Digalloyl Glycoside: A Potential Inhibitor of Trypanosomal PFK from Euphorbia abyssinica J.F. Gmel

Human African trypanosomiasis is an endemic infectious disease caused by Trypanosoma brucei via the bite of tsetse-fly. Most of the drugs used for the treatment, e.g., Suramin, have shown several problems, including the high level of toxicity. Accordingly, the discovery of anti-trypanosomal drugs from natural sources has become an urgent requirement. In our previous study on the anti-trypanosomal potential of Euphorbia species, Euphorbia abyssinica displayed significant anti-trypanosomal activity. Therefore, a phytochemical investigation of the methanolic extract of E. abyssinica was carried out. Twelve compounds, including two triterpenes (1, 2); one sterol-glucoside (4); three ellagic acid derivatives (3, 9, 11); three gallic acid derivatives (5, 6, 10); and three flavonoids (7, 8, 12), were isolated. The structures of isolated compounds were determined through different spectroscopic techniques. Compound (10) was obtained for the first time from genus Euphorbia while all other compounds except compound (4), were firstly reported in E. abyssinica. Consequently, an in silico study was used to estimate the anti-trypanosomal activity of the isolated compounds. Several compounds displayed interesting activity where 1,6-di-O-galloyl-d-glucose (10) appeared as the most potent inhibitor of trypanosomal phosphofructokinase (PFK). Moreover, molecular dynamics (MD) simulations and ADMET calculations were performed for 1,6-di-O-galloyl-d-glucose. In conclusion, 1,6-di-O-galloyl-d-glucose revealed high binding free energy as well as desirable molecular dynamics and pharmacokinetic properties; therefore, it could be suggested for further in vitro and in vivo studies for trypanosomiasis.


Investigation of N-Butanol Fraction of E. abyssinica J.F. Gmel
Chromatographic investigation of n-butanol fraction led to isolation of one compound. The structure of the isolated compound was elucidated using 1 H-NMR, and LC-HRMS.

Investigation of N-Butanol Fraction of E. abyssinica J.F. Gmel
Chromatographic investigation of n-butanol fraction led to isolation of one compound. The structure of the isolated compound was elucidated using 1 H-NMR, and LC-HRMS. Compound (12)

Docking Study for Anti-Trypanosomal Activity
The results of docking procedures (Table 1) contained binding free energies Kcal/mol, binding affinity constant (ki in nm) [24], distances (in Å) from the main residues, and type of interactions. Most compounds exhibited good affinity to the selected pocket according to binding affinity ( Figure 2) while suramin was represented in (Figure 3). Notably compounds 10, 7, 11, 8 and 12 in order, showed good binding affinity energies (from −18.9900 to −23.0767 Kcal/mol) when compared to the co-docked ligand suramin as a positive control ( Figure 4). The main residues involved in the interaction between compounds and T. brucei PFK enzyme were Arg173, Ser341, Asn343, Lys226, Thr201, and Gly107 residues as well as Mg Atom (MG1002) that mark them as good candidates for T. brucei PFK

Docking Study for Anti-Trypanosomal Activity
The results of docking procedures (Table 1) contained binding free energies Kcal/mol, binding affinity constant (ki in nm) [24], distances (in Å) from the main residues, and type of interactions. Most compounds exhibited good affinity to the selected pocket according to binding affinity ( Figure 2) while suramin was represented in (Figure 3). Notably compounds 10, 7, 11, 8 and 12 in order, showed good binding affinity energies (from −18.9900 to −23.0767 Kcal/mol) when compared to the co-docked ligand suramin as a positive control ( Figure 4). The main residues involved in the interaction between compounds and T. brucei PFK enzyme were Arg173, Ser341, Asn343, Lys226, Thr201, and Gly107 residues as well as Mg Atom (MG1002) that mark them as good candidates for T. brucei PFK inhibition, that could be used for the treatment of trypanosomiasis. Hydrogen acceptor and metal interactions were found to be the main formed interactions between compounds and the enzyme. 3D figures of the most active compounds via PyMOL 2.4 software were represented in ( Figure 5).

Molecular Dynamics Simulations
With the aim of proofing the reliability of molecular docking results, further computational validation was achieved through a number of MDS experiments and binding free energy (ΔG) calculations on compound 10 (1,6-di-O-galloyl-D-glucose), as well as suramin. As seen in Figure 6, compound 10 was able to achieve stable binding inside the enzyme′s (i.e., phosphofructokinase, PDB ID:3F5M) active site with an average RMSD from the initial docking pose of 3.1 Å; however, it showed higher fluctuation in comparison with the standard drug suramin. Accordingly, it obtained a binding free energy value (ΔG) of −7.1 kcal/mol (ΔG of suramin was −8.8 kcal/mol).

Molecular Dynamics Simulations
With the aim of proofing the reliability of molecular docking results, further computational validation was achieved through a number of MDS experiments and binding free energy (∆G) calculations on compound 10 (1,6-di-O-galloyl-D-glucose), as well as suramin. As seen in Figure 6, compound 10 was able to achieve stable binding inside the enzyme s (i.e., phosphofructokinase, PDB ID:3F5M) active site with an average RMSD from the initial docking pose of 3.1 Å; however, it showed higher fluctuation in comparison with the standard drug suramin. Accordingly, it obtained a binding free energy value (∆G) of −7.1 kcal/mol (∆G of suramin was −8.8 kcal/mol).

Prediction of the Pharmacokinetic Properties and Toxicological Properties Using ADMET
After the molecular docking studies of 12 isolated compounds, the absorption, distribution, metabolism, elimination, and toxicity (ADMET) of the best dock scored compound along with suramin were evaluated (

Prediction of the Pharmacokinetic Properties and Toxicological Properties Using ADMET
After the molecular docking studies of 12 isolated compounds, the absorption, distribution, metabolism, elimination, and toxicity (ADMET) of the best dock scored compound along with suramin were evaluated (Table 2).

Docking Study for Anti-Trypanosomal Activity
E. abyssinica methanolic extract was previously reported to exhibit potent anti-trypanosomal activity IC 50 17.3 and 19.4 µg/mL after 48 and 72 h incubation [23]. Phytochemical investigation of the methanolic extract was performed for isolation and identification of the major compounds. Twelve pure compounds were isolated and identified. Then, molecular docking was performed with T. brucei Phosphofructokinase (PFK) enzyme where most of them showed good affinity to the selected pocket according to binding affinity results. Interestingly, 1,6-di-O-galloyl-D-glucose (10), kaempferol-3-O-α-L-rhamnoside (7), 3,3 ,4tri-O-methyl-4 -O-rutinosyl-ellagic acid (11), and quercetin-3-O-α-L-rhamnopyrnosyl (8), luteolin-7-O-glucoside (12), in this order, showed good binding affinity energies when compared to the co-docked ligand suramin. Several reports highlighted the in vitro efficacy of some flavonoid compounds against T. brucei [19]. Moreover, penta-O-galloyl-β-D-glucose was revealed to have in vitro anti-trypanosomal activity [48]. Furthermore, gallic acid was cited to exert its effect on T. brucei via iron chelation that caused structural and morphological changes and stopping the cell cycle [49]. Most of the reported data informed that the biological activities of galloyl-glucose were related to the number of galloyl moiety [50]. Moreover, quercetin was reported to exhibit potent anti-trypanosomal activity [51]. Herein, the presented results confirmed the potential activity of the flavonoids and gallic acid derivatives against T. brucei (PFK) enzyme and highlighted the high effect of ellagic acid derivatives for further in vitro investigation.

Molecular Dynamics Simulations
Molecular dynamics (MD) simulation was scientifically used to confirm the reliability of physics-based methodology to evaluate protein-ligand binding interactions [52]. In the current study, MD simulations were carried out on the spike protein (PFK), viz., compound 10 (1,6-di-O-galloyl-D-glucose), and suramin. The results revealed that 1,6-di-O-galloyl-Dglucose held a structural role in modulating the conformational dynamics of the protein.
Elsewhere, it was able to solely shield the spike protein and stabilize PFK-like suramin.

Prediction of the Pharmacokinetic Properties and Toxicological Properties Using ADMET
1,6-di-O-galloyl-D-glucose (10) owns a low molecular weight of less than 500 Da that was considered a major advantage when compared with the larger molecular weight of suramin. This improved the absorption and decreases the toxicity over suramin, which was reported to cause renal impairment [11]. However, tannins were considered to be safe or even beneficial at low dietary levels [50]. The results of the ADMET prediction revealed that 1,6-di-O-galloyl-D-glucose showed high water solubility and respectable cellular permeability. 1,6-di-O-galloyl-D-glucose was likely to be a substrate for P-glycoprotein which was an ATP-binding cassette (ABC) transporter, so it was able to modulate the physiological functions of P-glycoprotein in limiting the active uptake. In addition, the prediction of the distribution properties showed poor blood-brain barrier (BBB) permeability and CNS permeability. Furthermore, 1,6-di-O-galloyl-D-glucose displayed good volume of distribution. However, there was no significant effect on cytochrome P450 metabolism or renal OCT2 substrate excretion. The total clearance as Log(CLtot) was also performed. It predicted the combination of hepatic clearance (metabolism in the liver and biliary clearance) and renal clearance (excretion via the kidneys) and it was found to be 0.47 mL/min/kg. Moreover, pkCSM software was used to predict the toxicological properties of 1,6-di-O-galloyl-D-glucose, such as mutagenicity, hepatotoxicity, cardiotoxicity, and skin sensitization. Herein, the bacterial mutagenic Ames toxicity testing showed that it was a non-mutagenic compound, but the toxicity in T. pyriformis and the cardiotoxicity, in the form of human ether-a-go-go-related gene II (hERG II) is high. Lastly, the maximum tolerated human dose is somewhat acceptable. Conclusively, 1,6-di-O-galloyl-D-glucose showed better-predicted safety and oral bioavailability than the synthetic drug "suramin".
Glass tanks for extraction and development of TLC chromatograms, Rotary evaporator (Buchi, labortechnik AG 9230 Flawil, Switzerland) for the concentration of extracts and fractions, micropipettes (0.1 mL), for spot application, glass columns for chromatography with different dimensions (120 × 5.5 cm, 100 × 5 cm, 30 × 5 cm, 50 × 3 cm, and 40 × 2 cm), an atomizer for spraying the chromatograms, sensitive electric balance (Sartorius, type 1712, West Germany), portable ultraviolet lamb for localization of spots on thin-layer chromatograms (λ max = 254 and 330 nm, Shimadzu), a product of Hanovia Lambs, UVvisible spectrophotometer, Shimadzu UV (P/N 204-58000) was used for recording UV spectra and measuring the absorbance in the UV range, and Bruker Ascend TM 400/R NMR spectrometer, 1 H-NMR, 400 MHZ, DEPT-Q NMR, 100 MHz spectra were recorded in a suitable deuterated solvent using TMS as internal standard and chemical shift values expressed in δ ppm (NMR Laboratory, Microanalytical unit) faculty of pharmacy, Beni-Suef University.

Extraction and Fractionation
The fresh non-flowering aerial parts of E. abyssinica J.F. Gmel. (6.5 kg) were cut into small pieces with a knife then with vegetable chopper and extracted by cold maceration with methanol (80%) till exhaustion. The methanolic extract was evaporated using the rotary evaporator to yield 300 g residue. About 250 g of the residue were suspended in 300 mL distilled water and partitioned successively with n-hexane (4 × 500 mL), methylene chloride (6 × 500 mL), ethyl acetate (5 × 500 mL), and n-butanol saturated with water (7 × 500 mL). Different fractions were evaporated to dryness using the rotary evaporator. The yield of the different extractives was 8, 12, 10, and 15 g residue of the n-hexane, methylene chloride, ethyl acetate, and n-butanol fractions, respectively.

Investigation of Ethyl Acetate Fraction of E. abyssinica J.F. Gmel
The ethyl acetate fraction (8 g) was chromatographed on 125 g polyamide column. Gradient elution was carried out using distilled water, then with distilled water containing 10% increment of methanol till 100% methanol. Fractions of 100 mL each were collected and monitored by TLC using methylene chloride, methanol (9:1 and 8.5:1.5), as a solvent system. Similar fractions were pooled together whereby 4 fractions (F1-F4) were obtained. Fraction 3 (3.0 g) was rechromatographed over 150 g silica gel H and gradient eluted with methylene chloride then with methylene chloride containing 10% increment of ethyl acetate till 100% ethyl acetate and then with washed by methanol. Fractions of 100 mL each were collected and monitored by TLC using methylene chloride: methanol (9:1, 8.5:1.5, and 8:2) as a solvent system, 5 subfractions (f1-f5) were obtained. After further purification of f2 (100 mg) on 25 g of Sephadex-LH 20 column using 100% methanol as a solvent system, compound (5) (17 mg) and compound (6) (14 mg) were obtained.

Docking Study for Anti-Trypanosomal Activity
To investigate the protein-ligand interactions, isolated compounds from E. abyssinica were drawn using Marvin sketch powered by Chem-Axon, and ChemBioDraw Ultra 14.0, and then they were applied to a molecular operating environment (MOE) platform to undergo energy optimization for each compound using the MMFF94× force-field. The crystal structure of ATP-bound phosphofructokinase from T. brucei (PDB ID:3F5M) contains 4 chains protein structure and co-crystallized with ATP ligand, snapshotted with X-ray diffraction at 2.70 Å resolution [5]. The structure was obtained from the RSCB protein data bank (http://www.rscb.org accessed on 17 November 2021) and the molecular docking was conducted using the MOE 2020.0101 package.
Visualization and generation of the 3D figures were performed using PyMOL 2.4 software. To ensure the validity of the docking protocol, re-docking of the co-crystallized native ligand into the active site was performed. The coordinates of the best scoring docking pose of the native ligand were compared with its coordinates in the co-crystallized PDB file based on the binding mode and root mean square deviation (RMSD). They showed an alignment with the original ligand as obtained from the X-ray resolved PDB file. The isolated 12 compounds from E. abyssinica and suramin were docked into PFK active domain, then 50 poses of each compound were scored by initial rescoring methodology (London dG) and the final re-scoring methodology (London dG) after placement using Triangle Matcher and post-placement refinement was force-field.

Molecular Dynamics Simulations
Molecular dynamic simulations (MDS) for the generated ligand-enzyme complexes were performed using the Nanoscale Molecular Dynamics (NAMD) 2.6 software [55], applying the CHARMM27 force field [56]. Hydrogen atoms were added to the protein structures using the psfgen plugin included in the Visual Molecular Dynamic (VMD) 1.9 software [57]. Afterward, the whole generated systems were solvated using water molecules (TIP3P) and 0.15 M NaCl. At first, the total energy of the generated systems was minimized and gradually heated to reach 300 K and equilibrated for 200 s. Subsequently, the MDS was continued for 50 ns, and the trajectory was stored every 0.1 ns and further analyzed with the VMD 1.9 software. The MDS output was sampled every 0.1 ns to calculate the root mean square deviation (RMSD). The parameters of compound 4 were prepared using the online software the VMD Force Field Toolkit (ffTK) [57]. Binding free energies (∆G) were calculated using the free energy perturbation (FEP) method [58]. The web-based software Absolute Ligand Binder was used to generate the input files for NAMD software which was performed the simulations required for ∆Gs calculations [58].

Prediction of the Pharmacokinetic Properties and Toxicological Properties Using ADMET
The online pkCSM pharmacokinetics prediction properties were used for the calculation of the pharmacokinetic properties of compound (10) and suramin (http://biosig. unimelb.edu.au/pkcsm/prediction accessed on 18 November 2021). The following properties were investigated: Absorption, (water solubility, Caco-2 permeability, intestinal human absorption (HIA), skin permeability, and P-glycoprotein interactions); distribution, (VDss, Fu, Log BB, and CNS permeability); metabolism; excretion. Furthermore, online pkCSM pharmacokinetics were used to predict the toxicity of the molecules, including skin sensitization, hepatotoxicity, and others. The results were analyzed and compared with the reference values of the pkCSM pharmacokinetics prediction properties.

Conclusions
Based upon the previous reports of the significant anti-trypanosomal activity of E. abyssinica methanolic extract, a phytochemical investigation of different fractions was carried out. Twelve compounds were isolated where 1,6-di-O-galloyl-D-glucose (10) was isolated for the first time from the Euphoria genus. Moreover, molecular docking of the isolated compounds with T. brucei PFK enzyme indicated compound (10) as a potent trypanosomal PFK inhibitor. The binding stability of (10) inside the pocket of the PFK proteins with time was further validated through molecular dynamics simulations involving root mean square deviation and estimated as~3.2 Å. Furthermore, ADMET showed satisfactory pharmacokinetic and toxicological properties. The predicted pharmacokinetic properties were within the standardized range for human use. Therefore, combining the docking results, ADMET predictions, and the biological activity of compound 10, we suggest this compound as a promising candidate for further in vitro, in vivo, and clinical studies.