 A) of s 2 R. Further, the phenyl ring of LACT5 established two Pi-Pi interactions with the active site residues Tyr103, Tyr206 of s 1 R. In case of LACT6, the protonated nitrogen atom was observed at cyclohexyl connected N-atom of the piperazine ring and this N-atom interact with Asp29 via salt-bridge. In addition, the protonated N-atom of the piperazine ring has made a Pi-cation interaction Tyr150 of s 2 R. Further, the protonated N-atom of the piperazine ring of LACT6 has established an H-bond and Pi-cation interaction with Glu172 and Phe107 of s 1 R, respectively. Compounds with electron withdrawing substituent's like CN  Ramachandran plot for the modeled sigma intracellular receptor 2 (s 2 ) of Homo sapiens. The plot is organized as follows: glycine, proline and all other residues are plotted as triangles, squares, and circles respectively. The red, yellow and white regions represent the favoured, allowed and the disallowed regions respectively.   and Val146 of s 2 R. Further, a salt-bridge and a Pi-cation interaction were observed for both LACT7, LACT8 with Glu172 and Phe107 of s 1 R, respectively. A Pi-Pi interaction was observed for 4-triuoromethyl substituted phenyl ring of LACT8, LACT9 with Tyr103 and Leu182 of s 1 R, respectively.  Compounds with electron donating substituent's like OMe (LACT10) and Me (LACT11) in the 4-position of the phenyl ring has shown good docking scores on s 2 R over s 1 R. LACT10 established two hydrogen bonds with the active site residues (Asp29, Glu73) of s 2 R. The protonated N-atom of phenyl piperazine has made an H-bond interaction with Asp29 and the carbonyl oxygen atom of butyrolactone shown H-bond interaction with Glu73. Further, the N-atom of piperazine is established a Pi-cation interaction with Tyr147. With respective to the s 1 R, compound LACT10 has formed an H-bond interaction with Glu172 and Pi-Pi interaction with His154. The detailed binding pose and protein-ligand interactions of LACT11 with s 2 R was depicted in Fig. 5. 4-Me substituted phenyl ring of LACT11 established a Pi-Pi interaction with Tyr50 and N-atom of phenyl piperazine has made a Pi-cation interaction Tyr147. Further, several hydrophobic interactions were observed for LACT11 and the active site residues of s 2 R, which stabilizes the lodging of LACT11 in the active pocket.
Compounds LACT12-LACT14 include electron withdrawing substituent's at 3-position of the phenyl ring based derivatives endowed with an improved docking scores with s 2 R if compared with the s 1 R. The carbonyl oxygen atom of butyrolactone of LACT12 has shown an H-bond interaction with Glu73. Further, the protonated N-atom of phenyl piperazine of both LACT12 and LACT13 has made a salt-bridge and a Pication interaction with Asp29 and Tyr147 of s 2 R, respectively. LACT14 established two hydrogen bonds with the active site residues Asp29 (d ¼ 2.29 A), Glu73 (d ¼ 2.3 A) and a Pi-cation interaction with Tyr147of s 2 R. Further, compound LACT15 and LACT16 with electron donating substituent's at 3-position of the phenyl ring showed almost similar docking scores on both s 1 & s 2 R.
Interestingly, compound LACT17-LACT19 with electron withdrawing substituent's at 2-position of the phenyl ring showed good docking scores on s 2 R if compared with the s 1 R. LACT17, LACT18 and LACT19 has shared a common H-bond interaction, as well as Pi-cation interaction with the active site residues Asp29 and Tyr147 of s 2 R, respectively. Additionally, compounds with electron donating substituent's at 2-position of the phenyl ring (LACT20 and LACT21) has also shown good docking scores on s 2 R over s 1 R. Mounting the steric bulk in the 2-position of the phenyl ring by placing an isopropyl group (LACT22) improved s 2 R selectivity. Fig. 6 displays the docking pose of LACT22 and its ligand-protein interactions in the active site of s 2 R. LACT23 with 2,4-di-Me substitution on phenyl ring displayed high docking score for both s 1 & s 2 R. LACT24 with 2pyridine ring showed poor docking score at s 2 R. In this analogue, the 2-pyridine ring was involved in the Pi-Pi stacking interaction with Tyr50 of s 2 R. With respective to the s 1 R, compound LACT24 has formed an H-bond interaction with Glu172 (d ¼ 2.55 A). In case of 3-pyridine ring containing analogue (LACT25) and 4-pyridine ring containing analogue (LACT26), we observed a common H-bond interactions (Asp29, Glu73) and Pi-cation interaction (Tyr147) with s 2 R. In addition, the 3-pyridine ring of LACT25 also involved in the Pi-Pi stacking interaction with Tyr50 of s 2 R. Further, LACT25 did not show any H-bond, Pi-Pi, salt-bridge and Pi-cation interactions with s 1 R. LACT26 exhibited an H-bond between protonated N-atom of phenyl piperazine and Glu172, and Pi-cation interaction between N-atom of 4-pyridine ring and His154 of s 1 R.
LACT27 with linker chain length of "3C" displayed good docking score on s 2 R over s 1 R. The protonated N-atom of phenyl piperazine ring of LACT27 established an H-bond, saltbridge, Pi-cation interactions with Val146 (d ¼ 2.56 A), Asp29 (d ¼ 4.68 A), and Tyr147 (d ¼ 4.51 A) of s 2 R, respectively. Compound LACT28 include linker chain length of "4C" also shown greater docking score on s 2 R over s 1 R. LACT28 established an H-bond with the active site residue Asp29 (d ¼ 2.34 A) and a Pi-cation interaction with Tyr147 (d ¼ 5.06 A) of s 2 R. 1-Naphthyl piperazine containing g-butyrolactone analogue (LACT29) displayed good docking score on s 2 R. LACT29 made an H-bond with the active site residue Asp29 (d ¼ 2.13 A) and a Pi-cation interaction with Tyr147 (d ¼ 4.98 A) of s 2 R. With respective to the s 1 R, the protonated N-atom of piperazine ring of LACT29 has formed an H-bond interaction with Glu172 (d ¼ 2.33 A) and the 1-naphthyl part made two Pi-Pi interactions with the active site residues Tyr103 and Tyr206 of s 1 R. The 4pyrimidine ring containing g-butyrolactone analogue (LACT30) displayed poor docking scores on both s 1 and s 2 R.
Captivatingly, compounds with prospective piperazine bioisosteres like homopiperazine analogue (LACT31), 2,6-diazaspiro [3.3]heptane analogue (LACT32), and hexahydropyrrolo [3,4-c]pyrrole analogue (LACT33) shown excellent docking scores on s 2 R over s 1 R. LACT31, LACT32 and LACT33 has shared a common H-bond interaction, as well as Pi-cation interaction with the active site residues Asp29 and Tyr147 of s 2 R, respectively. Further, LACT32 and LACT33 have shown additional Pi-cation interaction with Tyr150 of s 2 R. Replacing of phenyl ring in LACT33 with 4-pyridine led to increase in number interactions with the active site residues of s 2 R. The Natom of 4-pyridine of LACT34 established H-bond interaction with Gln77 (d ¼ 2.61 A), and also 4-pyridine formed a Pi-Pi interaction with His21 of s 2 R. Further, LACT34 has made Hbond interaction, as well as Pi-cation interaction with the active site residues Asp29 (d ¼ 2.03 A) and Tyr147 of s 2 R, respectively. According to our calculations, fascinatingly, compound LACT35-LACT38 with tetrahydroisoquinoline scaffold have shown admirable docking scores with the s 2 R. Compound LACT35-LACT38 has shared a common H-bond interaction, as well as two Pi-cation interactions with the active site residues Asp29 and Tyr147 & Tyr150 of s 2 R, respectively. The protonated N-atom of tetrahydroisoquinoline involved in both H-bond and Pi-cation interactions. In addition, the carbonyl oxygen atom of butyrolactone of LACT35 is acting as H-bond acceptor and made an H-bond interaction with Gln77 (d ¼ 3.38 A). Fig. 7 illustrates the binding pose of compound LACT37 and its ligand-protein interactions in the active site of s 2 R. Similarly, compound LACT39-LACT41 with pyridine type nitrogen atom in the tetrahydroisoquinoline ring also exhibited the good docking scores and interactions against both s 1 and s 2 R, but their actual biological K i values are not at all potent towards the both s 1 and s 2 R. The in silico docking scores for these compounds is probably due to the close structure resemblance with that of simple tetrahydroisoquinoline scaffold containing analogues (LACT35-LACT38).
The second set of compounds (OXAZ1-OXAZ17) consisted of a series of oxazolidinone-based derivatives with a heterocyclic primary core and various electron donating, electron withdrawing, hydrophobic, H-bond acceptor, and donor groups.
Within this series, compounds have various substituent's capped at amide group of oxazolidin-2-one ring. The unsubstituted oxazolidin-2-one ring containing compound OXAZ1 displayed very poor docking scores towards both s 1 and s 2 R. Compounds with 3 to 6 membered cycloalkane group attachments, i.e. cyclopropyl (OXAZ2), cyclobutyl (OXAZ3), cyclopentyl (OXAZ4) and cyclohexyl (OXAZ5) have shown good to moderate docking scores against s 2 R. Compound OXAZ2 established one hydrogen bond with the active site residue (Asp29) of s 2 R. The protonated N-atom of phenyl piperazine is acting as H-bond donor and made an H-bond interaction with Asp29 (d ¼ 1.80 A). Further, the N-atom of piperazine is established two Pication interactions with Tyr147 and Tyr150. Additionally, one of the phenyl groups of benzhydryl part established a Pi-Pi interaction with Tyr50. With respective to the s 1 R, the protonated Natom of piperazine ring of OXAZ2 has formed a salt-bridge with Glu172 and one of the phenyl groups of benzhydryl part established a Pi-Pi interaction with Tyr103. OXAZ3 and OXAZ4 have shared a common H-bond interaction, as well as Pi-cation interaction and Pi-Pi interaction with the active site residues Asp29, Tyr147 and Phe54 of s 2 R, respectively. Further, OXAZ3 have shown additional H-bond interaction with Glu73 of s 2 R. On the contrary, compound OXAZ3 and OXAZ4 was not docked at the active site of s 1 R. Compound OXAZ5 exhibited a Pi- cation interaction with Tyr147 of s 2 R and two Pi-Pi stacking interactions with Tyr103 and Tyr206 of s 1 R.
Compound with phenyl ring (OXAZ6) has showed good docking score against s 2 R but not docked to the active site of s 1 R. The protonated N-atom of piperazine ring of OXAZ6 has formed an H-bond with Asp29 (d ¼ 1.85 A) and a Pi-cation interaction with Tyr147 of s 2 R. Further, the phenyl group of benzhydryl part established a Pi-Pi interaction with Tyr50. OXAZ7 with benzyl substituent showed good docking score with s 2 R, but not docked at s 1 R active pocket. OXAZ8 with 4-Fbenzyl substituent showed increased docking score towards s 2 R, and poor score with s 1 R. Better activity of OXAZ8 with s 2 R is probably due to the strong salt-bridge formation between Natom of piperazine ring and Asp29 of s 2 R. OXAZ8 also exhibited a Pi-cation interaction with Tyr147 and Pi-Pi interaction with Phe54 of s 2 R. In contrast, the presence of electron donating groups, such as 4-OMe (OXAZ9) and 4-Me (OXAZ10) on benzyl part led to decrease in docking scores against s 2 R. Further, OXAZ9 and OXAZ10 showed very poor binding scores with s 1 R. Fig. 8 illustrates the docking pose of compound OXAZ8 and its ligand-protein interactions in the active site of s 2 R. Fig. 9 displays the ligand-protein interactions for the compounds LACT11, LACT22, LACT37 and OXAZ8 at the active site of s 1 R.
Compounds with a cyclohexane ring (OXAZ11) and tetrahydropyran ring (OXAZ12) also showed moderate binding with s 2 R and very poor binding with s 1 R. Compound OXAZ13 with phenethyl group displayed good docking score with s 2 R but not docked against s 1 R active pocket. The protonated N-atom of piperazine ring of OXAZ13 has formed an H-bond with Asp29 (d ¼ 1.95 A) and a Pi-cation interaction with Tyr147 of s 2 R. Further, one of the phenyl groups of benzhydryl part established a Pi-Pi interaction with Tyr150. Further, OXAZ14 with 4-F-phenethyl group showed poor binding with s 2 R and s 1 R. OXAZ15 with 4-OMe-phenethyl group showed an H-bond interaction with Asp29 (d ¼ 2.04 A) and a Pi-cation interaction with Tyr147 of s 2 R. OXAZ16 with 4-Me-phenethyl group showed poor docking with s 2 R and it exhibited two Pi-cation interactions with Tyr50 and Tyr147 of s 2 R. OXAZ17 with phenylpropyl also displayed an H-bond interaction with Asp29 (d ¼ 2.34 A) and a Pi-cation interaction with Tyr147 of s 2 R. Further, compound OXAZ15, OXAZ16 and OXAZ17 showed very poor binding score with the active site of s 1 R.

Prime MM/GBSA binding energy calculations
To further authenticate the binding affinity of docked g-butyrolactone and oxazolidinone-based ligands at the active sites of the sigma 1 and sigma 2 receptors, binding free energy was calculated using the molecular mechanics generalised born surface area (MM-GBSA) approach available in the Prime module. Table 2 summarizes the binding energies of certain gbutyrolactone and oxazolidinone-based ligands based on biological K i values and the s 2 /s 1 ratio.
The estimated binding free energy of docked poses of molecules with s 1 receptor ranged from À2.018 to À84.106 kcal mol À1 , whereas the BFG score for the s 2 receptor ranged from À84.578 to À114.210 kcal mol À1 . The study shows that produced chemicals bind to the s 2 receptor at a higher rate than those that bind to the s 1 receptor. The docking complexes for the chemicals OXAZ3, OXAZ4, OXAZ6, OXAZ7, and OXAZ13 were not identied. So, their binding energies were assumed to be 0 kcal mol À1 . The compound OXAZ3 with the s 2 receptor has the greatest BFG score of around À114.210 kcal mol À1 . The van der Waal energy (G vdW ¼ À50.162 kcal mol À1 ) and lipophilic energy (G lipo ¼ À65.374 kcal mol À1 ) terms are the major contributors to OXAZ3 binding activity in the active pocket of the s 2 receptor, while coulombic energy (G cou ¼ À12.314 kcal mol À1 ) moderately favours ligand binding, according to this study. Furthermore, the ligand's binding activity inside the active site of the s 2 receptor is deemed unfavourable due to covalent energy and electrostatic solvation energy terms. Nonetheless, when compared to the s 1 receptor, the compounds had lower BFG, van der Waal energy, lipophilic energy, and coulombic energy. The results show that the produced compounds had a higher affinity for the s 2 receptor than the s 1 receptor.

Conclusion
In this work, essential components responsible for the binding process between the s 2 receptor and its ligands were identied utilizing molecular docking and MM/GBSA binding energy calculations. Molecular docking study provides a useful prediction of structural features for certain series of compounds belonging to g-butyrolactone and oxazolidinones to bind at the active sites of s 1 and s 2 receptor. A 3D homology model for the s 2 receptor was built to perform the modeling studies. The Ramchandran plot examination was conducted to assess the correctness of modeled s 2 receptor. The putative binding site on the modeled s 2 receptor was identied using SiteMap analysis. In this present work, a total of 58 compounds have been used. The molecules were docked into both the active site of a homology-modeled s 2 receptor and the crystal structure of the s 1 receptor. The structural features required for ligand binding to s 1 and s 2 receptors were studied in 2D and 3D. With the s 2 receptor, the ligands showed higher BFG, van der Waal energy, lipophilic energy, and coulombic energy. The topranked molecules, according to the above study, had much more interactions and binding energy for the s 2 receptor than the s 1 receptor. Thus, the present study provides an understanding of the specic pharmacophoric features and interactions of ligands that are responsible for s 2 selectivity over s 1 receptor. Future research on these molecules might lead to the discovery of novel and potential selective s 2 ligands.

Conflicts of interest
The authors declare no conicts of interest.