Introduction

Nature seems to be the most efficient designer and producer of biologically active substances that are of great importance and value for the fabrication of drugs. Throughout the whole history of the mankind, nature has become a bottomless pantry of drugs or their components, which are used for the treatment or in therapy of different diseases. Just sufficient to say, nature was the only source of, e.g., quinine, strychnine, morphine, aspirin, penicillin and many other bioactive compounds called natural products. The latter three products are also known as key milestones in the development of commercial production of medicines. Even today, natural compounds have not lost their relevance, but on the contrary, they are attracting more and more attention due to both their unique properties and commercial perspectives [1].

Of a myriad of natural products, terpenes are a large class comprising several tens of thousands of compounds. According to the IUPAC Gold Book, terpenes are hydrocarbons of biological origin having carbon skeletons formally derived from isoprene. This class is subdivided into the C5 hemiterpenes, C10 monoterpenes, C15 sesquiterpenes, C20 diterpenes, C25 sesterterpenes, C30 triterpenes, C40 tetraterpenes (carotenoids) and C5n polyterpenes [2, 3]. These hydrocarbons are obtained primarily from plants [4] and are of particular interest for both industrial application and medicine. Nowadays, of the triterpene subclass representatives, betulin (3-lup-20(29)-ene-3β,28-diol) seems to be one of the most attractive compounds, especially for its pharmacological and anticancer properties, and as a precursor of new drugs [5,6,7]. Betulin, which is also known as betuline, betulinol or betulinic alcohol, is a pentacyclic lupane-type triterpene (Fig. 1). It was one of the first natural products obtained by Lowitz in 1788 [8], which structure was elucidated in 1952. The main natural source of betulin nowadays is the birch bark, from which the title compound can efficiently be extracted [9, 10]. The content of betulin in the birch bark can vary from about 10% up to about 35% depending on a number of different factors such as the type of birch, the age of the tree, growth conditions and place [9, 10]. Considering betulin as a chemical reagent, it has three functionalities, namely two hydroxyl groups and isopropenyl fragment (Fig. 1), which can be involved in chemical modifications, yielding a rich panel of derivatives with a wide spectrum of properties of value. Thus, betulin is an important and valuable precursor.

Fig. 1
figure 1

Diagram of betulin

Full size image

On the other hand, corrosion of metals and metallic materials is one of the main problems of industry, including medical industry especially where metal-based biomedical implants are used [11,12,13]. As one of the most efficient corrosion inhibitors, organic compounds have been of particular interest and quantum chemical calculations have become a powerful tool to probe molecule-surface bonding, that is crucial for corrosion inhibition [14,15,16,17,18].

With all this in mind, as well as in continuation of our ongoing interest in in silico studies of bioactive compounds [19,20,21,22,23,24,25,26,27,28,29], we have directed our attention to betulin. Theoretical calculations based on density functional theory (DFT) were performed to examine electronic properties of this compound. The global chemical reactivity descriptors were estimated from the energy of the HOMO and LUMO orbitals to examine the relative reactivity of the molecule. We have also applied the DFT calculations to probe betulin as a potential corrosion inhibitor for some important metals used in implants. ADMET properties of betulin were additionally evaluated. Molecular docking and molecular dynamics simulations were applied to probe interaction of betulin with a series of the SARS-CoV-2 proteins as well as one of the monkeypox proteins.

Experimental

Computational details

The ground state geometry of betulin in gas phase was fully optimized without symmetry restrictions. The calculations were performed by means of the GaussView 6.0 molecular visualization program [30] and the Gaussian 09, Revision D.01 program package [31], using the DFT method with Becke-three-parameter-Lee–Yang–Parr (B3LYP) hybrid functional [32, 33] and 6-311G++(d,p) [32, 34] basis set. The electronic isosurfaces of the molecular orbitals and molecular electrostatic potential (MEP) surface were generated from the fully optimized ground state geometry obtained by using the B3LYP/6–311++G(d,p) method. The density-of-states (DOS) plot was calculated using the GaussSum 3.0 software [35, 36].

In silico drug-likeness analysis

Bioavailability, druggability and toxicity properties of betulin were evaluated using the SwissADME [37], BOILED-Egg [38] and ProTox-II [39, 40] tools.

Molecular docking

Molecular docking simulations of the optimized structure of betulin with a series of the SARS-CoV-2 proteins and A42R profilin-like protein from monkeypox virus Zaire-96-I-16 were carried out with AutoDock Vina [41, 42], using the Lamarckian genetic algorithm (LGA) scoring function with number of GA runs = 100, population size = 500 and maximum number of evaluations = 25 000 000. The targeted protein structures were acquired via the RCSB PDB database [43] and were pretreated before the docking, including water removing, and inserting hydrogen atoms and missing residues and charges. Gasteiger charges were added to the ligand molecules prior converting to PDBQT format. AutoDock Tools (v. 1.5.7) was utilized to define the grid box with the dimensions of 30 × 30 × 30 Å with 0.375 Å grid spacing. Semi flexible docking was performed keeping the receptor molecule rigid and ligands flexible. During the docking procedure, 200 conformations for each ligand were left flexible, while the protein was held rigid. The lowest binding energy conformers and 2D interactions were filtered from 10 top-ranked poses. BIOVIA Discovery Studio 2020 [44] was utilized for visualization of the docked conformations and 3D target-ligand interactions.

Molecular dynamics simulation

Molecular dynamics (MD) simulations of the complex of betulin with papain-like protease (PLpro) were performed using the WebGRO on-line service [45]. Parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA) and intermolecular hydrogen bonds were assessed. The complex was prepared for MD using GROMOS96 43a1 forcefield and was equilibrated using the canonical (NVT) and the isothermal–isobaric (NPT) ensembles. The betulin topology was generated with the PRODRG tool [46]. Simple point charge (SPC) was used as a solvent model (triclinic water box with size 50 × 75 × 70 Å) for protein–betulin complex [47]. This system was neutralized by adding sodium or chlorine ions based on the total charges. For minimization of the system before MD, the steepest descent algorithm (5000 steps) was applied. The MD simulations were performed in the presence 0.15 M NaCl using the constant temperature (310 K) and pressure (1.0 bar). Approximate number of frame per simulation was 1000. The simulation time was set to 100 ns.

Results and discussion

We have applied the DFT calculations to probe the electronic properties of betulin. Firtsly, the structure of betulin was optimized in gas phase at 298.15 K, using the DFT/B3LYP/6–311++G(d,p) method. In the optimized structure, all the cyclohexane cycles adopt a chair conformation, while the cyclopentane fragment adopts an envelope conformation (Fig. 2). The obtained bond lengths and angles are typical. Particularly, the C–C bond lengths vary from 1.510 to 1.612 Å (Table 1), indicating their single bond nature. Interestingly, the longest C–C bond length was revealed for the C39–C82 bond (Table 1), which is formed between the two opposite quaternary carbon atoms of the central cyclohexane ring, thus possessing a certain degree of repulsion due to steric hindrance (Fig. 2). The C71–C79 bond length is 1.335 Å, characteristic for a double bond. Finally, the two C–O bonds are 1.432 and 1.434 Å (Table 1). The C–C–C bond angles, formed by two single bonds, vary from 100.80 to 120.15° (Table 1), with the lowest values corresponding to the cyclopentane intracyclic bond angles (Fig. 2 and Table 1). The sum of the C–C–C bond angles around the C71 carbon atom is almost 360° (359.91°), indicating its sp2-hybridization. In general, the geometrical parameters of the optimized molecule of betulin are in excellent agreement with those found in a number of solvate structures collected in the Cambridge Structural Database (CSD) [48].

Fig. 2
figure 2

The optimized structure of betulin, obtained using the DFT/B3LYP/6-311G++(d,p) method

Full size image
Table 1 Selected bond lengths (Å) and bond angles (°) in the optimized structure of betulin, obtained using the DFT/B3LYP/6-311G++(d,p) method (see Fig. 2 for atoms labeling)
Full size table

Analysis of the Mulliken atomic charges in the optimized structure of betulin revealed that all the hydrogen atoms are positively charged varying from 0.072 to 0.243 a.u. with the highest values corresponding to the hydroxyl hydrogen atoms (Fig. 3). Of non-hydrogen atoms, exclusively the C22, C26, and C71 carbon atoms carry the positive charge ranging from 0.074 to 0.115 a.u., while the C14 and C55 carbons are the most negatively charged and of −0.647 and −0.548 a.u., respectively (Fig. 3). Finally, the oxygen atoms are both negatively charged and of −0.152 and −0.198 a.u. (Fig. 3).

Fig. 3
figure 3

Mulliken atomic charges in the optimized structure of betulin, obtained using the DFT/B3LYP/6-311G++(d,p) method (see Fig. 2 for atoms labeling)

Full size image

The electrophilic and nucleophilic sites in a molecule of betulin were revealed using the molecular electrostatic potential (MEP) analysis. On the MEP surface of betulin, the most pronounced nucleophilic (red color) and electrophilic (blue color) centers are located, as expected, on the hydroxyl oxygen and hydrogen atoms, respectively (Fig. 4).

Fig. 4
figure 4

View of the molecular electrostatic potential surface of betulin, obtained using the DFT/B3LYP/6-311G++(d,p) method

Full size image

According to the DFT calculations, the dipole moment of betulin in gas phase is 0.7627 Debye with the highest contribution of the μz component (Table 2). Such low dipole moment is due to the overall balance in the charge from one side of a molecule to the other side as evidenced from the corrsponding MEP surface (Fig. 4). The energies of the highest occupied molecular orbital (HOMO) and lowest lying unoccupied molecular orbital (LUMO) for betulin are −6.71768 and −0.46069 eV, respectively, with the energy gap of 6.25699 eV (Table 2). The HOMO is mainly delocalized over the isopropenylcyclopentane fragment, while the LUMO is mainly spread around the CH2OH fragment (Fig. 5). The density-of-states (DOS) plot of betulin is shown in Fig. 6. Chemical potential (μ) was found to be −3.58919 eV, indicating electron donating ability and low electron accepting ability. The chemical hardness (η) describes the resistance towards deformation/polarization of the electron cloud of the molecule upon a chemical reaction, while softness (S) is a reverse of chemical hardness. The title compound is characterized by a relatively high value of the chemical hardness and a relatively low value of the chemical softness, indicating it exhibits a strong tendency to exchange its electron cloud with surrounding environment (Table 2). The electrophilicity index (ω), which is denoted as the energy of stabilization to accept electrons, is 2.05886 eV. This value is characteristic for strong electrophiles [49]. Finally, a molecule of betulin can accept about 1.15 electrons as evidenced from the ΔNmax value (Table 2).

Table 2 Dipole moment, frontier molecular orbitals, gap value, descriptors, and molecule-to-metal electron charge transfer values for betulin in gas phase, obtained using the DFT/B3LYP/6–311++G(d,p) method
Full size table
Fig. 5
figure 5

Energy levels and views on the electronic isosurfaces of the HOMO and LUMO of the ground state of betulin, obtained using the DFT/B3LYP/6-311G++(d,p) method

Full size image
Fig. 6
figure 6

The DOS plot for betulin, obtained using the DFT/B3LYP/6-311G++(d,p) method

Full size image

The HOMO and LUMO values are of importance to estimate corrosion inhibition properties of a compound [14,15,16,17]. This becomes even more crucial considering metal-based biomedical implants [11,12,13]. We have also probed potential corrosion inhibition properties of betulin toward a series of metals, which are prominent components of biomedical implants (Table 2) [13]. As such, we have used the most reliable equation, which includes the so-called work function (Ф) [50], to calculate electron charge transfer (Table 2) [18]. According to the obtained results, it was revealed electron charge transfer from the molecule of betulin to the surface of all the studied metals, of which the most efficient electron charge transfer was established for Ni, Au and Co (Table 2). Total negative charge (TNC) value is a parameter to indicate an adsorption of a molecule-inhibitor onto a metal surface and defined as a sum of negative Mulliken charges of atoms. As higher the absolute value of TNC as better a molecule will donate electrons to the metal surface, thus exhibiting better corrosion inhibition efficiency. For betulin, the TNC value was calculated as −7.70926 electrons (Table 2).

Interestingly, corrosion inhibition properties toward some of the metals estimated for betulin are comparable (and even superior) with those recently obtained for different tautomers of salen [29]. Notably, the latter compound belongs to salicylaldehyde based Schiff bases — a prominent class of corrosion inhibitors [51,52,53].

According to ProTox-II, a virtual lab for the prediction of toxicities of small molecules [39, 40], betulin belongs to the fourth class of toxicity (Fig. 7). As evidenced from the SwissADME [37] bioavailability radar, the title compound is highly preferred in four parameters, namely size, polarity, insaturation and flexibility, while it has a relatively poor result in the lipophilicity and insolubility parameters (Fig. 7). The latter features are obviously due to an extended polycyclic hydrocarbon structure of betulin. It was predicted that betulin is likely a pronounced inhibitor of enzyme, cytochrome P450, and nuclear receptor with the probabilities of 33.3%, 20.0% and 13.3%, respectively (Fig. 7). According to the toxicity model report, betulin was revealed to be inactive toward the listed targets (Fig. 7).

Fig. 7
figure 7

Toxicity results, calculated by ProTox-II (top-left and bottom), druggability predictions (top-right), BOILED-Egg model (top-middle) together with the bioavailability radar within the domain borders of ADME properties, calculated by SwissADME, of betulin (the colored zone of the radar is the suitable physicochemical space for oral bioavailability)

Full size image

The BOILED-Egg method was found to be efficient to predict the human blood–brain barrier (BBB) penetration and gastrointestinal absorption [38]. This approach is based on lipophilicity (WLOGP) and polarity (topological polar surface area, TPSA) (Fig. 7). Points located in the yellow region (BOILED-Egg’s yolk) are molecules predicted to passively permeate through the BBB, while points located in the white region (BOILED Egg’s white) are molecules predicted to be passively absorbed by the gastrointestinal tract. Blue (PGP+) and red (PGP–) dots are for molecules predicted to be effluated and not to be effluated from the central nervous system by the P-glycoprotein, respectively. As evidenced from the red dot position for betulin, both BBB and gastrointestinal absorption properties are negative with the negative PGP effect on the molecule (Fig. 7). This is explained by a high WLOGP value of 7.00 (Fig. 7).

We have further applied a molecular docking approach for betulin against a series of SARS-CoV-2 proteins. This approach allows to visualize and explicate interaction between a small compound as a ligand and biomolecule as a target [54]. This application is one of the most broadly exerted technique to examine the structure–activity relationship and biological activity in the drug discovery [55]. Docking is the best option to diminish the time and cost of synthesis of molecules of interest. In addition, it is considered a current and advantageous method to have insight information of the possible binding site of the ligand in the protein [56].

In this study, molecular docking was employed to rationalize betulin in the SARS-CoV-2 targets. The target structures were primarily selected in accordance with the structural features of the virus [57, 58] as well as based on biological mechanisms and functions that can be utilized to reduce, prevent or treat the virus [59] (Table 3).

Table 3 The best poses of betulin inside the binding sites of the listed proteins
Full size table

According to the docking analysis results, betulin was found to be active against all the applied SARS-CoV-2 proteins with the best binding affinity with papain-like protease (PLpro) and spike protein, RBD (native) (Fig. 8 and Table 3). Complexes of betulin with these proteins are exclusively defined with a conventional hydrogen bond with TYR268 and ASN343, respectively (Fig. 8 and Table 4). However, interaction of betulin with spike protein, RBD (mutated) is less efficient (Table 3), which is explained by the complex formation through a much weaker conventional hydrogen bond with GLY354, although two alkyl interactions with ALA386 and ALA387 are also formed (Fig. 8 and Table 4).

Fig. 8
figure 8

2D (left) and 3D (right) views on the interaction of betulin with (from top to bottom) papain-like protease (PLpro), spike protein (native), spike protein (mutated) and A42R profilin-like protein from monkeypox (strain Zaire-96-I-16)

Full size image
Table 4 The best types of interactions and distances of betulin with papain-like protease (PLpro), spike protein (native), spike protein (mutated) and A42R profilin-like protein from monkeypox (strain Zaire-96-I-16)
Full size table

The obtained molecular docking results for betulin are comparable with those found for initial redocked ligands [23], remdesivir [23], molnupiravir [25] and different tautomers of salen [29], and superior to those calculated for favipiravir [23]. Thus, betulin can be considered a promising agent against COVID-19.

Nowadays, besides the COVID-19 pandemic, the other infectious viral disease, namely monkeypox, is continuously rising [60]. With this in mind, we have also probed betulin as a potential inhibitor toward one of the monkeypox proteins, viz. A42R profilin-like protein from monkeypox (strain Zaire-96-I-16). It was found that betulin is also active against the applied monkeypox protein (Table 3) and interacts through a conventional hydrogen bond with THR126 and an alkyl interaction with ARG129 (Fig. 8 and Table 4).

We have also performed molecular dynamics (MD) simulations of 100 ns at 310 K to evaluate interactions in the betulin complex with papain-like protease (PLpro), which showed the most remarkable docking score. Particularly, the complex showed an RMSD below 0.476 nm with the average value of 0.381 nm (Fig. 9). The RMSF value for the same complex of PLpro with betulin was below 0.605 nm with the strongest fluctuations observed for GLU1 and LEU317, followed by VAL11, ASN13, HIS17, SER24, MET25, GLN29, LYS43, LYS45, ASN48, GLU51, GLN97, ASN99, THR191, LYS228, and LYS315 (Fig. 9). Rg values for this complex form a relatively stable profile from 2.457 to 2.603 nm (Fig. 9). The SASA profile was calculated for predicting the interaction between the complex of PLpro with betulin and solvents. It was established that the binding of betulin to PLpro did not impair the protein’s interaction with the solvent molecule and the stability of the protein (Fig. 9). During the 100 ns simulation time, the average SASA was calculated as 255.08 nm2. It was also observed that the complex forms 1 intermolecular hydrogen bond during almost the whole simulation time; 2 intermolecular hydrogen bonds at about 13–40, 50–75, 80–86, 94–97 ns; and 3 intermolecular hydrogen bonds at about 55–70 ns (Fig. 9).

Fig. 9
figure 9

RMSD, RMSF, Rg, SASA and intermolecular hydrogen bonds analysis profiles of the papain-like protease (PLpro)–betulin complex

Full size image

Conclusions

We report detailed computational studies of betulin, which is of great interest for its biological properties. The strcuture of betulin was optimized by the DFT/B3LYP/6–311++G(d,p) calculations to verify electronic properties. We have also established values of the global chemical reactivity descriptors, which allowed to reveal electron accepting and donating abilities of the reported compound. Bioavailability, druggability as well as absorption, distribution, metabolism, excretion and toxicity properties of betulin were evaluated using the SwissADME, BOILED-Egg, and ProTox-II tools. As evidenced from the red dot position in the BOILED-Egg plot, both BBB and gastrointestinal absorption properties are negative with the negative PGP effect on the molecule.

Betulin was also found to be of interest as a potential corrosion inhibitor for some important metals used in implants. Electron charge transfer from the molecule of betulin to the surface of all the examined metals (Ti, Fe, Zr, Co, Cu, Cr, Ni, Mn, Mo, Zn, Al, W, Ag, Au) was revealed, of which the best results were obtained for Ni, Au and Co.

In silico molecular docking was applied to probe interactions of betulin with a series of the SARS-CoV-2 proteins as well as one of the monkeypox proteins. It was established that betulin is active against all the applied SARS-CoV-2 and monkeypox proteins with the best binding affinity with papain-like protease (PLpro) and spike protein, RBD (native). The title compound also interacts with spike protein and RBD (mutated), and was also found to be active against the studied monkeypox protein. According to the molecular dynamics simulation data, PLpro forms stable complex with betulin.