In recent decades, the threat of fungal infections has risen due to the growing resistance of various fungal species to fungicides used in both human medicine and agriculture. To counter this challenge, researchers have explored plant-based medicines as potential disease-fighting agents due to their lower mammalian toxicity, reduced environmental risks, and societal acceptance [1,2].

Several plant families, such as Myrtaceae, Zingiberaceae, Mimosaceae, Anacardiaceae, Lauraceae, and Fabaceae, are recognized for their potent antifungal properties. One such example is the clove flower bud, derived from the Myraceae family. Clove, native to Indonesia, Zanzibar, and Madagascar, contains a high content of eugenol, an effective agent against both gram-positive and gram-negative microbes as well as numerous pathogenic fungi. Clove also boasts antiviral, analgesic, anesthetic, antitumor, and cardiovascular activities [2, 3, 4].

Sulfonamide compounds are renowned for their potent biological effects, finding applications not only in pharmaceuticals but also in agrochemicals such as herbicides and fungicides. Chesulfamide, a novel sulfonamide fungicide, stands out by targeting mycelium cell membranes, disrupting DNA, and inducing disease resistance in plants. Our study aimed to identify major secondary metabolites in a crude extract of cracked clove flower buds and evaluate its antifungal potential against fungi sourced from postharvest potatoes and tomatoes. The extract is rich in eugenol, which is known for its antioxidant, antimicrobial, antinociceptive, antiviral, and anesthetic properties [5].

Turning to synthetic approaches, a plethora of antifungal agents have been developed. In this context, we, as pharmaceutical chemists, focused on synthesizing potent fungicides in the form of new 2-thiouracil derivatives. The compounds 2-thiouracil and 6-methyl-2-thiouracil copper II complexes demonstrated significant antifungal effects against various Candida species [6].

Drawing inspiration from chesulfamide, which acts as a lead compound, we delved into the realm of sulfonamide compounds, which currently represent a major focus of fungicide research and development (R&D). Our research encompassed the design of newly synthesized compounds through two distinct structural modification strategies. The first approach involved replacing the cyclohexanone group with 6-methyl-2-thiouracil. The second objective centered around transforming chloro-(trifluoromethyl)benzene into nitropyrimidine, ((4-nitrophenyl)sulfonyl)thiazole, phenylbenzo[d]thiazole, thiadiazole, and triazole, all exhibiting various substituents (Figure 1).

Several 2-thiouracil-5-sulphonamides were synthesized through the condensation of 2-thiouracil-5-sulphonyl chloride and selected aryl amines. Pyridine was utilized as an acid scavenger in this process. These synthesized compounds were then evaluated against microsporium canis, sporotrichum schenckii, and candida albicans, revealing promising antifungal activity [5]. Building upon this foundation, we expanded our investigation by preparing five new 2-thiouracil-5-sulphonamide derivatives in addition to clove extract. This study aims to assess the antifungal efficacy of both natural and synthetic agents, as well as explore any potential synergistic effects they may exhibit.

Materials and Instrumentation

Melting points were determined using an electrothermal melting point apparatus. Infrared spectra were recorded on a PerkinElmer grating infrared spectrophotometer using KBr pellets. NMR spectra were measured using a BRUKER-500 MHz (\(^1\)H: 500 MHz) spectrometer, with SiMe4 serving as the internal reference for \(^1\)H NMR readings. Mass spectrometry was conducted on a JEOL JMSAx 500 model spectrometer. Standard drying methods were employed for solvents, and Merck 0.2 mm silica gel 60 F254 aluminum plates were used for thin-layer chromatography (TLC). Elemental analysis was carried out at the Microanalysis Laboratory, Cairo University, Egypt, yielding results in good agreement with expected values. The synthesis of 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulphonyl chloride (2) was performed [5].

General Procedure for Synthesis of 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulphonamides (3a-e)

The synthesis of 4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulphonamides (3a-e) involved refluxing 2 (1.13 mole) and the corresponding amino compound (1.13 mole) - namely, 2-amino-5-(4-nitrophenylsulphonyl)thiazole, 2-amino-5-nitropyrimidine, 2-(4-aminophenyl)-6-methylbenz- othiazole, 2-amino-1,3,4-thiadiazole, and 3-amino-1,2,4-triazole - in DMF (50 ml) with pyridine (0.016 mole) for 15 hours. After cooling, the resulting precipitate was collected, and recrystallization from DMF/water yielded the desired compound.

N-(4-(4-Nitrophenylsulfonyl)thiazol-2-yl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamide (3a)

(Yield: 73%). mp 261-263\(^\circ\)C; IR (KBr, cm\(^{-1}\)) \(V_{\text{max}}\): 3233, 3210, 3180 (3NH’s), 3176 (CH, aromatic), 1683 (C=O), 1350, 1560 (NO\(_2\)), 1174, 1322 (SO\(_2\)), 1277 (C=S); \(^1\)H NMR (500 MHz, DMSO): \(\delta\) = 7.59-8.06 (m, 6H, Ar-H, pyrimidine H-6 and thiazole H-5), 9.90, 10.59, 11.29 (3s, 3H, NH’s exchangeable \(D_2O\)) ppm; Mass spectrum: m/z 474 [M+]. Found, %: C, 32.84; H, 1.91; N, 14.73. \(C_{13}H_{9}N_{5}O_{7}S_{4}\) (475.50). Calculated, %: C, 32.77; H, 1.86; N, 14.84.

N-(5-Nitropyrimidin-2-yl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamide (3b)

(Yield: 70%). mp 255-257\(^\circ\)C; IR (KBr, cm\(^{-1}\)) \(V_{\text{max}}\): 3233, 3208, 2213 (3NH’s), 1665 (C=O), 1359, 1547 (NO\(_2\)), 1354, 1144 (SO\(_2\)), 1264 (C=S); \(^1\)H NMR (500 MHz, DMSO): \(\delta\) = 7.97 (2s, 2H, pyrimidine H’s), 8.02 (s, \(^1\)H, pyrimidine H-5), 9.10, 9.92, 10.83 (3s, 3H, NH’s exchangeable \(D_2O\)) ppm; Mass spectrum: m/z 329 [M+]. Found, %: C, 29.09; H, 1.83; N, 25.44. \(C_8H_6N_6O_5S_2\) (330.30). Calculated, %: C, 29.17; H, 1.92; N, 25.56.

N-(4-(6-Methylbenzo[d]thiazol-2-yl)phenyl)-4-oxo-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamide (3c)

(Yield: 67%). mp 290-292\(^\circ\)C; IR (KBr, cm\(^{-1}\)) \(V_{\text{max}}\): 3238, 3221, 3145 (3NH’s), 3187 (CH, aromatic), 1680 (C=O), 1170, 1325 (SO\(_2\)), 1271 (C=S); \(^1\)H NMR (500 MHz, DMSO): \(\delta\) = 2.01 (s, 3H, CH\(_3\)), 7.56-8.58 (m, 8H, Ar-H, pyrimidine H-6 and benzothiazole-H), 9.46, 9.95, 10.95 (3s, 3H, NH’s exchangeable \(D_2O\)) ppm; Mass spectrum: m/z 430 [M+]. Found, %: C, 50.22; H, 3.28; N, 13.01. \(C_{18}H_{14}N_4O_3S_3\) (430.52). Calculated, %: C, 50.30; H, 3.34; N, 13.11.

4-Oxo-N-(1,3,4-thiadiazol-2-yl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-sulfonamide (3d)

(Yield: 77%). mp 281-283\(^\circ\)C; IR (KBr, cm\(^{-1}\)) \(V_{\text{max}}\): 3251, 3221, 1129 (3NH’s), 3177 (CH, aromatic), 1686 (C=O), 1172, 1325 (SO\(_2\)), 1273 (C=S); \(^1\)H NMR (500 MHz, DMSO): \(\delta\) = 7.17 (s, \(^1\)H, thiadiazole-H-5), 8.19 (s, \(^1\)H, pyrimidine H-6), 9.95, 10.86, 11.30 (3s, 3H, NH’s exchangeable \(D_2O\)) ppm; Mass spectrum: m/z 290 [M+]. Found, %: C, 24.74; H, 1.73; N, 24.04. \(C_6H_5N_5O_3S_3\) (291.33). Calculated, %: C, 24.81; H, 1.63; N, 24.13.

4-Oxo-2-thioxo-N-(\(^1\)H-1,2,4-triazol-3-yl)-1,2,3,4-tetrahydropyrimidine-5-sulfonamide (3e)

(Yield: 79%). mp 244-246\(^\circ\)C; IR (KBr, cm\(^{-1}\)) \(V_{\text{max}}\): 3270, 3254, 3208, 1175 (4NH’s), 1687 (C=O), 1178, 1330 (SO\(_2\)), 1268 (C=S); \(^1\)H NMR (500 MHz, DMSO): \(\delta\) = 7.59 (s, \(^1\)H, triazole-H-5), 8.05 (s, \(^1\)H, pyrimidine H-6), 10.32, 10.87, 11.19, 11.42 (4br.s, 4H, NH’s exchangeable \(D_2O\)) ppm; Mass spectrum: m/z 274 [M+]. Found, %: C, 26.27; H, 2.20; N, 30.64. \(C_6H_6N_6O_3S_2\) (274.28). Calculated, %: C, 26.15; H, 2.29; N, 30.70.

Sample Gathering and Plant Preparation

The clove tree is commonly cultivated in coastal areas of Indonesia at elevations of up to 200 meters above sea level. Flower bud production, the commercially valuable part of the tree, typically begins four years after planting. Flower buds are harvested at the stage of maturity just before flowering. Harvesting can be done manually or chemically induced using a natural plant hormone that releases ethylene into plant tissues, accelerating ripening. Dried S. aromaticum flower buds were initially obtained from a vendor in Hawamdia, Giza governorate, Egypt. To clean them, the buds underwent multiple washes with tap water followed by sterile distilled water. Subsequently, they were finely powdered using a roll mill, sieved to remove coarse foreign particles, and then subjected to extraction using a Soxhlet apparatus with 70% (v/v) ethanol. The extracted solution underwent two filtration steps: first through sterile gauze and then through Whatman no. 1 filter paper. The filtrate was concentrated using a rotary evaporator at a temperature of 35-40\(^\circ\)C.

System Preparation

The crystal structure of Candida albicans sterol 14 alpha-demethylase, resolved at 2.0 \(\AA\) resolution, is available in the Protein Data Bank under sequence ID 5TZ1 [7]. These structures were imported into UCSF Chimera [8] for further use in molecular dynamics (MD) simulations. PROPKA was employed to adjust and optimize the pH to 7.5 [9]. The structure of compound 3e was drawn using ChemBioDraw Ultra 12.1 [10]. Both constructed systems were subjected to 20 ns MD simulations, as described in the dedicated simulation section.

Molecular Dynamic Simulation

Molecular dynamic (MD) models are employed to gain insights into otherwise inaccessible atomic and molecular motions in biological systems. These simulations provide a comprehensive view of the dynamic behavior of biological systems, encompassing changes in molecular conformation and interactions between molecules [11]. MD simulations were performed using the PMEMD engine from the AMBER 18 software package, GPU edition, which supports multiple platforms [12].

The General Amber Force Field (GAFF) approach, developed by ANTECHAMBER, was used to calculate the partial atomic charges of individual components [13]. The Leap module of the AMBER 18 software package solvated each system in an orthorhombic box containing TIP3P water molecules, with a minimum distance of 10 \(\AA\) from the box edges. Neutralization was achieved using the Leap module, incorporating counter ions such as Na+ and Cl-. The initial stage involved a 2000-step minimization with a maximum constraint of 500 kcal/mol, followed by a 1000-step minimization using the conjugate gradient algorithm with no constraints. During the MD simulation, each system’s temperature was gradually raised from 0 to 300 kelvin over 500 picoseconds, ensuring uniform volume and atom count. A collision frequency of 1 ps and a harmonic constraint of 10 kcal/mol were applied to the solutes.

Subsequently, each system was heated and equilibrated at a constant temperature of 300 K for 500 ps, maintaining the same atom count and system pressure. A Berendsen barostat was employed to maintain a constant pressure of 1 bar, simulating an isobaric-isothermal (NPT) ensemble [14]. A 20-ns MD simulation was conducted for each system, employing the SHAKE algorithm to constrain atoms involved in hydrogen bonds. A time step of 2 fs was used for each simulation, with an SPFP accuracy parameter. The simulations were performed in a random-seeded isobaric-isothermal ensemble (NPT) with a temperature of 300 K, a pressure of 1 bar, a pressure-coupling constant of 2 ps, and a collision frequency of 1 ps.

Post-MD Analysis

The CPPTRAJ component of the AMBER18 suite was employed to conduct post-MD analysis on the trajectory data, collected at 1 ps intervals throughout the simulation [15]. The data analysis tools Origin [16] and Chimera [8] were used for creating charts and figures.

Thermodynamic Calculation

Various techniques such as Poisson-Boltzmann, generalized Born, and surface area continuum solvation (MM/PBSA and MM/GBSA) have been established to estimate ligand-binding affinities [17, 18, 19]. These techniques utilize molecular simulations of protein-ligand complexes to calculate statistical mechanical binding free energies. These simulations are performed within a consistent force field. The binding free energy was obtained by averaging over 200 measurements collected during the entire 20 ns trajectory.

The estimated change in binding free energy (\(\Delta G\)) for each molecular species (complex, ligand, and receptor) is calculated as follows [20]:

\[\Delta G = \text{Egas} + \text{Eint} + \text{Eele} + \text{Evdw} + \text{GGB} + \text{Gsol},\] where Egas represents energy in the gas phase, Eint stands for internal energy, Eele for Coulomb energy, and Evdw for van der Waals energy. The FF14SB force field parameters were utilized to compute Egas. The solvation-free energy (Gsol) was determined by combining the polar (GGB) and non-polar (Gsol) state energies. The non-polar solvation free energy (GSA) was calculated from the solvent accessible surface area (SASA) using a water probe radius of 1.4 \(\AA\) [21, 22]. The polar solvation contribution (GGB) was calculated by solving the Generalized Born (GB) equation. The term S represents the total entropy of the solute, while T represents its temperature.

Collection of Specimens

Samples of potato tubers and tomato fruits exhibiting signs of rotting were prepared for infection. These samples were aseptically transferred to establish a population of fungal infection.

Isolation of Fungi

Infected tubers and fruits were cut into smaller fragments of size 1x1 cm\(^2\) and then soaked in a 70% solution of sodium hypochlorite, followed by a 5% solution for 3-5 minutes. Subsequently, the fragments were thoroughly washed with sterilized distilled water. These pieces were then placed onto petri dishes containing Czapek’s yeast extract agar medium (CYA), enriched with 0.5 g/L chloramphenicol as an antibacterial agent, following the method described by Gams, 1998 [23]. Two to three fragments were distributed onto the surface of the medium. The petri dishes were incubated at room temperature for 24 hours. Over the course of a week, the dishes were regularly inspected to ensure further purification. Portion of the growing mycelia were transferred to fresh sterile petri plates containing CYA medium.

All purified fungal isolates were re-inoculated into healthy potato tubers and tomato fruits to confirm their ability to cause infection. Subsequently, a pathogenicity test was conducted to verify the infectivity of the investigated fungi. Finally, each fungal isolate was examined for both macroscopic and microscopic properties it exhibited [24].

Antifungal Activity Test

The antifungal activity was assessed using the well diffusion method, as described in the study by Rojas, 2006 [25]. Following the procedure outlined by Beever, 1970, a petri dish containing 20 mL of potato dextrose agar (PDA) medium was prepared, and then inoculated with 260 \(\mu\)L of spore suspension (0.75 x 10\(^5\) cfu/mL) from a one-week-old fungal culture [26]. Wells with a diameter of 6 millimeters were created using a sterile cork borer, and then 55 microliters of the plant crude extract, target compounds (3a-e), the antibiotic Ciclopirox Olamine as a positive control, and ethanol as a blank were added to each well using sterilized dropper micropipettes.

The petri dishes were incubated at 28\(^{\circ}\)C for one week. Both the clove alcoholic extract and the newly synthesized compounds (3a-e) were dissolved in 95% ethanol to achieve various concentrations (50%, 25%, 12.5%, 5%, 2.5%, and 1%). Their inhibitory effects were then determined using the well-diffusion method [27]. In this experiment, 250 \(\mu\)L of spore suspension (0.75 x 10\(^5\) cfu/mL) from one-week-old fungal cultures was used to inoculate 20 mL of PDA medium in each petri dish. Wells with a diameter of 7 mm were created using a sanitized cork borer. Subsequently, 50 \(\mu\)L of each dilution was added to the respective wells (triplicate). After incubating the plates at 28\(^{\circ}\)C for a week, the diameter of the inhibition zones around each well was measured in millimeters. The experimental setup was conducted in triplicate (Table 1).

Clove extract exhibited variable potent wide spectrum activity against all fungal isolates. Compounds 3b and 3e were the most potent antifungal agents, showing wide spectrum activity. Compound 3d showed moderate antifungal activity compared to clove extract. Compound 3a showed weak antifungal activity and was inactive against F. oxysporum and G. candidum. Compound 3c was the weakest antifungal agent, being inactive against A. alternata, F. oxysporum, and G. candidum. Both clove extract and 3e exhibited synergistic antifungal activity.

The phenolic components carvacrol and eugenol in clove have demonstrated fungicidal action in vitro and in vivo, respectively (Figure 2) [28]. Combination therapy with clove is highly useful in treating fungal infections resistant to fluconazole and other drugs due to widespread fungal resistance. Research indicates that eugenol can be synergistically co-administered with fluconazole or amphotericin B to achieve maximum antifungal activity [29]. This enhancement of antifungal potency is due to a significant reduction in ergosterol, a crucial membrane component of fungal cells [30].

It is plausible that antifungal drugs like Griseofulvin, azole derivatives, and allyl amines will lose their efficacy as antifungal agents due to fungal resistance development and the requirement for prolonged therapy, alongside their adverse effects. The antifungal action of several aromatic compounds derived from medicinal plants has garnered significant interest recently. One such compound is eugenol, the main active constituent of clove buds. It has been thoroughly investigated, particularly when combined with antifungal agents like ketoconazole, where their synergistic activity has been studied. They have demonstrated potent activity against worldwide pathogenic fungi of the Trichophyton species, which cause dermatophytosis such as tinea unguium, tinea pedis, tinea corporis, and tinea capitis. Studies have shown that eugenol inhibits mycelial development and conidia germination, induces irregularities in the dermatophyte’s structure, and hampers ergosterol biosynthesis, disrupting the integrity and functionality of the fungal membrane. As eugenol is a phenolic compound with a lipophilic structure, it enhances the fluidity and permeability of the fungal cell membrane, thereby disrupting ion transport, osmotic balance, and the effectiveness of associated membrane proteins. These actions prevent or hinder fungal growth and can even cause cell rupture (Figure 2).

Analogously, 2-thiouracil in its tautomeric form could be considered as a phenolic antifungal agent in addition to its antimetabolic activity, thus inhibiting the nucleic acid synthesis of fungi. Therefore, from our perspective, it could be used in combination antifungal therapy. In our targeted protocol, we involved the chlorosulphonation of 2-thiouracil at the 5th position through refluxing with chlorosulphonic acid using a gradient temperature technique to obtain a high yield of the corresponding sulphonyl chloride derivative (2) [5].

Chlorosulphonation of pyrimidines is generally challenging due to the strong deactivation of the pyrimidine ring by the -I/-M effects of the two nitrogen atoms. One method to activate the pyrimidine ring is the introduction of two activating groups with +M effects. An example of such activation is 2-thiouracil, which possesses two activating groups (OH and SH groups). As a result, 2-thiouracil readily undergoes common aromatic electrophilic substitution reactions, including chlorosulphonation using chlorosulphonic acid. Chlorosulphonation reactions are widely used in pharmaceutical chemistry to prepare bioactive sulphonamides. Thus, 2-thiouracil-5-sulphonyl chloride was previously synthesized in recent publications as an intermediate in various reactions. Data on this reaction was relatively available, and it was initially prepared by directly chlorosulphonating 2-thiouracil through refluxing at 120\(^{o}\)C, yielding an acceptable yield. However, a more reliable and efficient technique, called the gradient temperature technique, was adopted to increase the yield. In this technique, the reaction conditions were initiated at 30\(^{o}\)C with no significant reaction occurring. Practically, the reaction only starts at 35\(^{o}\)C with a 15% yield. By gradually increasing the temperature at regular intervals (55\(^{o}\)C, 75\(^{o}\)C, 100\(^{o}\)C, and finally 125\(^{o}\)C), the maximum yield of 75% was obtained. The partial hydrolysis of some of the final product to 2-thiouracil-5-sulfonic acid was a challenge in this reaction and occurred during work-up using ice to obtain the final compound. The hydrolysis was prevented by pouring the reaction mixture onto an equal volume of ice and acetic acid. Additionally, a reliable method to increase the yield was employed by combining chlorosulphonic acid with thionyl chloride (in a 1.5:1 ratio). Other reagents such as sulphonyl chloride and phosphorus oxychloride were examined, but no noticeable effects were observed. The product yield is temperature-dependent; for example, it was chlorosulphonated at 120\(^{o}\)C with a 60% yield. The yield was improved using the gradient temperature technique. The produced sulphonyl derivative 2 was employed as a blocking unit for the preparation of the target compounds (3a-e) through refluxing with a series of aromatic amines, namely: 2-amino-5-(4-nitrophenylsulphonyl)thiazole, 2-amino-5-nitropyrimidine, 2-(4-aminophenyl)-6-methylbenzothiazole, 2-amino-1,3,4-thiadiazole, and 3-amino-1,2,4-triazole, in DMF containing pyridine as an acid scavenger (Scheme presented in Figure 3).

All natural and synthetic samples were evaluated against fungal isolates of Geotrichum candidum, Alternaria alternata, Fusarium oxysporum, and Mucor hiemalis, which correspond to the plant infections found in harvested potatoes and tomatoes. The synergistic activity of clove extract and compound 3e (a triazole derivative) might be attributed to the ability of both eugenol and the triazole ring of compound 3e to form a lipophilic complex with divalent cations such as Zn\(^{+2}\) and Cu\(^{+2}\), thereby increasing cell permeability of the fungus, leading to cell leakage and subsequent death (Figure 4) [31].

Molecular dynamics and system stability

The effectiveness of the developed molecule in binding to the protein’s active site, along with its binding interaction and stability, was predicted using molecular dynamics simulation [32, 33]. Ensuring the stability of a system is crucial for tracking motion disturbances and preventing the emergence of artifacts during simulation. In this study, system stability was assessed over 20 ns simulations using the Root-Mean-Square Deviation (RMSD). The average RMSD values for the apo-protein and 3e-complex systems were 1.38 and 1.63, respectively (Figure 5A). These results indicated that the complex system with 3e bound to the protein achieved a more stable confirmation compared to other investigated systems. Analyzing protein structural flexibility upon ligand interaction is essential during MD simulation to study residue behavior and ligand interaction [34].

The effect of inhibitor binding on protein residue variations was examined over 20 ns simulations using the Root-Mean-Square Fluctuation (RMSF) method. The calculated average RMSF values for the apo-protein and 3e-complex systems were 1.03 and 1.02 , respectively (Figure 5B). Figure 5B illustrates the overall residue fluctuations across all systems. These findings suggest that the system where 3e is bound to the protein complex exhibits a greater diversity in the number of residues present. The radius of gyration (Rg) serves as a measure of protein structure compactness and stability during simulation. As depicted in Figure 5C, the Rg values for the apo-protein and the complex with compound 3e were 22.75  and 22.88, respectively. The Rg analysis indicated that the ligand-bound protein structure exhibited greater flexibility compared to the apo-protein structure (Figure 5).

Binding interaction mechanism based on binding free energy calculation:

To estimate the free binding energies of small compounds to biological macromolecules, the molecular mechanics energy methodology (MM/GBSA) is often employed, which can provide more accurate results than docking scores [35, 36, 37]. The MM-GBSA tool in AMBER18 was utilized to compute the binding free energies by capturing snapshots of the systems and applying a Monte Carlo method to the results. As shown in Table 2, large negative values were obtained for all estimated energy components (except Gsolv), indicating favorable interactions. The binding affinity of 3e to the protein was determined to be -10.20 kcal/mol.

A detailed analysis of each individual energy contribution leading to the reported binding free energies reveals that the interactions between 3e and the Candida albicans sterol 14 alpha-demethylase protein receptor residues are primarily driven by the more positive van der Waals energy components (Table 2).

Identification of crucial binding residues

To gain a better understanding of the role of key residues involved in the inhibition of these enzymes, the overall energy associated with the binding of 3e to the Candida albicans sterol 14 alpha-demethylase receptor was decomposed into the contributions of different site residues (Figure 6). The most significant interactions of the 3e molecule with the Candida albicans sterol 14 alpha-demethylase receptor are observed at residues Phe 14 (-0.355 kcal/mol), Ala 17 (-1.679 kcal/mol), Ala 18 (-0.812 kcal/mol), Tyr 20 (-1.379 kcal/mol), Gly 21 (-0.491 kcal/mol), Gln 22 (-0.348 kcal/mol), Leu 43 (-0.631 kcal/mol), Leu 44 (-0.735 kcal/mol), Pro 186 (-1.225 kcal/mol), and Ile 187 (-0.278 kcal/mol).

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