1,2,4-Triazole Derivatives Targeting Fungal 14-Alpha Demethylase Enzyme: An Insilico Approach

Fungal infection is growing health concern, with an estimated 1.5million deaths annually caused by invasive fungal infection. These infections are caused by a variety of fungal species, including Candida, Aspergillus, and Cryptococcus. Antifungal therapies are used to treat these infections, but rise of antifungal resistance has become a major challenge in the field. Fungal infections are caused by fungi that invade the tissues of humans and other animals. There are many different types of fungi that can cause infections, including yeasts and Molds. Some common fungal infections including athlete’s foot, jock itch, ringworm, and thrush. Fungal infections can affect different parts of the body, including the skin, nails and lungs. Other types of fungal infections can affect the lungs, like aspergillosis and histoplasmosis. Some types of fungal infections can be serious and even life-threatening, especially in people with weakened immune systems, such as those with HIV/AIDS or cancer or those taking immunosuppressive medications. Some of the common fungal infections: Athlete’s foot: It is also known as tinea pedis, is a common fungal infection that affects the skin on the feet. Athlete’s foot can cause symptoms such as itching, burning and cracking of the skin between the  toes and the soles of the feet. Ringworm: Ringworm is a common fungal infection that affects the skin, hair and nails. Ringworm is highly contagious and can spread through direct contact with an infected person, animal or object. The azole antifungal includes two broad classes, imidazole and triazoles. The systemic triazoles are more slowly metabolized and have less effect on human sterol synthesis than do the imidazole’s. Because of these advantages, new congeners under the development are mostly triazoles, not imidazole’s(1).

MECHANSIM OF ACTION

The major effects of triazoles on fungi is inhibition of 14-alpha-demethylase, a microsomal cytochrome 450-dependent enzyme system. Triazoles thus impair the biosynthesis of ergosterol for the cytoplasmic membrane and lead to the accumulation of 14-alpha-methylsterols.These methyl sterols may disrupt the close packing  acyl chains of phospholipids, impairing the functions of certain membrane bound enzyme systems such as ATPase and enzymes of the electron transport system and thus inhibiting growth of the fungi(2,3,4).

INTRODUCTION ABOUT TRIAZOLE

In the last few decades, the chemistry of 1,2,4 triazoles and their fused heterocyclic derivatives. Have received considerable attention owing to their synthetic and effective biological importance.1,2,4 triazole moiety has been incorporated into a wide variety of therapeutically interesting drug candidates including antifungal, antibacterial, analgesics and anti-inflammatory, Antineoplastic, anticonvulsant, antiviral etc. The triazole is the five-membered three nitrogen containing heterocyclic aromatic ring. Triazole play a key role in various biological mechanism related to infections, cancer, Convulsions, inflammation and neurodegeneration. The 1,2,4-triazole are five membered and sp³ hybridization molecule. The synthesis and development of new 1,2,4-triazoles with low toxicity and inhibit the fungal Growth.

CHEMISTRY AND STRUCTURE ACTIVITY RELATIONSHIP

• 1,2,4 triazole is one of a pair of isomeric chemical compounds.
• Molecular formula: C₂H₃N₃
• It has five-membered ring of two carbon atoms and three nitrogen atoms.
• 1,2,4 triazole is a basic aromatic heterocycle(5).

DRUG DISCOVERY- Drug discovery is a multidisciplinary scientific process aimed at identifying new therapeutic compounds that can prevent, cure or manage diseases. The modern drug discovery process integrates principles from chemistry, biology, pharmacology, and computational sciences to design and develop novel molecules with optimal efficacy and safety profiles.(6)

MOLECULAR DOCKING -Molecular docking is a powerful computational technique used to predict the interaction between a ligand and a target molecule, typically a protein or nucleic acid. The fundamental principle of molecular docking is to stimulate the binding of a ligand to the active site of a target molecules to evaluate the strength of this interaction using scoring functions(7).

SWISS ADME- Swiss ADME is a free, web-based computational tool, for predicting the physicochemical properties, pharmacokinetics properties. It is widely used in drug discovery and development to assess the Absorption, Distribution, ,Metabolism, and Excretion. One of its standout features is the BOILED -EGG model, which provides a visual prediction of gastrointestinal absorption and blood brain permeability.

PROTOX III-Protox is online tool used for toxicity prediction in drug discovery and chemical research. It helps researcher predict the potential toxicity of small molecules based on their chemical structure. It is designed to predict LD ₅₀ values (lethal dose for 50% of population),Toxicity classes(I to VI),Possible toxicological pathways, Organ toxicity. The objective of this study was to compare the docking efficiency of PyRx , a free molecular docking software with that of an Auto dock 4.2.6, which is used and to find a potent 14-alpha demethylase using the best docking software found(8).

MATERIALS AND METHODS

Table 1: Derivative structure and its IUPAC name

SR.NO	STRUCTURE	IUPAC NAME
1.		N-[3-(4-tert-butylphenyl)-1H-1,2,4-triazol-5-yl]acetamide
2.		N-[3-(4-nitrophenyl)-1H-1,2,4-triazol-5-yl]acetamide
3.		N-[3-(3-bromophenyl)-1H-1,2,4-triazol-5-yl]acetamide
4.		N-{3-[3-(trifluoromethyl)phenyl]-1H-1,2,4-triazol-5-yl}acetamide
5.		N-[3-(3,5-dimethylphenyl)-1H-1,2,4-triazol-5-yl]acetamide
6.		N-[3-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazol-5-yl]acetamide
7.		3-(4-methoxyphenyl)-N-(4-methylphenyl)-1H-1,2,4-triazol-5-amine
8.		3-(4-fluorophenyl)-N-(4-methylphenyl)-1H-1,2,4-triazol-5-amine
9.		N-(4-methylphenyl)-3-(4-nitrophenyl)-1H-1,2,4-triazol-5-amine
10.		3-(3-bromophenyl)-N-(4-methylphenyl)-1H-1,2,4-triazol-5-amine
11.		N-(4-methylphenyl)-3-[3-(trifluoromethyl)phenyl]-1H-1,2,4-triazol-5-amine
12.		3-(3,5-dimethylphenyl)-N-(4-methylphenyl)-1H-1,2,4-triazol-5-amine
13.		N-(4-methylphenyl)-3-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazol-5-amine
14.		N-cyclohexyl-3-(4-methylphenyl)-1H-1,2,4-triazol-5-amine
15.		3-(4-tert-butylphenyl)-N-cyclohexyl-1H-1,2,4-triazol-5-amine
16.		N-cyclohexyl-3-(4-methoxyphenyl)-1H-1,2,4-triazol-5-amine
17.		N-cyclohexyl-3-(4-fluorophenyl)-1H-1,2,4-triazol-5-amine
18.		3-(4-chlorophenyl)-N-cyclohexyl-1H-1,2,4-triazol-5-amine
19.		N-cyclohexyl-3-(4-nitrophenyl)-1H-1,2,4-triazol-5-amine
20.		3-(3-bromophenyl)-N-cyclohexyl-1H-1,2,4-triazol-5-amine
21.		N-cyclohexyl-3-[3-(trifluoromethyl)phenyl]-1H-1,2,4-triazol-5-amine
22.		N-cyclohexyl-3-(3,5-dimethylphenyl)-1H-1,2,4-triazol-5-amine
23.		N-cyclohexyl-3-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazol-5-amine
24.		3-(4-tert-butylphenyl)-N-phenyl-1H-1,2,4-triazol-5-amine
25.		3-(4-methoxyphenyl)-N-phenyl-1H-1,2,4-triazol-5-amine
26.		3-(4-fluorophenyl)-N-phenyl-1H-1,2,4-triazol-5-amine
27.		3-(4-chlorophenyl)-N-phenyl-1H-1,2,4-triazol-5-amine
28.		N-phenyl-3-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazol-5-amine
29.		3-(4-nitrophenyl)-N-phenyl-1H-1,2,4-triazol-5-amine
30.		3-(3-bromophenyl)-N-phenyl-1H-1,2,4-triazol-5-amine
31.		N-phenyl-3-[3-(trifluoromethyl)phenyl]-1H-1,2,4-triazol-5-amine
32.		3-(3,5-dimethylphenyl)-N-phenyl-1H-1,2,4-triazol-5-amine
33.		N-[3-(4-tert-butylphenyl)-1H-1,2,4-triazol-5-yl]pyridin-2-amine
34.		N-[3-(4-methoxyphenyl)-1H-1,2,4-triazol-5-yl]pyridin-2-amine
35.		N-[3-(4-fluorophenyl)-1H-1,2,4-triazol-5-yl]pyridin-2-amine
36.		N-[3-(4-chlorophenyl)-1H-1,2,4-triazol-5-yl]pyridin-2-amine
37.		N-[3-(4-nitrophenyl)-1H-1,2,4-triazol-5-yl]pyridin-2-amine
38.		N-[3-(3-bromophenyl)-1H-1,2,4-triazol-5-yl]pyridin-2-amine
39.		N-{3-[3-(trifluoromethyl)phenyl]-1H-1,2,4-triazol-5-yl}pyridin-2-amine
40.		N-[3-(3,5-dimethylphenyl)-1H-1,2,4-triazol-5-yl]pyridin-2-amine
41.		N-[3-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazol-5-yl]pyridin-2-amine
42.		3-(4-methylphenyl)-N-(trichloromethyl)-1H-1,2,4-triazol-5-amine
43.		3-(4-tert-butylphenyl)-N-(trichloromethyl)-1H-1,2,4-triazol-5-amine
44.		3-(4-methoxyphenyl)-N-(trichloromethyl)-1H-1,2,4-triazol-5-amine
45.		3-(4-fluorophenyl)-N-(trichloromethyl)-1H-1,2,4-triazol-5-amine
46.		3-(4-chlorophenyl)-N-(trichloromethyl)-1H-1,2,4-triazol-5-amine
47.		3-(4-nitrophenyl)-N-(trichloromethyl)-1H-1,2,4-triazol-5-amine
48.		3-(3-bromophenyl)-N-(trichloromethyl)-1H-1,2,4-triazol-5-amine
49.		N-(trichloromethyl)-3-[3-(trifluoromethyl)phenyl]-1H-1,2,4-triazol-5-amine
50.		3-(3,5-dimethylphenyl)-N-(trichloromethyl)-1H-1,2,4-triazol-5-amine
51.		N-(trichloromethyl)-3-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazol-5-amine
52.		N-[3-(4-methylphenyl)-1H-1,2,4-triazol-5-yl]quinolin-6-amine
53.		N-[3-(4-tert-butylphenyl)-1H-1,2,4-triazol-5-yl]quinolin-6-amine
54.		N-[3-(4-methoxyphenyl)-1H-1,2,4-triazol-5-yl]quinolin-6-amine
55.		N-[3-(4-fluorophenyl)-1H-1,2,4-triazol-5-yl]quinolin-6-amine
56.		N-[3-(4-nitrophenyl)-1H-1,2,4-triazol-5-yl]quinolin-6-amine
57.		N-[3-(3-bromophenyl)-1H-1,2,4-triazol-5-yl]quinolin-6-amine
58.		N-{3-[3-(trifluoromethyl)phenyl]-1H-1,2,4-triazol-5-yl}quinolin-6-amine
59.		N-[3-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazol-5-yl]quinolin-6-amine
60.		N-[3-(3,5-dimethylphenyl)-1H-1,2,4-triazol-5-yl]quinolin-6-amine /0

• • Chemsketch Ultra
  • Discovery Studio
  • PyRx
  • Autodock 1.3.7
  • SWISS ADME
  • PROTOX III

Preparation Of Ligand

Synthetic 1,2,4 triazole derivatives are drawed using chemsketch against 14 alpha demethylase enzymes. Total 60 derivative were selected all of these derivative analogues were added with hydrogens, energy minimization is done.

Preparation Of Protein Structure

The crystal structure of 3JUV and 5FRB protein fragment retrieved from Protein Data Bank(PDB) have the pharmacological targets for development of new drugs to treat fungal disease. We removed all of the heteroatoms of both receptors such as water molecules, bound ligands and any other co crystallized solvent from the PDB file.

Figure 1: 5FRB protein structure

Figure 2: 3JUV protein structure

PyRx

PyRx is a user-friendly open-source virtual screening software that integrates several computational tools for drug discovery. It is widely used in academic and research settings for molecular docking and virtual screening studies. PyRx utilizes Autodock and Autodock Vina as its docking engines. Ligand structures can be prepared by minimizing their energy using open babel, which is integrated into PyRx. The software allows flexible ligand docking by treating the ligand as a rotatable torsion tree, while the protein is usually kept rigid. The binding site of the protein can be defined by setting up a grid box, which focuses the docking simulation in a specific region of interest. PyRx supports batch docking of multiple ligands, which is particularly useful for screening phytochemicals. Each ligand is docked into the proteins active site, and binding affinities(binding energy scores) are calculated using the Autodock vina scoring function. The conformations with the lowest binding energies are considered the most favourable and further analysed to assess their interactions with the active site residue of the target protein.

Autodock 4.2.6

Autodock 4.2.6 is a widely used molecular docking tool designed for predicting the binding of small molecules (ligands) to a receptor(usually a protein). It utilises a Lamarckian Genetic Algorithm, which is a combination of a genetic algorithm and local search method, to explore the conformational space of the ligand. In this study, flexible ligand docking was performed  using Autodock. The ligand molecules were drawn and energy minimized and converted to PDBQT format using Autodock Tools. The protein structure of the 3JUV and 5FRB was retrieved from the RCSB Protein Data Bank and the receptor was prepared by removing water molecules and adding polar hydrogens. Kollman charges were added to the protein. The grid box was set around the active site of the receptor to define the docking region. Autodock uses an energy-based scoring function to evaluate binding poses, accounting for electrostatic interactions, hydrogen binding, desolvation effects, and van der Waals forces(9,10).

RESULTS AND DISCUSSION

Table 2 :Comparison of docking score using PyRx and Autodock 4.2.6 with standard antifungal drugs

Molecular docking was performed to evaluate the binding affinities of synthesized 1,2,4-triazole derivatives (Deri1-Deri60) compared to standard antifungal drug(fluconazole, Isavuconazole, Itraconazole, Posaconazole and Voriconazole). Docking studies were carried out using two different software platforms, PyRx and 4.2.6 against two fungal target proteins 3JUV and 5FRB. The standard drugs exhibited docking score ranging from PyRx:-5.3 to -8.8kcal/mol. Autodock:-6.211 to -9.231kcal/mol. Among them, isavuconazole and posaconazole showed the most favourable binding energies both targets.

The synthesised derivatives displayed a wide range of binding affinities. Notably, many derivatives exhibited better docking scores than standard drugs. Derivative 28 showed a docking score of -9.203(3JUV) and -9.445(5FRB) using auto dock, surpassing even the reference drugs. Derivative 34, derivative 35 and derivative 38 also demonstrated strong binding affinities, with values around -10.590 and-10.249(3JUV), suggesting excellent interaction with the protein binding sites. The data confirms that several synthesized triazole derivatives particularly deri28,deri34 and deri38,may possess promising antifungal agents(11,12).

Table 3: Comparing the pharmacokinetic properties with standard antifungal drug and 1,2,4 triazole derivatives

Table 4: Predicted pharmacokinetic properties and enzyme interaction with standard antifungal drug and 1,2,4 triazole derivatives