Chemdraw professional

The statistical analysis and illustration were carried out using GraphPad Prism 8 (version 8.0.0 (224)) software. Results were expressed as mean ± standard deviation. Mean values were compared by either unpaired t-test (with Welch’s correction when no homoscedasticity) or an ordinary one-way Welch’s ANOVA test (with Welch’s correction when no homoscedasticity) followed by Games-Howell’s multiple comparisons post-hoc test. A p value < 0.05 was considered to be statistically significant. Prior to ANOVA, normal distribution using Shapiro–Wilk test (α = 0.05) and homoscedasticity using Brown-Forsythe test (p < 0.05) were asserted. ChemDraw Professional (version 19.0.0.26) was used to generate the chemical structures. Principle component analysis (PCA) was performed on fourth root calculated data. The dataset was composed of the commercial (n = 11) and primary pine EOs (n = 54) characterized by 39 compounds and ( ±)-α-pinene (Supplementary Information, Table A3 and A4). PCA was performed with Rstudio (version 1.2.5019; packages: ggbiplot, version 0.55; pca3d, version 0.10).

The hybrid compound 8g was rendered in two-dimensional (2D) images using the ChemDraw Professional
(Version 19.1.1.21) software package, converted to SDF format, and
then prepared for docking using the Molecular Operating Environment
(MOE 2019.01) software package. After loading the SDF files, it was
processed as follows: the compound was energy-minimized and partial
charges were added (Amber10 force field) using QuickPrep. To prepare
the enzyme, the PvNMT PDB file was loaded into MOE
and processed using QuickPrep. The docking simulation was set up by
setting the receptor to “receptor+solvent”. The SDF
file containing the processed ligands to be docked was loaded. Ligand
placement and refinement were performed using the α PMI and
induced fit methods, with 30 and 3 poses, respectively.

The hybrid compound 12e was rendered in the form of 2D images using the ChemDraw Professional (Version 19.1.1.21) software package, converted to SDF format, and then prepared for docking using the Molecular Operating Environment (MOE 2019.01) software package. After loading the SDF files, it was processed as follows: the compound was energy-minimized, and partial charges were added (Amber10 forcefield) using QuickPrep. To prepare the receptor protein, the PvNMT PDB file (PDB: 6MB1) was loaded into MOE and processed using QuickPrep. The docking simulation was set up by setting the receptor to “receptor+solvent.” The SDF file containing the processed ligands to be docked was loaded. Ligand placement and refinement were performed using the Alpha PMI and rigid receptor methods, with 30 and 3 poses, respectively.

The structure of PPH, polyphyllin VI and polyphyllin VII were drawn by using ChemDraw Professional, and was then converted into Mol2 format files by Chem3D 17.0. To optimize the molecular structures and obtain energetically favourable conformations, energy minimization calculations were performed using the Tripos force field. This conformation process was carried out using SYBYL‐X 2.0. The crystal structure of KEAP1 (PDB code: 4l7b) was obtained from the PDB database (http://www.pdb.org). Prior to the docking procedure, all water molecules and co‐crystallized ligands were removed from the KEAP1 structure. Subsequently, hydrogen atoms were added and gasteiger charges were computed. The protein structure was saved as Mol2 format files. KEAP1 and corresponding small molecules were then docked in SYBYL‐X 2.0 by using Surflex‐Dock docking mode. The binding energy was calculated and the molecular interaction was visualized by Pymol 2.4.0 visualization tool.

All the structures included in the 3D-plot were converted into SMILES using ChemDraw Professional 16.0.0.82 and uploaded to the ChemGPS-NP_Web tool (http://chemgps.bmc.uu.se) [25 (link)]. The resulting coordinates were plotted using Grapher 2.5 distributed together with MacOS X. All the pyrimidines reported in this article were included. The following list comprises all the other compounds in alphabetical order and relevant/available K_i values for PKCα are indicated in parenthesis: 9-decyl-benzolactam-V8 (3.8 nM) [32 (link)]; bryostatin-1 (1.35 nM) [33 (link)], bryostatin-18 (4.8 nM) [34 (link)]; (E)-DAG-lactone 31 (2.7 nM) [16 (link)], (Z)-DAG-lactone 9 (11 nM) [35 (link)]; HMI-1a1 and -1a2, HMI-1a3 (205 nM), HMI-1b1–1b10, HMI-1b11 (319 nM), HMI-1b12–1b21, HMI-15e, -22c and -24a [12 (link)]; indolactam-V (11 nM) [36 (link)]; ingenol 3-angelate (0.1 nM) [37 (link)]; iripallidal (75.6 nM) [38 (link)]; mezerein (0.27 nM) [36 (link)]; phorbol 13-acetate (120 μM) [39 (link)], phorbol 12,13-dibutyrate (0.3 nM) [37 (link)], phorbol 12-myristate-13-acetate (2 nM) [39 (link)]; prostratin (4.83 nM) [36 (link)]. The full list of the compounds, ChemGPS-NP raw data, SMILES and structures are available in S1 File.

A bar diagram was plotted for the cytotoxicity test to visualize the “mean percent of inhibition ± standard error” of cell proliferation using Microsoft^® Excel 2019 software. The significance of the lactucin doses was compared with control sample data and calculated by “one-way ANOVA” using “IBM^® SPSS^® Statistics 26”. Significant difference at p 0.05, 0.01, 0.001, and 0.0001 was calculated and expressed with different Asterix “*” numbers on the chart or the data table legend. IC₅₀ was calculated by plotting the Log10 dose on X-axis versus the Normalized response on the Y-axis in Graph Pad Prism 7. Cell cycle cytometry data were analyzed using ModFit LT™ (Version 5.0.9). Apoptosis compensation calculation and data analysis were done using CytExpert™ (Version 2.3.0.84). Protein concentrations were calculated in Microsoft^® Excel 2019. WB bands were quantified using ImageJ^® (Version 1.52a) and analyzed using Graph Pad Prism 7. NMR data was analyzed, the structure was predicted using MestReNova™, and the chemical structure was made using ChemDraw^® Professional. LC-MS/MS results analyzed in MASCOT were combined with WB markers data, and functional enrichment was performed to predict the involved molecular pathway using DAVID Bioinformatics resources [36 (link),37 (link)].