Cannabis sativa L. chemical compositions as potential plasmodium falciparum dihydrofolate reductase-thymidinesynthase enzyme inhibitors: An in silico study for drug development

2.1 Protein and ligand preparation

Among 14 structures of enzyme pfDHFR-TS available from Protein Data Bank (RCSB PDB), the best structure with PDB ID: 1J3I was selected for research with a resolution of 2.33 Å [10,11]. The protein structure was prepared using the Graphical User Interface program named Autodock Tools to produce accurate representation of amino acid residues in terms of ionization and tautomeric states [12]. Procedures of the protein preparation process included removal of water molecules, addition of polar hydrogen atoms, and assignment of Kollman united atom partial charges and salvation parameters. Obtained atomic coordinates of the protein were then exported into a PDBQT file which will be used for execution of AutoGrid and AutoDock.

The chemical structure of 125 compounds isolated from Cannabis Sativa L. were collected from published literature [8,9,13] (Figure S1). Chemical structures of these compounds were visualized using Marvin software. The 3D structure of the compounds were built using Pymol 2.2.2 [14]. The energy minimization was carried out using Gabedit 2.5.0 [15]. Open bioactivity prediction online server Molinspiration and ProTox-II were utilized to evaluate the drug-like properties and the acute toxicity of all the research compounds.

3.1 Validation docking

Two co-crystallized ligand cycloguanil and WR99210 redocked as references with pfDHFR-TS resulted in dock score of −9.32 and −9.92 kcal/mol, respectively. These two inhibitors were both shown to form hydrogen bonds with Ile14, Asp54, and Ile164, as indicated previously in the literature [20] (Figure 1).

Figure 1

Hydrogen bonding patterns of cycloguanil and WR99210 inhibitors with enzyme pfDHFR-TS (PDB ID: 1J3I). (a) Cycloguanil and (b) WR99210.

Obtained results show that these inhibitors share the common residues interaction similar to previously published studies, suggesting that the procedure and the set parameters were suitable for docking simulation and are reproducible.

3.2 Drug-like and pharmacokinetic properties assessment

As part of the in silico screening, the drug-like properties of 125 compounds were assessed by subjecting them to Lipinski’s rule of five (Table S1). This rule includes criteria that determine which compound is considered to be drug-like in nature, such as molecular weight (MW) < 500 Da, number of hydrogen bond donors (HBD) ≤5, number of hydrogen bond acceptors (HBA) ≤10, octanol-water partition coefficient (log P) < 5, and molar refractivity (MR) value between 40–130. Compounds that satisfy these rules would be considered to be drug-like in nature. The obtained results help in providing essential information regarding the development and discovery of new drugs.

The outcomes indicate that amongst the studied molecules, 65 candidates were sorted out as favorable for drug development (Table 1). These compounds were then further evaluated for pharmacokinetic properties and toxicity prediction using Molinspiration and ProTox-II cheminformatic server (Table S2) [21]. In general, the studied compounds showed interesting results regarding calculated toxicity. From Table S2, compounds 5, 54, and 60 were classified as non-toxic with very high LD₅₀ (6,000, 10,000 and 13,500 mg/kg, respectively). Ten compounds were positioned at rank 5 and could be considered as safe. Forty-five compounds were classified as compounds with low toxicity (rank 4) which is equivalent to the toxic prediction of cycloguanil and WR99210. On the other hand, it is observed that three compounds 5, 17, and 63 had their milog P value lain in minus values (−0.9, −1.99, and −4.3, respectively) which suggest their inability to bind with pfDHFR-TS enzyme. In addition, the enzyme inhibitory potential value of compounds 29 and 62 were −0.14 and −0.32, respectively, indicating that these compounds are not likely to exhibit inhibition activity toward target enzyme. Thus, these five ligands were excluded and only 60 amongst 65 compounds possess pharmacokinetic properties and toxic ranking suitable for further docking studies.

3.3 Docking studies

To explore the inhibition potential of 60 selected ligands with enzyme pfDHFR-TS model, AutoDock4.2.6 and AutoDock Vina were utilized for docking studies. Table 2 exhibits the dock score of the studied compounds.

Cycloguanil and WR99210 were used as reference inhibitors with dock score obtained from AutoDock4.2.6 being −7.77 and −8.91 kcal/mol and dock score of AutoDock Vina being −8.50 and −9.10 kcal/mol, respectively. Thus, ligand has docking energy fall within these range or more negative would be considered as potential inhibitor of pfDHFR-TS. In general, according to the obtained results, 16 out of 60 screened compounds were identified as potential inhibitors (Table 2). Compounds 9 and 61 are the top two ligands with docking energy far more exceeding those of standard compounds. The rest 14 hits presented dock scores that matched the selection criteria and had dock score ranging from −7.85 to –9.06 kcal/mol, and from −8.5 to −9.4 kcal/mol, obtained from AutoDock and AutoDock Vina, respectively. On the other hand, a high correlation coefficient between the docking energy of AutoDock4.2.6 and AutoDock Vina was recorded with the value of R ² = 0.74 (Figure 2) that supports for the accuracy of the docking study.

Figure 2

Correlation graph between dock score of AutoDock4.2.6 and AutoDock Vina.

The potential hits were further analyzed for ligand efficiency (LE) and binding poses (Table 3). LE is a useful metric for the selection of lead compounds in drug discovery and has been widely used as a measurement for the binding energy of the ligand per atom, which is calculated according to the equation (1):

where Δg is the ligand efficiency and ΔG is the docking energy.

Statistically, compounds with LE varying within 0.3 < LE < 0.5 are more potential for further optimization. Calculated LE of 16 hit compounds in this study ranged from −0.39 to −0.47, suggesting their considerable potential for drug development.

For inhibition to occur, interaction is needed on key amino acid residues Ile14, Asp54, and Ileu164 at the active site of the enzyme [20]. As indicated in Table 3, although having better docking energy than standard cycloguanil and WR99210, compounds 13, 28, 45, 61, and 65 did not form hydrogen bonds with any of three key residues, therefore, these ligands were assumed as non-specific for pfDHFR-TS inhibition potential. Regarding the toxicity prediction (Table 2), compound 41 was excluded due to high toxicity (LD₅₀ value of 3 mg/kg). Compounds 30 and 61 were highlighted for their safe class of toxicity (LD₅₀ value of 3,919 and 2,647 mg/kg, respectively) (Table S1). The hydrogen bonding patterns and stereo view of binding mode of ten potential pfDHFR-TS inhibition compounds are shown in Figures S2 and S3.