Expansion of the antifungal activities through in silico docking study of compounds from Albizia lebbeck fruits

The development of new computational and experimental techniques that are efficient, fast, and accurate has become a priority for researchers in all domains around the world. The present manuscript deals with the computational investigation of quercitrin ( 1 ), lebbeckisoetin A ( 2 ), quercetin-3-O-β -D-glucopyranoside ( 3 ), ( E )- p -coumaric acid ( 4 ), eugenol ( 5 ), eugenol, 1-acetate ( 6 ), chiakine ( 7 ), hexancosan-1',26'-dioate of bis[(2 S ), 2,3-dihydroxypropyl] ( 8 ), oleanolic acid ( 9 ), betulin ( 10 ), hopan-29-ol ( 11 ), hopan-30-ol ( 12 ), 22-hydroxyhopan-3-ol ( 13 ), and lupeol ( 14 ). These compounds were all isolated from the fruit of Albizia lebbeck . Only lebbeckisoetin A ( 2 ) and chiakine ( 7 ) were previously evaluated for their experimental antimicrobial activities, which both revealed potent antifungal activities. The virtual screening of the antifungal activity was performed on Maestro Schr ӧ dinger software using sterol 14-alpha demethylase (CYP51) from Candida albicans in complex with the tetrazole-based antifungal drug candidate VT1161 (VT1) (PDB Id: 5TZ1 ) and sterol 14-alpha demethylase (CYP51) from the pathogenic yeast Candida albicans in complex with the antifungal drug posaconazole (PDB Id: 5FSA ). This


Introduction
The development of new computational and experimental techniques, which are efficient, fast, and accurate, has become a priority for researchers in all domains around the world.The development of high-performance computers with great capacities has led computers to be used in both experimental and theoretical research.Nowadays, almost all research areas have both experimental and theoretical parts.This has shifted the paradigms in drug discovery.Most often, theoretical research is faster than experimental research and is used to either predict or support the experimental results (Santamouris et al. 20012 Nowadays, the modelization/production of new drugs is an easy task.However, the challenge incurred for this development is not long and tedious like two decades ago because the targeted drug is firstly designed in a computer and then suggested for molecular docking in order to better predict the ligand-receptor interactions.The term for the computational schemes that attempt to search for the "best" atomic coordinates/connectivity or bound association between two complex molecules is known as "docking" (Ghasemi et al. 2017).Docking study is a multidisciplinary science that is applied in a wide range of areas, particularly in drug discovery (Sultana et al. 2024).The prediction with a reasonable accuracy of the binding affinity between the ligand(s) and the protein is crucial in drug discovery and helpful in the optimization of the compound to establish a perfect connectivity with the targeted protein (Rezaei et al. 2020;Sultana et al. 2024).Molecular docking or computational simulation drug design calls upon the calculation of the binding strength between ligand and the protein which are two biological homologs.This manuscript deals with the computational investigation of some naturally isolated compounds against C. albicans.Therefore, the most prevalent human yeast  3), (E)-p-coumaric acid (4), eugenol (5), eugenol, 1-acetate (6), chiakine (7), hexancosan-1',26'-dioate of bis[(2S), 2,3-dihydroxypropyl) (8), oleanolic acid (9), betulin (10), hopan-29-ol (11), hopan-30-ol (12), 22-hydroxyhopan-3-ol (13), and lupeol (14) (Figure 1) were all isolated from the fruit of Albizia lebbeck.Unfortunately, only lebbeckisoetin A (2) and chiakine (7) were evaluated for their experimental antimicrobial activities.The antimicrobial activities of lebbeckisoetin A (2) and chiakine (7) were assayed against five microbial strains (fungal: Candida albicans, and bacteria: Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus) (Leutcha et al. 2022).This theoretical study was undertaken in the framework to support and better understand the experimental results at the atomic level, and to expand the antimicrobial assays of the other compounds reported from Albizia lebbeck that were not tested experimentally.The virtual screening of the antimicrobial activity performed on the crystal structure of sterol 14-alpha demethylase (CYP51) from Candida albicans in complex with the tetrazole-based antifungal drug candidate VT1161 (VT1) (PDB Id: 5TZ1) and the crystal structure of sterol 14-alpha demethylase (CYP51) from a pathogenic yeast Candida albicans in complex with the antifungal drug posaconazole (PDB Id: 5FSA) with natural compounds isolated from the fruit of Albizia lebbeck have shown promising antimicrobial results.This is the first docking study using natural compounds as ligands with the 5TZ1 and 5FSA proteins.In addition, this survey has exhibited promising antifungal activities most of the docked compounds against Candida albicans.

General Experimental Procedures
The technical characteristics of the spectroscopic and spectrosmetric machines, as well as the experimental antifungal assays of the studied compounds, were already reported by Leutcha et al. ( 2022).ChemDraw 16.0 software was used for the firstly used to draw the study compounds and secondly in the preparation of the docked ligands, while Schrӧdinger 4.2.1 software (Maestro 4.2.1) was used for the second preparation of the ligands and for the final docking.

Plant Material, Extraction and Isolation, and Experimental Characterization
A. lebbeck dry fruits were sampled in 2018 in Maroua city and identified by comparison with voucher specimen 58964/NHC in the Cameroon herbarium.The secondary metabolites theoretically studied here were all obtained from the fruits of Albizia lebbeck, a plant species of the Fabaceae family.The experimental antifungal assays, as well as all the reported compounds in this study, were obtained as previously reported by Leutcha et al. (2022).

Docking Material
The in silico docking investigation was performed in order to have a better view and understanding of the binding interaction strength between the natural ligands and the proteins at the atomic level.So, the 3D crystal complex structures of the two proteins were directly downloaded from the Protein Data Bank (PDB) website.The crystal structure of sterol 14alpha demethylase (CYP51) from Candida albicans in complex with the tetrazole-based antifungal drug candidate VT1161 (VT1) (PDB Id: 5TZ1) and the crystal structure of sterol 14-alpha demethylase (CYP51) from the pathogenic yeast Candida albicans in complex with the antifungal drug posaconazole (PDB Id: 5FSA) were used as proteins.The structures of the isolated compounds were firstly drawn and prepared in the "MDL SDfile V3000 ( ⁎. sdf)" format using ChemDraw 16.0.Secondly, the software Maestro Schrӧdinger 4.2.1 Ligprep module was used for the preparation of ligands.A receptor grid was generated around the co-crystallized ligands for both proteins, and the ligands were docked using the glide XP module.In addition, the protein preparation wizard was used for the protein preparation and minimization as reported by Zafar et al. (2023).Finally, to visualize and better understand the ligand-protein binding effects, the 3D and 2D docked poses were captured and examined.

Results and Discussion
Computational or molecular docking drug design calls upon the calculation of the binding strength between two biological homologs, which are ligands and proteins.This manuscript deals with the computational investigation of some naturally isolated compounds.Therefore, quercitrin (1), lebbeckisoetin A (2), quercetin-3-O-β-D-glucopyranoside (3), (E)-pcoumaric acid (4), eugenol (5), eugenol, 1-acetate (6), chiakine (7), hexancosan-1',26'-dioate of bis[(2S), 2,3dihydroxypropanone) (8), oleanolic acid (9), betulin (10), hopan-29-ol (11), hopan-30-ol (12), 22-hydroxyhopan-3-ol (13), and lupeol (14) (Fig. 1; Table 1) were all isolated from the fruit of Albizia lebbeck.From all the above-listed compounds, only lebbeckisoetin A (2) and chiakine (7) were experimentally assayed for their antimicrobial activities against five microbial strains (one fungal: Candida albicans and four bacterial: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis) (Table S1) (Leutcha et al. 2022); the reason being the least quantities of most of the isolated compounds.This theoretical study was undertaken in the framework to support and better understand the experimental results at the atomic level, and to expand the antimicrobial assays of the other compounds reported from Albizia lebbeck that were not tested experimentally.(Title: Crystal structure of sterol 14-alpha demethylase (CYP51) from Candida albicans in complex with the tetrazole-based antifungal drug candidate VT1161 (VT1)); PDB ID: 5FSA (Title: Crystal structure of sterol 14-alpha demethylase (CYP51) from a pathogenic yeast Candida albicans in complex with the antifungal drug posaconazole).structure of Candida albicans CYP51 as a suitable complex agent for a docking study was previously reported by Hargrove et al. (2017).This is the first docking study performed on the 5TZ1 and 5FSA proteins using natural compounds as ligands.However, these two proteins were targeted for their in silico and in vitro antifungal activities against C. albicans Before docking, the most stable conformation of the docked compounds was firstly searched, and after docking, some poses were also captured to better explain the ligand-protein interactions.Compounds (1-14) were docked for their antifungal potencies against the sterol 14-alpha demethylase (CYP51) from Candida albicans in complex with the tetrazole-based antifungal drug candidate VT1161 (VT1) (PDB Id: 5TZ1) and sterol 14-alpha demethylase (CYP51) from the pathogenic yeast Candida albicans in complex with the antifungal drug posaconazole (PDB Id: 5FSA).Compounds (1-10) reveal interesting docking scores ranging from -7.923 to -3.516, while compounds ( 11-14) reveal docking scores less than -2 on the two proteins (5TZ1 and 5FSA) (Table 2 and S2).Generally, phenolic compounds were more potent, which may be due to their capacity to form H-bonds, aromatic interactions, π-πinteractions, and hydrophobic interactions, while other compounds were forming H-bonds, salt bridges, and hydrophobic interactions.In addition, quericitrin (1) (-7.892 and -7.923), lebbeckisoetin A (2) (-7.815 and -7.756), and quercetin-3-O-β-D-glucopyranoside (3) (-5.528 and -7.717), which are all flavonoids, were the most active compounds against 5TZ1 and 5FSA, respectively (  On the other hand, chiakine (7) was the only compound that established the salt bridge with Leu 124, Tyr 183, H 2 O 803, and HEM 601 at the atomic level, with a docking score of -5.273 on 5FSA.This might be due to its amphionic structure.From the above mentions, it appears that 5FSA was more interactive with all the docked ligands as compared to 5TZ1.Like previous theoretical investigations undertaken on the above-listed compounds, we can report, to the best of our Indeed, the virtual screening of the antimicrobial activity performed on the crystal structure of sterol 14-alpha demethylase (CYP51) from Candida albicans in complex with the tetrazole-based antifungal drug candidate VT1161 (VT1) (PDB Id: 5TZ1) and the crystal structure of sterol 14-alpha demethylase (CYP51) from a pathogenic yeast Candida albicans in complex with the antifungal drug posaconazole (PDB Id: 5FSA) or natural compounds isolated from the fruit of Albizia lebbeck has shown promising antimicrobial results.In addition, this is the first docking of natural compounds used as ligands with the 5TZ1 and 5FSA proteins.

Conclusion
The aim of this research was the computational investigation of the naturally isolated compounds against C. albicans, which is the most prevalent human yeast pathogen.This theoretical study was undertaken in the framework to better understand the previous experimental results.So, the antifungal performed using 5TZ1 and 5FSA as protein with fourteen natural compounds (1-14) reported from Albizia lebbeck was in agreement with the experimental results.

Conflicts of Interest
No financial or conflict of interests are declared by the authors.

Qeios
has exhibited promising antifungal activities of the two naturally isolated compounds against Candida albicans with an MIC value of 32 µg/mL (Leutcha et al. 2022).On the other hand, the most prevalent human yeast pathogen is C. albicans because it is always present throughout the entire mammalian life cycle ( Hargrove et al. 2017).The cytochrome P450 enzyme [Sterol 14α-demethylase (CYP51)] is the required enzyme used by eukaryotic cells in steroidal biosynthesis and is mostly targeted by antifungal clinical drugs (Hargrove et al. 2017).The X-ray crystal and a π-π interaction with Phe 233, and compound (3) was establishing H-bonds with Lys 90, Ser 507, Pro 210, and Leu; aromatic interactions with Phe 380 and Tyr 132, and π-π interactions with Hie 377, Tyr 132, and Phe 126, and a metallic connection with HEM 601 (heme group), which supports their high docking scores.These results are in agreement with the experimental results assayed on the C. albican fungal strain.The interesting in silico and in vitro potencies of flavonoids against C. albican are not amazing.According to Silva et al. (2022), flavonoids are reported to have potent antifungal activities against C. albican with interesting docking scores.The moderate activities of eugenol derivatives (compounds 5 and 6) against the sterol 14-alpha demethylase (CYP51) of C. albican were in agreement with the reported data of Lima et al. (2020).Among all the non-phenolic compounds, hexancosan-1',26'-dioate of bis[(2S), 2,3dihydroxypropyl)] (8) has revealed the most interesting docking score of -7.425 on 5FSA; this might be due to its capacity to form a large number of H-bonds (H-bonds with Hie 120, ALA 117, Asp 225, Asp 502, Ser 507, Gly 303, and Pro 503).