Anti-Helicobacter Pylori Activity of Phytochemicals from Artocarpus spp.: In Silico Analysis ^†

Abstract

Peptic ulcer disease, affecting up to 20% of the global population, poses a significant health challenge with limited treatment options due to side effects and the inefficiency of existing drugs. This study explores the potential of penicillin-binding proteins (PBPs) as targets for peptic ulcer treatment. PBPs, critical for bacterial cell wall integrity, are inhibited by beta-lactam antibiotics, leading to bacterial vulnerability. Flavonoids, prominent in plants, exhibit antimicrobial and gastroprotective properties against peptic ulcers. A docking analysis of 35 phytochemicals from the Artocarpus plant against PBP (PDB code: 1QMF) revealed artocarpin as a promising candidate (docking score: −148.24 Kcal/mol). Artocarpin exhibited interactions with key amino acids and demonstrated favourable in silico pharmacokinetics, including high absorption and good drug-likeness. Additionally, engeletin 5 and rutin showed significant docking scores (−134.89 and −148.07 kcal/mol, respectively). Artocarpin, identified as a potential H. pylori inhibitor, presents a promising avenue for peptic ulcer treatment, warranting further exploration of its therapeutic application. This study contributes valuable insights into the molecular interactions of phytochemicals with PBPs, paving the way for novel and effective approaches in peptic ulcer therapy.

1. Introduction

The World Health Organisation classifies Helicobacter pylori, a spiral-shaped, Gram-negative bacillus that affects 50% of people worldwide, as a class 1 carcinogen [1]. Helicobacter pylori is one of the primary causative agents of chronic gastritis, which in turn causes the development of peptic ulcers [1]. By interacting with gastric epithelial cells, H. pylori directly colonises the gastric mucosa. It is commonly known that H. pylori causes intestinal metaplasia, severe atrophy, increased levels of mononuclear and neutrophilic infiltrates, apoptosis, and changes in the gastric epithelial cell cycle.

Antimicrobial resistance has been shown to reduce the global rates of H. pylori infection eradication, according to evidence from clinical practice [2]. Furthermore, there may be toxicity concerns with the main medications used to treat H. pylori.

As an alternative to triple therapy, a number of plant-based treatment approaches, including as plant extracts and isolated phytochemicals, have been explored recently to address drug resistance and side effects [3]. The focus of recent research on natural goods has particularly concerned plant extracts that are high in flavonoids. These substances have demonstrated encouraging outcomes in addressing several modes of action of the anti-H. pylori. Flavonoids also strengthen the mucosal defences against peptic ulcers by enhancing their cytoprotective, antioxidative, anti-inflammatory, and antibacterial properties (Figure 1 and Figure 2) [3]. Typically, a single kind of flavonoid can function as an anti-ulcer through a variety of ways. Numerous studies have shown how flavonoids protect the intestinal epithelium [3]. These effects include preserving the integrity of the intestinal barrier, absorbing fats and carbohydrates, modifying the activities of enzymes, controlling secretions from the stomach, regulating the immune system, and interacting with pathogenic microorganisms. PBPs, or penicillin-binding proteins, have a critical role in the bacterial cell wall’s maintenance. The penicillin-binding proteins that have an allosteric site binding mechanism cause the active site to slightly open during binding, increasing the accessibility of the site for substrate binding. Finding inhibitors that are able to bind to both the active and allosteric sites shows promise as a clinical approach [4]. Both the active and allosteric binding mechanisms had to be examined in order to accurately assess the affinity of flavonoids from Artocarpus spp. for PBPs, as doing so would produce a different and extra inhibitory pattern. In the present study, we have evaluated the set of phytomolecules reported from Artocarpus against the PBP (Figure 1). This study puts forward the usefulness of flavonoids to assist the conventional drug therapy [4]. We further also evaluated in-silico pharmacokinetics to gain more insights into the absorption, distribution, metabolism, excretion (ADME) characteristics.

2. Materials and Methods

2.1. Molecular Docking Simulations

The 3D crystal structure of the penicillin-binding protein (PDB code: 1QMF) was downloaded from the Protein Database Bank. The concerned 3D structure was then processed to add missing H atoms and missing amino acid residues. The binding site for this protein was referred to from the earlier publication. The docking was then performed with the software ‘iGemDock’ V. 2.1 [5,6,7,8]. Finally, the visualization was performed with the ‘Discovery Studio 2020 Visualizer’ [8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26].

2.2. In Silico Drug-Likeness and ADMET Analysis

The top 3 hits were further analysed for in silico pharmacokinetics using ‘SwissADME’ (http://www.swissadme.ch, accessed on 1st June 2023). The toxicity parameters were then assessed using toxicity assessments, conducted using ‘admetSAR’ (http://lmmd.ecust.edu.cn:8000/, accessed on 1st June 2023) [6].

2.3. Boiled Egg Model Analysis

At different phases of the drug development process, it is essential to estimate two pharmacokinetic behaviours: brain access and gastrointestinal absorption. For this reason, an accurate predictive model called the Brain Or IntestinaL EstimateD permeation method (BOILED-Egg) was developed. It computes the lipophilicity and polarity of tiny compounds. The same two physicochemical characteristics yield concurrent predictions for intestine and brain penetration, which may be easily translated into the molecular design due to the model’s quickness, precision, conceptual simplicity, and easily understandable graphical output. The BOILED-Egg model can be used in many different contexts, such as evaluating drug candidates for development or filtering chemical libraries in the early stages of drug discovery.

3. Results and Discussion

3.1. Molecular Docking Simulations

The dataset of 35 molecules were analysed against penicillin-binding proteins (PBPs). Out of 35 phytomolecules, the phytomolecule artocarpin depicted strong interaction with PBP (PDB code: 1QMF) via THR 526, TRP 374, Ser 337, Ser 395, Thr 550, Met 527, and Tyr 595 (

Table 1. Docking interaction energies* of selected bio-active molecules and 3 FDA-approved drugs for target protein 4P8C.

Molecules	-iGemDock Interaction Energy	Molecules	-iGemDock Interaction Energy
Quercetin	−111.11	Artonin A	−117.1
Ascorbates	−84.29	Artocarpanone	−98.62
Catechine	−94.99	Caffeic acid	−78.51
Lupeol acetate	−90.55	Cycloheterophyllin	−125.23
Bita -sitosterol	−97.62	Cyclocommunol	−106.54
Kaempferol	−107.95	Isobavachalcone	−121.11
Gallic acid	−90.35	Artonin E	−128.21
Engeletin 5	−134.89	Heterophyllin	−131.91
Oxyresveratrol	−72.08	Cyclomorusin	−120.05
Artocarpin	−48.24	Cryptoxanthin	−92.1799
Cycloartocarpin	−111.69	Myricetin	−82.5936
Cudraflavone	−111.35	Ascorbic acid	−85.54
Vanillic acid	−79.88	Cinnamic acid	−69.96
Isorhamnetin	−113.3	Ferulic acid	−79.6
Psoralenoside	-107.8	Betulinic acid	−95.9
Epigallocatechin Gallate	−140	Resorcinol	−59.9
Morin	−106.1	Norartocarpetin	−105
Ursolic acid	−99.6	Pyrogallol	−69.1
Artocarpesin	−112.9	Rutin	−148.07
Albanin A	−107.9	Arbutin	−98.2
Engeletin	−116.79	Diethyl phthalate	−83.86
α-amyrin acetate	−92.9	β-amyrin acetate	−95.27
Cycloartenol	−92.66
		Amoxicillin *	−109.20

* Docking scores have been provided only for the higher affinity-scored target protein.

and

Table 2. Energy contribution of the key residues computed by the docking methodology.

Sr. No.	Molecules	Residues with Contribution Energy
1.	Amoxicillin	THR 526, TRP 374, Ser 337, Ser 395, Thr 550, Met 527, and Tyr 595
2.	Artocarpin (Best docked)	THR 526, TRP 374, Ser 337, Ser 395, Thr 550, Met 527, and Tyr 595

, Figure 3). The docking interaction showed H-bonding, van der Waals and π-π types interactions. The standard amoxicillin demonstrated major interactions with Trp 374, Ser 571, Gly 549, Thr 526, and Ser 395. The standard amoxicillin (docking score: −109.20 kcal/mol) had lesser affinity as compared with artocarpin (docking score: −148.24 kcal/mol) against the PBPs [7]. PBPs, or penicillin-binding proteins, have a critical role in the bacterial cell wall’s maintenance. The penicillin-binding proteins that have an allosteric site binding mechanism cause the active site to slightly open during binding, increasing the accessibility of the site for substrate binding (

Table 3. In silico ADMET profiling for the top 3 best docked hits against the target 1QMF.

Properties	Artocarpin *	Rutin	Engeletin 5
GI absorption	High	High	High
BBB permeant	Low	Low	Low
P-gp substrate	NO	NO	NO
CYP1A2 inhibitor	NO	NO	NO
CYP2C19 inhibitor	Yes	Yes	Yes
CYP2C9 inhibitor	NO	NO	NO
CYP2D6 inhibitor	NO	NO	NO
CYP3A4 inhibitor	NO	NO	NO
Lipinski	Yes	Yes	Yes
Ghose	NO	NO	NO
Veber	Yes	Yes	Yes
Egan	Yes	Yes	Yes
Blood–Brain Barrier	-	-	-

* Best docked.

) [7]. Finding inhibitors that are able to bind to both the active and allosteric sites shows promise as a clinical approach. It was vital to look into both the active and allosteric binding mechanisms in order to accurately assess artocarpin’s affinity for PBPs, as doing so may reveal a different and additional inhibitory pattern. Moreover, our re-docking validation protocol also resulted in an RMSD value below 1.4 Å, suggesting valid docking results.

3.2. In Silico ADMET Studies

Cytochrome P450 (CYPs) enzymes play a crucial role in diverse metabolic processes within the human body, serving as key catalysts for the transformation of various endogenous and exogenous compounds. These enzymes, predominantly located in the liver, contribute significantly to the biotransformation of drugs, xenobiotics, and endogenous substances, influencing their absorption, distribution, metabolism, and excretion. Our study focused on understanding the implications of CYP-mediated metabolism on the pharmacokinetics of potential drug candidates. Through in silico calculations, we evaluated the ADMET (absorption, distribution, metabolism, excretion, toxicity) properties of the top three docked hits, shedding light on their behaviour within the intricate landscape of human physiology. Notably, artocarpin exhibited favourable characteristics, such as positive human intestinal absorption profiles, while also demonstrating a lack of carcinogenicity, absence of AMES toxicity, and a class IV acute oral toxicity profile (Figure 4). These findings provide valuable insights into the interplay between molecular docking and the intricate network of cytochrome enzymes, offering a comprehensive understanding of the potential therapeutic relevance of the studied compounds.

4. Conclusions

In the present study, we analysed the potential of Artocarpus phytochemicals against the PBP, as indicated by a higher docking score of the same phytochemicals. Moreover, among the list of 35 known phytocompounds, it had a low human ether-a-go-go-related gene inhibition, no AMES toxicity, and no carcinogens. This study may provide further directions to develop more potent anti-H. pylori compounds.