Multi-Target In Silico Evaluation of New 2-Pyrazolines as Antimicrobial Agents ^†

Abstract

The world today is being ravaged by the emergence and re-emergence of microbial infections caused by antimicrobial-resistant strains, brought about primarily by the frequent and perhaps unnecessary use of antimicrobial agents. A need therefore arises to develop new antimicrobial drugs that can combat these pathogens resistant to currently available antibiotics. This present study has adopted a multi-enzyme in silico approach in evaluating new 2-pyrazolines as antimicrobial agents, targeting and aiming to inhibit three pivotal enzymes in the bacteria’s life cycle. A library of 2-pyrazolines was tailored to achieve the desired activity. The library of compounds and amoxicillin, a standard antimicrobial drug, were docked into the molecular target enzymes. They were also subjected to toxicity and drug-likeness tests, using PROTOX and swissADME, respectively. A moderate toxicity profile was indicated, as more than 90% of the ligands were in ProTox class 4. The majority exhibited advantageous ADME characteristics. A significant number of them demonstrated a binding affinity for the target proteins that was stronger than both the native ligand and the binding affinity of amoxicillin. Ligands 30, 20, and 8 are the notable ones across all target enzymes. These results suggest that these novel ligands may be powerful inhibitors, particularly when it comes to interfering with the formation of bacterial cell walls, folic acid, and nucleotide metabolism. Additional in vivo and in vitro research is required to confirm these results and evaluate their therapeutic potential.

1. Introduction

Life is impacted by microorganisms. These microbes can either be non-pathogenic, assisting ecology, or pathogenic, causing harm to life [1]. Pathogens are primarily responsible for infectious diseases by exploiting a host’s immune system. However, since Fleming, A., in 1929 reported his accidental discovery of Penicillin, their first combatant, other drugs classed as antibiotics (i.e., ciprofloxacin, ampicillin, and sulfamethazine) have been adopted in clinical use.

Despite the immense progress made in the discovery of drugs to treat microbial infections, antimicrobial resistance (AMR) still poses a significant challenge to this day [2]. Hence, these AMRs or emerging and re-emerging microbial diseases continue to compel scientists to search for newer antibiotics with exceptional therapeutic safety and efficacy [3,4].

Among the numerous classes of compounds being investigated, Refs. [5,6] recognize pyrazoline derivatives as fascinating chemical entities with diverse biological activities and great promise for drug development. Ref. [7] also pointed out that over a decade ago, published papers reported 2-pyrazolines as pharmacophore scaffolds with paramount pharmacological potency. A number of them have been identified to possess biological activities, such as antimicrobial, antitumor, and anticancer activities.

Refs. [7,8] emphasized that Glucosamin-6-Phosphase synthase, GlcN-6-P, is a crucial enzyme in the synthesis of peptidoglycan, a primary component of the bacterial cell wall. Its inhibition can prevent cell wall synthesis and subsequently, bacterial cell death. Likewise, inhibition of the bacteria enzyme Dihydrofolate Reductase, DHFR, which is responsible for folic acid synthesis, can equally be detrimental to the bacteria [9]. Similarly, Thymidine Phosphorylase, TP, which is responsible for DNA synthesis and repair, can be targeted so that antibiotics can disrupt the nucleotide metabolism in bacteria, leading to impaired DNA synthesis and ultimately bacterial cell death [10].

2. Methods

In creating the 2D structures of the library of compounds, Chem Draw was used. Spartan 14 was used to convert the structures to 3D, minimize their energies, and save them as .mol2 files. The target enzymes were Glucosamine-6-Phosphase synthase, Thymidine Phosphorylase, and Dihydrofolate Reductase, with the respective PDB IDs 1MOQ, 4EAD, and 7MQP, which were downloaded from rcsb.org. The deletion of solvent molecules and co-crystallized ligands was performed using UCSF Chimera. Subsequently, UCSF Chimera was used for the dock prep of the receptor and ligands, including the native ligand and amoxicillin. AutoDock was used to change the receptor and ligands’ file format to pdbqt and to set the grid box. Binding energies were determined using Cygwin Terminal. And postdock analysis was performed using UCSF Chimera and Discovery Studio 2020.

3. Results and Discussion

3.1. Drug-Likeness and Bioavailability

Figure 1 illustrates the general structure of the library of compounds. Where R₁ and R₂ represents the respective substituents. None of the ligands have more than one violation of Lipinski’s rule, with most of them complying fully with it, demonstrating desired drug-likeness properties. The ligands’ bioavailability score is 0.55, indicating they can be orally administered.

3.2. Synthetic Accessibility

As seen in

Table 1. Show extracts from SwissADME output.

Lig	Formula	MW	H-Acceptors	H-Bond Donors	Water Solubility	XLOGP3	Bioavailability Score	Synthetic Accessibility
2	C18H17N3O3	323.35	4	1	Soluble	2.31	0.55	3.57
3	C19H18N2O3	322.36	4	0	Soluble	2.88	0.55	3.54
5	C23H20N2O2	356.42	3	0	Moderately soluble	5.2	0.55	3.7
7	C19H19N3O3	337.37	4	1	Soluble	2.67	0.55	3.69
8	C20H20N2O3	336.38	4	0	Soluble	3.24	0.55	3.66
9	C20H20N2O4	352.38	5	0	Moderately soluble	3.65	0.55	3.8
10	C24H22N2O2	370.44	3	0	Poorly soluble	5.57	0.55	3.82
12	C17H14ClN3O3	343.76	4	1	Soluble	2.57	0.55	3.46
13	C18H15ClN2O3	342.78	4	0	Soluble	3.14	0.55	3.43
15	C22H17ClN2O2	376.84	3	0	Moderately soluble	5.46	0.55	3.6
17	C17H13Cl2N3O3	378.21	4	1	Moderately soluble	3.2	0.55	3.48
18	C18H14Cl2N2O3	377.22	4	0	Moderately soluble	3.77	0.55	3.45
19	C18H14Cl2N2O4	393.22	5	0	Moderately soluble	4.17	0.55	3.58
20	C22H16Cl2N2O2	411.28	3	0	Poorly soluble	6.09	0.55	3.61
25	C22H18N2O3	358.39	4	1	Moderately soluble	4.48	0.55	3.64
27	C18H16N2O5	340.33	6	2	Soluble	1.8	0.55	3.53
29	C18H16N2O6	356.33	7	2	Soluble	2.21	0.55	3.67
30	C22H18N2O4	374.39	5	2	Moderately soluble	4.13	0.55	3.7
35	C23H20N2O3	372.42	4	0	Moderately soluble	4.81	0.55	3.75
40	C24H22N2O4	402.44	5	0	Moderately soluble	4.78	0.55	3.93

, their synthetic availability scores are well within the range of 3.43 to 3.93, which underscores their plausible synthesis in the laboratory with ease.

3.3. LogP and Water Solubility

From Table 1 below, their logP value ranges from 2.35 to 4.52, and most of them are moderately soluble or of higher solubility in water, which means that they exhibit the crucial balance between hydrophilicity and lipophilicity, necessary for drug absorption within the biological entity.

3.4. Binding Affinity and Ligand–Receptor Interaction

The binding affinity data from

Table 2. Showing ligands’ substituents, report, protox class, and the binding energies across the three targets.

LIGANDS	SUBSTITUENTS		REPORT	PROTOX CLASS	BINDING ENERGY
	R1	R2			1MOQ	4EAD	7MQP
NATIVE LIGAND					−7.5	−6.8	−9.6
AMOXYCILLIN					−7.9	−8.6	−6.8
2	4-CH3	ONH2	REPORTED	4	−8.9	−8.7	−9.2
3	4-CH3	(CO)CH3	REPORTED	4	−8.9	−8.7	−9.2
5	4-CH3	C6H5	NEW	4	−7.9	−9.5	−9.9
7	3,5-CH3	ONH2	NEW	4	−9.1	−8.9	−9.3
8	3,5-CH3	(CO)CH3	NEW	4	−9.2	−9.0	−9.8
9	3,5-CH3	(CO)OCH3	NEW	4	−8.7	−8.9	−9.1
10	3,5-CH3	C6H5	NEW	4	−7.9	−9.6	−9.2
12	4-Cl	ONH2	NEW	4	−8.8	−8.2	−9.2
13	4-Cl	(CO)CH3	NEW	4	−8.8	−8.6	−9.1
15	4-Cl	C6H5	NEW	4	−7.9	−9.3	−9.8
17	3,5-Cl	ONH2	NEW	4	−8.9	−8.5	−9.2
18	3,5-Cl	(CO)CH3	NEW	4	−9.0	−8.7	−9.5
19	3,5-Cl	(CO)OCH3	NEW	4	−8.7	−8.7	−9.5
20	3,5-Cl	C6H5	NEW	4	−7.8	−9.4	−10.1
25	4-OH	C6H5	NEW	4	−7.6	−9.1	−9.2
27	3,5-OH	ONH2	NEW	4	−8.6	−8.7	−9.5
29	3,5-OH	(CO)OCH3	NEW	4	−8.2	−8.7	−9.3
30	3,5-OH	C6H5	NEW	4	−8.2	−9.2	−10.2
35	4-(CO)OCH3	C6H5	NEW	4	−7.5	−9.1	−8.6
40	3,5-(CO)OCH3	C6H5	NEW	4	−7.8	−8.9	−8.5

below indicate that several ligands have a stronger binding affinity to the target enzymes compared to the native ligand and amoxicillin. Ligands 8, 20, and 30 notably showed the highest binding affinities across the three targets. These binding energies suggest that these ligands have a strong potential to inhibit the target enzymes more effectively than the current standard drug, amoxicillin. The data indicate that certain substituents, particularly CH₃ and Cl groups, significantly enhance binding, which is consistent with findings in the existing literature.

4. Conclusions

This study demonstrates that 2-pyrazoline derivatives show promising potential as antimicrobial agents. These compounds exhibited strong binding affinities to target enzymes, surpassing the standard drug, amoxicillin. The ligands also displayed favorable drug-likeness and bioavailability properties, indicating their potential for oral administration. However, further in vivo and in vitro studies are necessary to confirm these findings and evaluate their therapeutic potential.