In silico-based identification of some selected phytoconstituents in Ageratum conyzoides Leaves as potential inhibitors of crucial proteins of Blastomyces dermatitidis :Maxwell Mamfe Sakyiamah, Evans Boakye Larbi, Samuel Kojo Kwofie, Biomedical and Biotechnology Research Journal (BBRJ)

ORIGINAL ARTICLE
Year : 2022 | Volume : 6 | Issue : 4 | Page : 501--509

In silico-based identification of some selected phytoconstituents in Ageratum conyzoides Leaves as potential inhibitors of crucial proteins of Blastomyces dermatitidis

Maxwell Mamfe Sakyiamah¹, Evans Boakye Larbi¹, Samuel Kojo Kwofie²,
¹ Department of Phytochemistry, Centre for Plant Medicine Research, Mampong-Akuapem, Accra, Ghana
² Department of Biomedical Engineering, University of Ghana, Legon, Accra, Ghana

Correspondence Address:
Maxwell Mamfe Sakyiamah
Centre for Plant Medicine Research, P.O. Box 73, Mampong-Akuapem
Ghana

Abstract

Background: Blastomyces dermatitidis poses health threats to humans due to the frequency of infections (blastomycosis) and the increasing resistance to existing standard antifungal drugs. Moreover, the use of experimental in vitro and in vivo approaches in search for potent drug candidates is costly and time-consuming. The aim of this study was to evaluate the pharmacological properties of some reported phytoconstituents of Ageratum conyzoides against key enzymes of B. dermatitidis using in silico approach. Methods: A total of 29 reported bioactive compounds previously isolated from the leaves of A. conyzoides were randomly selected by a literature survey and their 3D Structure Data File (SDF) structures were downloaded from PubChem database. Applying molecular docking and dynamics simulation techniques, the phytoconstituents (ligands) were docked with the binding ligand pocket of three simulated enzymes; Saccharomyces cerevisiae lanosterol 14-alpha demethylase, human squalene epoxidase, and thymidylate synthase from Pneumocystis carinii using AutoDock 4.0 software and the poses that showed lowest binding energies were visualized using LigPlot⁺. Results: The results obtained from the docking studies of the selected phytoconstituents in A. conyzoides leaves showed that 4 out of the 29 ligands (sitosterol, catechin, stigmasterol, and 5-benzamido-4-oxo-6-phenylhexanoic acid) interacted with and showed very good binding affinity toward the 3 crucial antifungal drug target receptors, and exhibited significant inhibition compared to the standard drugs. Conclusion: Therefore, sitosterol, catechin, stigmasterol, and 5-benzamido-4-oxo-6-phenylhexanoic acid from A. conyzoides leaves hold a promising potential to be explored for their antifungal activities.

How to cite this article:
Sakyiamah MM, Larbi EB, Kwofie SK. In silico-based identification of some selected phytoconstituents in Ageratum conyzoides Leaves as potential inhibitors of crucial proteins of Blastomyces dermatitidis.Biomed Biotechnol Res J 2022;6:501-509

How to cite this URL:
Sakyiamah MM, Larbi EB, Kwofie SK. In silico-based identification of some selected phytoconstituents in Ageratum conyzoides Leaves as potential inhibitors of crucial proteins of Blastomyces dermatitidis. Biomed Biotechnol Res J [serial online] 2022 [cited 2023 Apr 1 ];6:501-509
Available from: https://www.bmbtrj.org/text.asp?2022/6/4/501/363574

Full Text

Introduction

Blastomycosis is an infection of the skin, bones, and central nervous system caused by a dimorphic spore-forming fungus called Blastomyces dermatitidis which thrives on moist soil and decomposing matter. The infection occurs when a person inhales the microscopic fungal spores (conidia) from the air after engaging in activities that disturb contaminated soil, and takes about 30–45 days to manifest.[1] The clinical manifestation of B. dermatitidis infections is protean, imitating viral and bacterial respiratory infections. Nonetheless, general symptoms include fever, cough, body pains (especially muscle and joint), tiredness, cough with phlegm, and skin sores. The progress of the disease is largely dependent on the immunocompetence of the infected person, where the infection can spread from the lungs to other parts of the body such as the central nervous system, bones, joints, and skin in immunocompromised people.[2] In the lungs, the transition from conidia of B. dermatitidis to the yeast form may be inhibited through phagocytosis by alveolar macrophages, neutrophils, and monocytes.[3]

Although blastomycosis can be fatal, patients are cured through antifungal agents that target thymidylate synthase (for example, fluorinated pyrimidine analogs like 5-fluorocytosine), squalene epoxidase (for example, terbinafine and naftifine), and/or lanosterol 14-α-demethylase (for example, imidazoles such as fluconazole, itraconazole, and voriconazole).[4] However, the use of orthodox medicine is saddled with some limitations which include cost, untoward side effects, and a propensity of resistance.[5] Medicinal plants, therefore, provide a safe, cheap, and effective alternative.

Ageratum conyzoides is a ubiquitous plant found in Africa, Asia, Australia, and South America, known for curing many diseases. Its leaves have been reported to have anti-inflammatory, hemostatic, insecticidal, antifungal, antibacterial, anti-snake venom, wound healing, and anti-leucorrhoea properties, while the root has been reported to have an antilithic property and nematicidal activity.[6] Previous studies have reported the antifungal properties of bioactive compounds from A. conyzoides. These compounds include caryophyllene oxide, phytol, sinapic acid, catechin, β-caryophyllene, p-coumaric acid, protocatechuic acid, gallic acid, benzoic acid, coumalic acid, stigmasterol, and sitosterol.[7],[8],[9],[10] Deba et al. also reported that caryophyllene oxide and β-caryophyllene are fungitoxic against Fusarium solani and Fusarium oxysporum, disrupting their membrane integrity.[10] According to Ilondu, phytol causes inhibitory effects on infectious agents by disrupting their cell membrane which causes efflux of K+ out of the cell.[7] Although the potentials of some of the abovementioned compounds are known, the targets and possible mechanisms of action of these compounds are still not fully understood, thus the need to be investigated.

Owing to the lengthy time and high costs involved in performing experimental in vitro and in vivo studies on potential drug candidates, computer-aided drug design (CADD) has proven a useful strategy.[11],[12],[13],[14] For instance, Talele et al. have reported successful drugs such as captopril, saquinavir, and zanamivir that have been developed using this computer-aided drug discovery strategy.[15] This study, therefore, evaluated the pharmacological properties of some reported phytoconstituents of A. conyzoides against key enzymes of Blastomyces dermatitidis using in silico approach as a result of the efficacy, effectiveness, safety, and cost-effectiveness of phytomedicines.

Methods and Material

Selection of target receptors

Saccharomyces cerevisiae lanosterol 14-alpha demethylase bound to lanosterol (PDB ID: 4 LXJ) with a resolution of 1.9Ầ, R-value free 0.227, and R-value work 0.195; human squalene epoxidase (PDB ID: 6C6N) with a resolution of 2.3Ầ, R-value free 0.220, and R-value work of 0.189; and ternary complex thymidylate synthase from Pneumocystis carinii (PDB ID: 1CI7) with a resolution of 2.6 Ầ, R-value free 0.294, and R-value work 0.221 were downloaded from protein data bank where crystal waters and other residues which were not amino acids were stripped out using a plain text editor.

Preparation of their structure

The enzymes were prepared by removing every molecule which was not an amino acid residue. The topology parameters of the proteins were created using GROMACS program. A molecular simulation was performed for each of the proteins using GROMACS. A simulation system was made up of the enzyme in a box with water and the required ions, ensuring total neutrality of the simulated system. The energy of the system was minimized. To equilibrate the system, the enzymes (solutes) were subjected to 100ps position-restrained dynamic simulation (Constant temperature constant volume - NVT and Constant temperature constant pressure - NPT) at 300K, stabilizing the simulation system. Finally, the full system was subjected to MD production run at 300K temperature, 1 bar pressure for 20000ps. The simulated proteins were then analyzed, where trjconv, a postprocessing tool was used to strip out coordinates and correct periodicity in the system. The radius of gyration, a measure of the compactness (folding) of the simulated proteins, was determined.

Molecular docking

Selection of ligands

A total of 29 bioactive compounds previously isolated from the leaves of A. conyzoides were randomly selected by a literature survey. They include 1(2H)-naphthalenone, 2,4,6-tri-tert-butylphenol, 2-methoxy-4-vinylphenol, 2-methylbutanoic acid, 2-monopalmitin, 3,7,11,15-tetramethyl-2-hexadecen-1-ol, 5-benzamido-4-oxo-6-phenylhexanoic acid, 7-tert.-butyl-3,3-dimethyl-1-indanone, alpha muurolene, benzoic acid, β-caryophyllene, β-sesquiphellandrene, caryophyllene oxide, catechin, Coumalic acid, 1-Dotriacontene, gallic acid, glycerin, linolenic acid ethyl ester, palmitic acid ethyl ester, palmitic acid, p-coumaric acid, phytol, precocene 1, protocatechuic acid, sinapic acid, sitosterol, stigmasterol, and the standard antifungal drugs, fluconazole, terbinafine, voriconazole, 5-flucytosine, itraconazole, naftifine. The 3D Structure Data File (SDF) structures were downloaded from PubChem database (www.pubchem.ncbi.nlm.nih.gov).

These ligands were docked with the simulated enzymes using AutoDock 4.0 software and the poses that showed the lowest binding energies were visualized using LigPlot+.

Analyzing protein–ligand interaction using LigPlot+

Hydrogen bonding and hydrophobic interactions between ligands and active sites of protein–ligand complexes were analyzed using LigPlot+, where schematic diagrams of protein–ligand interaction were generated.[16]

The imported ligands in AutoDock Vina were saved. PyMOL was then used to open the simulated enzymes where the saved ligands were added to the respective enzymes in PyMOL forming enzyme–ligand pose. The enzyme–ligand poses were then opened in LigPlot+ and ran. The schematic diagrams of protein–ligand interaction were then saved.

Pharmacological profiling using SwissADME and admetSAR

Canonical smiles formats of the ligands were obtained from PubChem (www.pubchem.ncbi.nlm.nih.gov) and subjected to the free web tools of SwissADME/admetSAR to evaluate the pharmacokinetics, drug-likeness, and medicinal chemistry friendliness of small molecules; absorption, distribution, metabolism, excretion, and toxicity of the bioactive compounds (http://lmmd.ecust.edu.cn/admetsar2/).

Results

From the study, sitosterol, stigmasterol, catechin, and 5-benzamido-4-oxo-6-phenylhexanoic acid produced higher binding energy when docked with S. cerevisiae lanosterol 14-alpha demethylase (PDB id: 4lxj), P. carinii thymidylate synthase (PDB id: 1ci7), and chain A of squalene epoxidase (PDB id: 6c6n), as shown in [Table 1]. Flucytosine, a standard antifungal drug that targets fungal thymidylate synthase, showed lower binding energy (−5.8 kcal/mol) when docked with simulated fungal thymidylate synthase compared to energy values obtained from sitosterol, catechin, stigmasterol, and 5-benzamido-4-oxo-6-phenylhexanoic acid interacting with thymidylate synthase (−9.1 kcal/mol, −7.9 kcal/mol, −9.4 kcal/mol, and −7.6 kcal/mol, respectively). Similarly, the interaction of sitosterol, catechin, stigmasterol, and 5-benzamido-4-oxo-6-phenylhexanoic acid with squalene epoxidase showed either higher or comparable energy values (−8.3 kcal/mol, −6.9 kcal/mol, −8.6 kcal/mol, and −7.6 kcal/mol, respectively) relative to the standard antifungal drug, terbinafine (with a binding energy of −6.9 kcal/mol), which targets squalene epoxidase in the ergosterol biosynthesis. The interaction of sitosterol, catechin, stigmasterol, and 5-benzamido-4-oxo-6-phenylhexanoic acid with S. cerevisiae lanosterol 14-alpha demethylase also showed high energy values (−7.4 kcal/mol, −7.0 kcal/mol, −7.7 kcal/mol, and −7.1 kcal/mol, respectively) although lower than itraconazole, a standard antifungal drug that targets lanosterol 14-alpha demethylase in the ergosterol biosynthetic pathway which showed a binding energy of −8.4 kcal/mol [Table 1].{Table 1}

The binding interactions between the ligands (sitosterol, stigmasterol, catechin, and 5-benzamido-4-oxo-6-phenylhexanoic acid) and the enzymes (lanosterol 14-alpha demethylase, thymidylate synthase, and squalene epoxidase) are shown in [Figure 1], [Figure 2], [Figure 3]. All four ligands interacted with amino acid residue at the active pocket of its respective enzymes via hydrogen bonding and/or hydrophobic interactions.{Figure 1}{Figure 2}{Figure 3}

[Table 2] shows the drug-likeliness of sitosterol, stigmasterol, catechin, and 5-benzamido-4-oxo-6-phenylhexanoic acid using the Lipinski rule of five model, Ghose model, and Veber filter. Altogether, the four ligands were identified as drug-like compounds.{Table 2}

The pharmacokinetic and physicochemical properties of stigmasterol, sitosterol, catechin, and 5-benzamido-4-oxo-6-phenylhexanoic acid are shown in [Table 3] and [Table 4], respectively. From [Table 3], sitosterol and stigmasterol were localized in the lysosome while catechin and 5-benzamido-4-oxo-6-phenylhexanoic acid were localized in the mitochondrion. From the results, with the exception of stigmasterol which served as a substrate to cytochrome P450 isoforms (CYP2C9), none of the other ligands from A. conyzoides leaves is a substrate to P-gp, CYP1A2, and CYP2C19 and CYP2C9. Furthermore, none of the four ligands from A. conyzoides leaves is permeable to the blood–brain barrier. Sitosterol and stigmasterol showed poor aqueous solubility. From [Table 4], all the four ligands have a high molecular weight.{Table 3}{Table 4}

The toxicity profile of sitosterol, catechin, 5-benzamido-4-oxo-6-phenylhexanoic acid, and stigmasterol is shown in [Table 5]. From the results, sitosterol and stigmasterol were not hepatotoxic and carcinogenic and had no mutagenic effect. Catechin showed Ames mutagenesis but no hepatotoxic and carcinogenic effects while 5-benzamido-4-xo-6-phenylhexanoic acid showed hepatotoxicity but no carcinogenic and Ames mutagenic effects.{Table 5}

Discussion

CADD has become a useful tool in the discovery and development of drugs in recent years. This study aimed at computationally analyzing and evaluating lead compounds from A. conyzoides geared toward the optimization or development of potent drugs against B. dermatitidis. This has become necessary because existing standard antifungal drugs are susceptible to resistance and are prone to many side effects.

In this study, the phytoconstituents sitosterol, stigmasterol, catechin, and 5-benzamido-4-oxo-6-phenylhexanoic acid from A. conyzoides leaves showed higher activity against the three fungal enzymes even though other bioactive compounds of A. conyzoides leaves has been reported to also have some level of antifungal properties.[7],[8],[9],[10] Flucytosine and terbinafine, standard antifungal drugs that target fungal thymidylate synthase and squalene epoxidase, respectively, showed either lower or comparable binding energy values relative to sitosterol, stigmasterol, catechin, and 5-benzamido-4-oxo-6-phenylhexanoic acid. This implies that thymidylate synthase and squalene epoxidase showed higher binding affinities for sitosterol, catechin, stigmasterol, and 5-benzamido-4-oxo-6-phenylhexanoic acid than their respective standard antifungal drugs. The higher the affinity (ka), the lower the substrate concentration at half the Vmax (km). This means that lower concentrations (mg) of sitosterol, catechin, stigmasterol, and 5-benzamido-4-oxo-6-phenylhexanoic acid are required to exhibit their inhibitory activities against thymidylate synthase and squalene epoxidase as compared to the standard antifungal drugs.

Itraconazole, a standard antifungal drug that targets lanosterol 14-alpha demethylase in the ergosterol biosynthetic pathway, on the other hand, showed a higher binding energy value compared to sitosterol, stigmasterol, catechin, and 5-benzamido-4-oxo-6-phenylhexanoic acid [Table 1]. In general, maximum interaction between protein–ligand complex occurs when the ligand in its transition state complementarily binds the active site of the enzyme giving rise to maximum binding energy which then is used to decrease the activation energy of a reaction resulting in high catalytic rate.[17] The higher binding energy of the itraconazole–lanosterol 14-alpha demethylase interaction may be attributed to the high complementarity of the transition state of itraconazole to the active site of lanosterol 14-alpha demethylase compared to sitosterol, catechin, stigmasterol, and 5-benzamido-4-oxo-6-phenylhexanoic acid.

To understand the characteristics of the binding, the interactions between the ligands and the active binding sites of the respective receptors were assessed using the LigPlot+ analyses. From the analyses of the binding interactions between the ligands and lanosterol 14-alpha demethylase, itraconazole formed a hydrogen bond with the active pocket of lanosterol 14-alpha demethylase via two amino acid residues, namely Arg430 and Val441, with a bond length of 3.06Å and 3.33Å, respectively. There were also hydrophobic interactions between Tyr419, Phe420, Pro421, Asn427, Trp431, Ser440, Gly442, Ser453, Gly455, Ser457, Ser458, and Tyr460 of the amino acid residues at the active site of lanosterol 14-alpha demethylase and hydrophobic parts of itraconazole [Figure 1]. Stigmasterol also showed hydrophobic interactions with similar amino acid residues as itraconazole, namely Tyr419, Phe420, Trp431, Ser458, Gly455, and Ser453. The other amino acid residues at the active site of lanosterol 14-alpha demethylase involved in the hydrophobic interaction with stigmasterol are Arg430, Ser438, Tyr439, Val441, Tyr460, Leu461, and Arg469 yielding the binding energy of −7.7 kcal/mol [Figure 1] and [Table 1]. Furthermore, sitosterol showed hydrophobic interactions with amino acid residues, Pro38, Tyr41, Asn42, Trp45, Tyr61, Pro64, Trp65, Val94, and Gly97, which are closer to the N-terminal of S. cerevisiae lanosterol 14-alpha demethylase, probably accounting for the binding energy of −7.4 kcal/mol [Figure 1] and [Table 1].

Catechin and 5-benzamido-4-oxo-6-phenylhexanoic acid interact with amino acids at the active site of lanosterol 14α-demethylase via hydrogen bonding and hydrophobic interactions accounting for the binding energies of −7.0 and −7.7 kcal/mol, respectively. Catechin formed a hydrogen bond with the active pocket of lanosterol 14-alpha demethylase via three amino acid residues, namely Lys371, Tyr460, and Arg469, while 5-benzamido-4-oxo-6-phenylhexanoic acid formed a hydrogen bond with Thr507 at the active site of lanosterol 14-alpha demethylase [Figure 1]. Again, catechin was involved in hydrophobic interactions with Ser438, Tyr439, Ser453, Ser458, Leu461, and Pro462 at the binding site of the enzyme while 5-benzamido-4-oxo-6-phenylhexanoic acid showed hydrophobic interactions with Ala69, Val70, Tyr72, Gly73, Leu95, Leu96, Arg98, Thr237, Pro238, Ille239, Phe241, Ser508, and Met509 [Figure 1].

LigPlot+ analysis of the binding interaction also revealed that terbinafine formed a hydrophobic bond with the active pocket of squalene epoxidase via Arg161, Gly132, Val133, Pro159, Asp160, Val163, Gly164, His226, Asp408, Pro415, Gly418, Gly419, and Gly420 [Figure 2]. Stigmasterol also showed hydrophobic interaction with five of the amino acid residues associated with terbinafine, namely Gly132, Val133, Pro159, Arg161, and Gly164. Other amino acid residues of squalene epoxidase involved in the hydrophobic interaction with stigmasterol are Val129, Glu153, Arg154, Ile230, Val249, Val250, Ala284, Asp285, Lys290, and Phe291 [Figure 2]. Further, stigmasterol also formed hydrogen bonding with squalene epoxidase via His226 and Val163 showing bond lengths of 1.27Å and 2.96Å, respectively.

Similar to stigmasterol, sitosterol also showed hydrophobic interaction with some amino acid residues of squalene epoxidase associated with the standard drug, terbinafine. These are Gly132, Val133, Pro159, Arg161, and Gly164. The other amino acid residues involved in the hydrophobic interaction between sitosterol and the active pocket of squalene epoxidase are Val129, Glu153, Arg154, Ile230, Ala284, Asp285, and Phe291 [Figure 2]. In addition, sitosterol, like stigmasterol, also formed two hydrogen bonds with Val163 and His226 at the active site of squalene epoxidase but with bond lengths of 3.02Å and 3.24Å, respectively.

Catechin and 5-benzamido-4-oxo-6-phenylhexanoic acid both formed hydrogen bonds with the active pocket of squalene epoxidase via four amino acids (Glu153, Asp155, Lys157, and Arg234) and three amino acids (Asp408, Asn411, and Arg413), respectively [Figure 2]. In addition, whereas catechin was involved in six hydrophobic bonding with Val133, Leu156, Pro159, His226, Asp285, and Gly286 at the active site of squalene epoxidase, 5-benzamido-4-oxo-6-phenylhexanoic acid was involved in ten hydrophobic interactions with Val133, Arg161, Val163, Gly164, Gly407, Pro415, Gly418, Gly420, Met421, and Ala424, accounting for the binding energies of −6.9 and −7.6 kcal/mol of catechin and 5-benzamido-4-oxo-6-phenylhexanoic acid, respectively [Figure 2] and [Table 1].

LigPlot+ analysis of the binding interaction also revealed that flucytosine formed hydrophobic interaction with the active pocket of thymidylate synthase via Gly72, Glu73, Thr74, and Cys127 [Figure 3]. Contrarily, in addition to forming various hydrogen bonds with amino acid residues at the binding pocket of thymidylate synthase, 5-benzamido-4-oxo-phenylhexanoic acid, catechin, stigmasterol, and sitosterol also showed hydrophobic interaction but with different amino acid residues altogether at the active pocket of thymidylate synthase. These may underpin the lower energy level of flucytosine (−5.8 kcal/mol) compared to 5-benzamido-4-oxo-phenylhexanoic acid (−7.6 kcal/mol), catechin (−7.9 kcal/mol), stigmasterol (−9.6 kcal/mol), and sitosterol (−9.1 kcal/mol). Catechin exhibited hydrogen bonding with Arg153, His240, Asp238, Ser200, Asn210, and Asp202 and hydrophobic interactions with Leu170, Cys173, Phe209, Gly206, and Leu205 at the binding pocket of thymidylate synthase. 5-benzamido-4-oxo-6-phenylhexanoic acid also formed a hydrogen bond with Asp238, Ser200, Asn210, His240, and Tyr242 which are similar to the amino residues that interacted with catechin via hydrogen bond as stated above. Further, 5-benzamido-4-oxo-6-phenylhexanoic acid showed hydrophobic interaction with three amino acid residues similar to those that interacted with catechin: Cys173, Phe209, and Gly206. The other amino acid residues involved in the hydrophobic interaction with 5-benzamido-4-oxo-6-phenylhexanoic acid and thymidylate synthase are Ile86, Trp87, Asn90, Cys201, Asp202, and Leu205. The similarities in the amino residues involved in the interaction between catechin and 5-benzamido-4-oxo-6-phenylhexanoic acid, with the binding pocket of thymidylate synthase, probably justify the comparison between their energy levels: −7.9 kcal/mol and −7.6 kcal/mol, respectively. Although sitosterol and stigmasterol showed a hydrogen bond via different amino acid residues: Thr290 and Tyr113, respectively, the two ligands shared some similarities in the amino acid residues involved in the hydrophobic interaction with thymidylate synthase. These residues are Lys55, Phe58, Ile86, Trp87, Leu205, Phe209, and Met295. This probably accounts for the similarities in the energy levels of sitosterol and stigmasterol upon interacting with thymidylate synthase: −9.1 kcal/mol and −9.6 kcal/mol, respectively. The other amino acid residues involved in the hydrophobic with sitosterol are Glu65, Tyr113, His174, Ile291, and Lys292 while the amino acid residues involved in the hydrophobic interaction with stigmasterol are Arg56, Val57, Leu170, and Cys173.

The four lead compounds in A. conyzoides leaves and the three standard drugs were evaluated for their drug ability likeness using the Lipinski rule of five model, Ghose model, and Veber filter [Table 2]. Lipinski rule states that an orally active drug has no more than one violation of the following criteria: octanol–water partition coefficient of the drug should be less than calculated log P (ClogP) of 5 (or MilogP should not exceed 4.15), molecular weight should be <500 Da, hydrogen bond donors (such as the combined OH and NH group count) should not exceed 5, and hydrogen bond acceptors (such as the combined nitrogen and oxygen atom count) should not exceed 10.[18] Based on the Lipinski filter, sitosterol, catechin, stigmasterol, 5-benzamido-4-oxo-6-phenylhexanoic acid, terbinafine, and flucytosine are identified as drug-like compounds even though sitosterol, stigmasterol, and terbinafine violated one of the rules which was logp >4.15 while itraconazole was not drug like since it violated two of Lipinski's rule of five, that is, itraconazole has a molecular weight of 705.63 Da, logp >4.15, and NorO >10. According to Srimai, drug compounds with a molecular weight <500 Da are easily transported, diffused, and absorbed as compared to compounds of drug molecules with a molecular weight more than 500 Da.[19]

Another model for evaluating the drug-likeness of a compound is Ghose rule which states that a molecule is drug like if the molar refractivity of the molecule is 40–130, the number of atoms is 20–70, Logp is −0.4 to +5.6, and molecular weight from 180 to 480 Da.[20] With this model sitosterol, stigmasterol, itraconazole, and flucytosine could be classified as nondrug. This is because the logp of sitosterol, stigmasterol, and itraconazole was >5.6, their molar refractivity was also more than 130, and their number of atoms exceeded 70. Flucytosine was classified as nondrug based on Ghose model because the molecular weight of flucytosine was <160 Da, molar refractivity was <40, and the number of atoms was <20.

Veber's model was also employed in evaluating the drug-likeness of the ligands. Veber's rule classified sitosterol, stigmasterol, catechin, 5-benzamido-4-oxo-6-phenylhexanoic acid, terbinafine, and flucytosine as drug-like compounds except for itraconazole with rotatable bonds more than 10. Good bioavailability of a drug or lead compound is more promising for compounds with 10 or fewer rotatable bonds and polar surface area not >140 Å.[21]

Itraconazole was the only ligand that failed to be drug-like molecule based on Lipinski, Ghose, and Veber filters. Lipinski rule of five, however, is the most common filtering method that looks for compounds that can be absorbed, and eliminates those that do not have reasonable absorption characteristics.[22] Thus, based on the results of the Lipinski rule of five, stigmasterol, sitosterol, catechin, and 5-benzamido-4-oxo-6-phenylhexanoic acid can be envisioned as potential ligands that could interfere with the activities of squalene epoxidase, thymidylate synthase, and lanosterol 14-alpha demethylase.

The pharmacokinetics of stigmasterol, sitosterol, catechin, 5-benzamido-4-oxo-6-phenylhexanoic acid, itraconazole, terbinafine, and flucytosine are shown in [Table 3]. The pharmacokinetics data of these molecules were retrieved from (http://www.swissadme.ch/index.php). The results showed that sitosterol, stigmasterol, and terbinafine were localized in the lysosome while catechin, 5-benzamido-4-oxo-6-phenylhexanoic acid, itraconazole, and flucytosine were localized in the mitochondrion. The subcellular localization of drugs and the mode of delivery of drugs are essential factors that influence their biological activity and also the efficiency of drug metabolism depends on the compartment in which the reaction is taking place.[23] Catechin, 5-benzamido-4-oxo-6-phenylhexanoic acid, itraconazole, terbinafine, and flucytosine have high gastrointestinal absorption while sitosterol and stigmasterol have low gastrointestinal absorption. According to Devadasu et al., poor aqueous solubility and poor permeability of a drug molecule are the factors that limit drug absorption from the gastrointestinal tract.[24] The water solubility result of the sitosterol and stigmasterol from SwissADME, a free web tool, confirmed that sitosterol and stigmasterol have poor aqueous solubility, hence their low gastrointestinal absorption. Hydrophobicity and low molecular weight are factors that promote blood–brain barrier penetration.[25]

From the physicochemical properties of all the ligands [Table 4], it could be observed that all the compounds are large, that is, they have a high molecular weight which makes them nonpermeable to the blood–brain barrier. The tested ligands were not P-glycoprotein (P-gp) except itraconazole which proved to be a substrate for P-gp that effluxes drugs and other xenobiotic compounds to undergo further metabolism and clearance.[26] The extrusion of xenobiotic compounds leads to decrease in the concentration of the compounds resulting in therapeutic failure.[27]

The inhibition of the cytochrome P450 isoform enzymes has also been reported. Cytochrome P450 isoform enzymes are drug-metabolizing enzymes and their inhibition results in drug–drug interaction where affected drug may lead to an increase in the toxic levels due to the accumulation of unmetabolized drugs.[28] From the results, itraconazole was an inhibitor of CYP1A2, an enzyme that plays an important role in drug metabolism,[29] while sitosterol, stigmasterol catechin, 5-benzamido-4-oxo-6-phenylhexanoic acid, terbinafine, and flucytosine did not inhibit CYP1A2 [Table 3]. Itraconazole and stigmasterol also inhibited CYP2C9, an enzyme in cytochrome P450 superfamily that plays a role in the oxidation of both xenobiotic and endogenous compounds,[28] while sitosterol, catechin, 5-benzamido-4-oxo-6-phenylhexanoic acid, terbinafine, and flucytosine did not inhibit CYP2C9. Itraconazole was an inhibitor of CYP2C19, CYP2D6, and CYP3A4. Terbinafine also inhibited CYP2D6.

Toxicity testing is key in drug discovery and development in order to establish the safety and also characterize the level of possible toxicity of a drug. [Table 5] also shows the toxicity profile of four reported secondary metabolites from the leaf extract of A. conyzoides: sitosterol, catechin, 5-benzamido-4-oxo-6-phenylhexanoic acid, and stigmasterol, and three standard antifungal drugs: itraconazole, terbinafine, and flucytosine. Catechin did not show hepatotoxicity and carcinogenicity but showed Ames mutagenesis. Itraconazole and 5-benzamido-4-oxo-6-phenylhexanoic acid showed hepatotoxicity but did not show carcinogenicity and Ames mutagenesis. Terbinafine and flucytosine were hepatotoxic and showed mutagenic effects but were not carcinogenic. The results also showed that sitosterol and stigmasterol were not hepatotoxic and carcinogenic and had no mutagenic effect. The compounds that showed either hepatotoxicity, Ames mutagenesis, or carcinogenicity, need to be administered cautiously. The acute oral toxicity category of sitosterol, catechin, stigmasterol, 5-benzamido-4-oxo-6-phenylhexanoic acid, itraconazole, terbinafine, and flucytosine was classified based on the criterion of Globally Harmonized System of Classification and Labelling of chemicals which is widely used for classification and labeling of chemicals worldwide according to a numeric cutoff of its lethal dose (LD50).[30] The lethal dose (LD50) is a statistical estimate of the number of mg of toxicants per kg of bodyweight required to kill 50% of test animals.[31] Category 1 consists of compounds with LD50 values <5 mg/kg and such compounds are fatal if swallowed. Sitosterol and stigmasterol were classified under category 1. This means a small amount of sitosterol and stigmasterol in mg (approximately, 1/10th of its LD50) is required for their antifungal potency. Category 2 contains compounds with LD50 from 5 mg/kg to 51 mg/kg and such compounds are fatal if swallowed. Itraconazole had an acute oral category of 2. This means that <5 mg/kg of itraconazole needs to be administered in humans. Administering 5 mg/kg to 51 mg/kg of itraconazole will be fatal. Category 3 consists of compounds with LD50 from 50 mg/kg to 300 mg/kg and such compounds are toxic if swallowed. 5-benzamido-4-oxo-6-phenylhexanoic acid, terbinafine, and flucytosine fell under category 3 of the classification. Less than 50 mg/kg of 5-benzamido-4-oxo-6-phenylhexanoic acid, terbinafine, and flucytosine are required to be administered in humans to reduce their toxicity. The acute oral category of catechin was category 4. Category 4 consists of compounds with LD50 from 300 mg/kg to 2000 mg/kg and such compounds are harmful if swallowed. Category 5 consists of compounds with LD50 from 2000 mg/kg to 5000 mg/kg and compounds may be harmful if swallowed.

Conclusion

B. dermatitidis poses health threats due to the frequency of fungal infections and the increasing resistance to standard antifungal drugs. Effective drugs with no or minimal side effects need to be developed and one such way is the use of phytomedicine. The results obtained from the docking studies of some selected phytoconstituents in A. conyzoides leaves showed that the ligands (sitosterol, catechin, stigmasterol, and 5-benzamido-4-oxo-6-phenylhexanoic acid) interacted with and displayed very good binding affinity toward the three crucial antifungal drug target receptors (lanosterol 14-alpha demethylase, thymidylate synthase, and squalene epoxidase) with significant inhibition as compared to the standard drugs, and thus hold a promising potential to be explored for their antifungal activities. However, further studies in terms of both in vitro and in vivo tests should be carried out to assess and optimize the therapeutic efficacy of A. conyzoides in treating blastomycosis.

Limitation of study

The results obtained are theoretical predictions and may differ from in vivo and in vitro studies.

Ethical approval

No ethical approval was required for this study

Financial support and sponsorship

The Centre for Plant Medicine Research, Mampong-Akuapem; Biochemistry Department, Kwame Nkrumah University of Science and Technology; Department of Biomedical Engineering, School of Engineering Sciences; and West African Centre for Cell Biology of Infectious Pathogens, University of Ghana, Accra, Ghana, all provided indirect financial support toward this study.

Conflicts of interest

There are no conflicts of interest.

References

1	Miceli A, Krishnamurthy K. Blastomycosis. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2022.
2	Lohrenz S, Minion J, Pandey M, Karunakaran K. Blastomycosis in Southern Saskatchewan 2000-2015: Unique presentations and disease characteristics. Med Mycol 2018;56:787-95.
3	McBride JA, Gauthier GM, Klein BS. Turning on virulence: Mechanisms that underpin the morphologic transition and pathogenicity of Blastomyces. Virulence 2019;10:801-9.
4	McCarthy MW, Kontoyiannis DP, Cornely OA, Perfect JR, Walsh TJ. Novel agents and drug targets to meet the challenges of resistant fungi. J Infect Dis 2017;216:S474-83.
5	Nketia A, Nakua E, Tetteh AW, Thomford KP, Boadu KO, Thomford AK, et al. Herbal medicine practice in Ghana: A cross-sectional study to understand the factors influencing patient utilization of herbal medicine services. Int Res J Public Environ Health 2022;9:16-23.
6	Yadav N, Ganie SA, Singh B, Chhillar AK, Yadav SS. Phytochemical constituents and ethnopharmacological properties of Ageratum conyzoides L. Phytother Res 2019;33:2163-78.
7	Chahal R, Nanda A, Akkol EK, Sobarzo-Sánchez E, Arya A, Kaushik D, et al. Ageratum conyzoides L. And its secondary metabolites in the management of different fungal pathogens. Molecules 2021;26:2933.
8	Batish DR, Kaur S, Singh HP, Kohli RK. Nature of interference potential of leaf debris of Ageratum conyzoides. Plant Growth Regulation 2008;57:137-44.
9	Sidra J and Uzma B. Antifungal activity of different extracts of Ageratum conyzoides for the management of Fusarium solani. Afr J Biotechnol 2012;11:11022-9.
10	Adesanwo JK, Egbomeade CO, Moronkola DO, Akinpelu DA. Chemical, toxicity and antibacterial studies on methanol extracts of Melanthera scandens, Ageratum conyzoides, Aspilia Africana and Synedrella nodiflora. J Explor Res Pharmacol 2019;4:1-7.
11	Kherade DS, Tambe VS, Wagh AD, Kothawade PB. A comparative molecular docking study of crocetin with multiple receptors for the treatment of Alzheimer's disease. Biomed Biotechnol Res J 2022;6:230-42.
12	Kwofie SK, Broni E, Yunus FU, Nsoh J, Adoboe D, Miller WA, et al. Molecular docking simulation studies identifies potential natural product derived-antiwolbachial compounds as filaricides against Onchocerciasis. Biomedicines 2021;9:1682.
13	Muddukrishnaiah K, Vijayakumar V, Thavamani BS, Shilpa VP, Radhakrishnan N, Abbas HS. Synthesis, characterization, and in vitro antibacterial activity and molecular docking studies of N4, N4'-dibutyl-3,3'-dinitro-[1,1'-biphenyl]-4,4'-diamine. Biomed Biotechnol Res J 2020;4:318-22.
14	Halder ST, Dhorajiwala TM, Samant LR. Molecular docking studies of filarial β-tubulin protein models with Antifilarial phytochemicals. Biomed Biotechnol Res J 2019;3:162-70.
15	Talele TT, Khedkar SA, Rigby AC. Successful applications of computer aided drug discovery: Moving drugs from concept to the clinic. Curr Top Med Chem 2010;10:127-41.
16	Verma R, Agarwal M, Jatav V. Computer-aided screening of therapeutic ligands against KLF8 protein (Homo sapiens). Int J Comput Bioinform In silico Model 2014;3:479-82.
17	Schwartz SD, Schramm VL. Enzymatic transition states and dynamic motion in barrier crossing. Nat Chem Biol 2009;5:551-8.
18	Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev 2001;46:3-26.
19	Srimai V, Ramesh M, Parameshwar KS Parthasarathy T. Computer-aided design of selective Cytochrome P450 inhibitors and docking studies of alkyl resorcinol derivatives. Med Chem Res 2013;22:5314-23.
20	Ghose AK, Viswanadhan VN, Wendoloski JJ. A knowledge-based approach in designing combinatorial or medicinal chemistry libraries for drug discovery. 1. A qualitative and quantitative characterization of known drug databases. J Comb Chem 1999;1:55-68.
21	Veber DF, Johnson SR, Cheng HY, Smith BR, Ward KW, Kopple KD. Molecular properties that influence the oral bioavailability of drug candidates. J Med Chem 2002;45:2615-23.
22	Turner JV Agatonovic-Kustin S. In silico prediction of oral bioavailability. Compr Med Chem 2007;5:669-724.
23	Studzian K, Kik K, Lukawska M, Oszczapowicz I, Strek M, Szmigiero L. Subcellular localization of anthracyclines in cultured rat cardiomyoblasts as possible predictors of cardiotoxicity. Invest New Drugs 2015;33:1032-9.
24	Devadasu VR, Deb PK, Maheshwari R, Sharma P Tekade RK. Physicochemical, pharmaceutical, and biological considerations in GIT absorption of drugs. In: Dosage Form Design Considerations. United Kingdom: Academic Press; 2018. p. 149-78.
25	Bertrand L, Velichkovska M, Toborek M. Cerebral vascular toxicity of antiretroviral therapy. J Neuroimmune Pharmacol 2021;16:74-89.
26	Amin ML. P-glycoprotein inhibition for optimal drug delivery. Drug Target Insights 2013;7:27-34.
27	Nishino K, Yamasaki S, Nakashima R, Zwama M, Hayashi-Nishino M. Function and inhibitory mechanisms of multidrug efflux pumps. Front Microbiol 2021;12:737288.
28	Srinivas M, Thirumaleswara G, Pratima S. Cytochrome P450 enzymes, drug transporters and their role in pharmacokinetic drug-drug interactions of xenobiotics: A comprehensive review. Peertechz J Med Chem Res 2017;3:1-11.
29	Obach RS, Isoherranen N. Pathways of drug metabolism. In: Huang S, Lertora JJ, Vicini P, Atkinson AJ, editors. Atkinson's Principles of Clinical Pharmacology. 4^th ed., Ch. 10. U.S.: Academic Press; 2022. p. 151-68.
30	WHO. WHO Recommended Classification of Pesticides by Hazard and Guidelines to Classification, 2019 edition. Geneva: World Health Organization; 2020.
31	Ochoa R. Pathology issues in the design of toxicology studies. In: Haschek WM, Rousseaux CG, Wallig MA, editors. Handbook of Toxicologic Pathology. 2^nd ed. USA: Academic Press; 2002. p. 307-26.