This readme file was generated on 2025-12-09 by Nina Kobe


# GENERAL INFORMATION

* Title of Dataset: Data of In silico characterisation of a novel SARS-CoV-2 Envelope protein inhibitor and in vitro validation against Murine coronavirus
*provide at least two contacts*
## Author/Principal Investigator Information
Name: Nina Kobe
ORCID: 0009-0008-3351-408X
Institution: National institute of Biology Slovenia
Address: Večna pot 121, 1000 Ljubljana, Slovenia
Email: nina.kobe@nib.si

## Author/Associate or Co-investigator Information
Name: Polona Kogovšek
ORCID: 0000-0002-4035-0115
Institution: National institute of Biology Slovenia
Address: Večna pot 121, 1000 Ljubljana, Slovenia
Email: polona.kogovsek@nib.si

# SHARING/ACCESS INFORMATION

* Licenses/restrictions placed on the data: open
* Links to publications that cite or use the data: In silico characterisation of a novel SARS-CoV-2 Envelope protein inhibitor and in vitro validation against Murine coronavirus (doi)
* Links to other publicly accessible locations of the data: /
* Links/relationships to ancillary data sets: /
* Was data derived from another source? /
	* If yes, list source(s): 
* Recommended citation for this dataset: Kobe N, Dreisewerd L, Lukšič M, Pavlin M, Grošelj U, Podlipnik Č, Janc M, Lengar Ž, Mrak P, Tušek Žnidarič M, Pompe Novak M, Kuhar U, Hostnik P, Dattola F, Carletti T, Marcello A, Kogovšek P. Raw data from the experiments. Zenodo.


# DATA & FILE OVERVIEW

## File List: *list all files (or folders, as appropriate for dataset organization) contained in the dataset, with a brief description*

* Relationship between files, if important: /
* Additional related data collected that was not included in the current data package: /
* Are there multiple versions of the dataset? /


# METHODOLOGICAL INFORMATION

## Description of methods used for collection/generation of data: 

Cytotoxicity assay
The cell counting kit-8 (CCK-8, Abcam) test was conducted following the protocol provided by the manufacturer. Cells were seeded at a density of 3 × 105 /mL in 96-well plates and treated with different concentrations of the compounds after 24 hours at 37 °C. In all experiments, a solvent control (growth medium containing 0.5 % DMSO), a negative control (growth medium), and assay-specific positive controls were included. After 48 hours at 37 °C, 10 µL of CCK-8 reagent was added to each well and the cells were cultured in the dark at 37 °C for 1.5 hours. The absorbance was measured at 460 nm using Cytation 5 (Agilent BioTek). The relative cell viability was determined using the following formula: Cell viability (%) = OD of sample/average OD of negative control × 100 %. Three independent biological replicates were performed for each individual compound.

Time of addition assay and Plaque reduction assay
L929 cells at a density of 3 × 105 /mL were seeded in 12-well culture plates and incubated at 37 °C for 24 h. Dilutions of the compounds were added 2 h before infection (pre-treatment) or together with the virus – after 1 h of co-incubation of virus and compound at room temperature (co-treatment) or after 1 h of infection (post-treatment) (Fig. 3). The cell culture supernatants collected after 48 h were used to determine number of plaques by plaque reduction assay. Extended protocol is available in Supplementary material (S2-1 and S2-2) and raw data at Zenodo.

Real-time quantitative polymerase chain reaction
L929 cells were seeded in 12-well culture plates and after 24 hours inoculated with 0.001 multiplicity of infection (MOI) MHV for 1 h at 37 °C and 5 % CO2. The inoculum was replaced by complete EMEM with the highest non-toxic concentration of the inhibitor (LC, HMA, or RMT) or at infected non-treated cells (positive control) only by vehicle (0.5 % DMSO) (post-treatment conditions). Negative control samples were not infected nor treated. Presence and amount of MHV RNA was monitored at different time points (0, 24, and 48 h) post treatment. Primers, probes and extended protocol are available in Supplementary material (S2-3).

Light microscopy and transmission electron microscopy  
The impact of MHV and the LC on L929 cells was observed using light and transmission electron microscope. Four samples were prepared simultaneously. First sample named L929 represented control (72 h culture); second sample, named L929+MHV, grew at the same conditions for 24 h, then it was infected with MHV for 1 h at MOI 0.001 and was incubated for 48 h at the same conditions. Third and fourth samples were treated with LC (post-treatment conditions), L929+LC without and L929+MHV+LC with infection with MHV for 1 h at MOI 0.001. All samples were prepared in duplicates. At 48 hours post infection (hpi) cultures were harvested by scraping and immediately fixed in 3 % glutaraldehyde in 0.1 M PBS (phosphate buffer saline), postfixed in 1% solution of osmium tetroxide for 1 hour, followed with dehydration with ethanol and finally embedded in Agar 100 resin. Semithin and ultrathin sections were prepared with ultracut UC7 (Leica), using glass and diamond knife, respectively. Semithin sections were stained with toluidine blue and examined with light microscope AxioSkop Opton (Zeiss) equipped with digital camera Leica DFC290HD and software Leica Application Suite 4. 0. 0. Ultrathin sections were stained with 1 % (w/v) water solution of uranyl acetate and lead citrate. The grids were than examine with transmission electron microscope TALOS L120C (Thermo Scientific), operating at 100 kV equipped with Ceta 16M camera. 


## Methods for processing the data: 
The sigmoidal dose-response curve function (variable slope) was used to calculate the half maximal effective (EC50) and cytotoxic (CC50) concentrations. The ratio between CC50 and EC50 determines the selective index (SI). All data were plotted and analysed using GraphPad Prism software version 10.4.0 (USA). Results are presented as means ± standard deviations. To calculate the statistical significance between control (infected samples, treated with 0.5 % DMSO) and treatment (infected samples, treated with compounds) for qPCR results, a two-way analysis of variance (ANOVA) with Dunnett’s test, a multiple comparison test, was conducted with GraphPad Prism software. Significance was reported for p-value < 0.05 (*), < 0.01 (**), < 0.001 (***) and ≤ 0.0001 (****).


## Instrument- or software-specific information needed to interpret the data: 
The 2-EPRO NMR structure of Mandala et al. (PDB ID 7K3G) with a resolution of 2.1 Å was selected [2]. The Schrödinger Protein Preparation Wizard [3,4] was used to pre-process the protein structure. A restrained minimisation of the protein structure was performed, followed by an optimisation of the protein's hydrogen bond network, after which a receptor grid for the C- and N-terminal vestibule was generated. The docking step was conducted with Schrödinger software involving an extra-precision step via Glide, using standard settings [5]. Herein, docking was performed at the C- and N-terminal binding side of the EPRO ion channel for the LC and RMT, yielding three complexes: RMT-C-EPRO, RMT-N-EPRO and LC-N-EPRO. We note that docking of LC to the C-terminal of EPRO did not produce any results to incorporate, due to unfavourable steric properties of the ligand. Specifically, the ligand seems to have been unable to access the binding pocket due to excessive size, limited flexibility, and steric clashes with the receptor.
The three ligand-protein complexes were used as inputs for classical MD simulations. Each complex was embedded in a 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) lipid bilayer (the total number of lipid molecules in both layers was 778) located in a rectangular simulation box (edge lengths of the box 165 x 165 x 97 Å). The assembled ligand-protein-lipid systems were solvated with ∼46,490 TIP3P water molecules. 126 Na+ and 127 Cl− ions were added to neutralise the system and mimic a physiological 0.150 mol/L NaCl solution. The complexes were embedded in the membrane, and the system was solvated using the CHARMM-GUI web server [6–13]. Each system consisted of a total of ∼325,000 atoms. GROMACS software [14] was used to perform the molecular dynamics simulations of the equilibrated 2-EPRO ligand complexes at 37 °C and 1 bar. Amber FF19SB was used to describe the protein [15] and the membrane was described with the Lipid21 force field [16]. GAFF2 was used to describe ligands, which were parametrised using Amber21 module ANTECHAMBER [17,18]. The protein-ligand binding free energy and the per-residue contributions were calculated via single-trajectory Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) calculations using gmxMMPBSA [19]. MD simulations were performed using the GROMACS (v2022.3) software [20]. The preparation protocol consisted of a restrained minimisation step followed by five equilibration steps, which were designed to gradually relax the system while maintaining its original coordinates. First, steepest descent minimisation was carried out for a maximum of 50,000 steps. Positional restraints were applied with force constants of 4000 kJ mol⁻¹ nm⁻² for backbone atoms, 2000 kJ mol⁻¹ nm⁻² for sidechain atoms, and 1000 kJ mol⁻¹ nm⁻² for both lipid and dihedral atoms. Next, a 1-nanosecond simulation in the isothermal–isochoric ensemble (NVT) was conducted using the same force constants. The temperature was set to 310.15 K, ensuring that the POPC membrane remained above its main phase transition temperature [16,21]. Temperature coupling was handled via the Nosé–Hoover thermostat [22,23] with a time constant of 1 ps. The integration time step was 2 femtoseconds. Periodic boundary conditions were applied, and the Verlet cutoff scheme was used. The cutoff distance for the short-range neighbour list was 1.2 nm, and the van der Waals cutoff was also 1.2 nm. Electrostatics were computed using the Particle Mesh Ewald (PME) method with a Coulomb cutoff of 1.2 nm [24]. All bonds involving hydrogen atoms were constrained using the LINCS algorithm. The neighbour list was updated every 20 steps. Following the NVT simulation, four equilibration steps were performed in the isothermal–isobaric (NpT) ensemble, during which restraints were gradually reduced. The duration and force constants used in each NpT step are summarised in Table S1. Pressure was maintained at 1 bar using the Parrinello–Rahman barostat [25] with a pressure coupling time constant of 2 ps and a compressibility of 4.5 × 10⁻⁵ bar⁻¹.
