In-Silico Investigation of Phyllanthus niruri Phytochemicals as Hepatic Fibrosis Modulators ^†

Abstract

The liver is a pioneer internal organ that orchestrates major metabolic, detoxification, and endocrine roles. Acute factors like hepatitis and drug allergy and chronic causes like metabolic dysfunction-associated fatty liver disease (MASLD) and Hepatocellular carcinoma (HCC) drive hepatic wellness imbalances. Liver fibrosis is a reversible and curable anomaly, but the limited availability of safe and higher-specificity therapeutics is a challenging quest in hepatology. This study investigates the hepato-protective effect of Phyllanthus niruri compounds against liver fibrosis targets like lysyl oxidase-like 2 (LOXL2), heat shock protein 47 (HSP47), bromodomain-containing protein 4 (BRD4) and inhibitory kappa B kinase beta (IKKβ) and compare their anti-hepatic fibrosis activity against known inhibitors. Potential plant compounds from P. niruri were retrieved from the literature repositories, and the top 35 compounds were screened based on molecular weight, Lipinski’s rule of 5, and bioavailability score. The in silico molecular docking and in silico ADMET results provide valuable insights into hit compounds of P. niruri, namely quercitrin and hinokinin, to have good binding scores (BE) below −7 kcal/mol threshold and molecular interactions with many key residues of all the four liver fibrosis targets namely the BRD4, HSP47, LOLX2, and IKKB proteins explored in this research. Quercitrin has been identified to have BE values of −8.1, −8.3, −8.2, and −9.1 kcal/mol scores against the BRD4, HSP47, LOLX2, and IKKB proteins, respectively. Similarly, hinokinin also shows BE values of −8.8, −7.4, −6.7, and −9.0 kcal/mol scores with BRD4, HSP47, LOLX2, and IKKB proteins individually. Further, in vitro and animal model-based in vivo experimental analysis needs to be explored to validate the potential of quercitrin and hinokinin for anti-liver fibrosis in the future.

1. Introduction

Chronic liver injury, liver inflammation, and hepatic stellate cell (HSC) activation are some of the key features of liver fibrosis. HSCs are precursor fibroblastic, liver-resident cells that transdifferentiate to become actively proliferating fibroblastic cells to secrete extracellular matrix (ECM) components like collagen and proteoglycans [1]. ‘Perpetuation phase’ is a series of HSCs phenotypic activation events, which includes uncontrolled proliferation, retinoid reduction, fibrogenesis, cholesterol metabolism, changes in matrix degradation, contractility, endoplasmic reticulum (ER) and oxidative stresses, epigenetics, chemotaxis, inflammatory responses and receptor-mediated signaling [2,3]. While elevated proliferation and activation of HSCs majorly contribute to liver fibrosis [4], enhanced stabilization of ECM components, especially collagen, along with the promotion of collagen synthesis [5,6], also contributes to matrix stiffness and recurrence of fibrotic injury [7]. Likewise, chronic inflammatory cues precede hepatic fibrosis, and its persistence drives various signaling cascades of liver fibrosis [8].

Phyllanthus niruri, an Indian herb belonging to the Euphorbiaceae family, is lauded for its anti-cancer [9] and hepato-protective effects [10]. Numerous studies have vouched for the potential of multiple natural plant-derived phytochemicals to alleviate or improve hepatic fibrosis via an array of molecular mechanisms. Key transcription cum epigenetic regulator protein, Bromodomain protein 4 (BRD4), is found to be overexpressed in HSCs in promoting its trans-activation via inhibition of its apoptosis through the P300/H3K27ac/PLK1 axis [11,12]. The heat shock protein 47 (HSP47) is a collagen-specific ER-chaperon, which aids collagen triple helix formation and efficient folding. Additionally, the interaction between HSP47 and the NLRP3 inflammasome further promotes HSC activation and fibrosis progression [13,14]. The ECM crosslinking cum stabilizing protein lysyl oxidase-like 2 (LOXL2) has been identified to have prime mediation in collagen remodeling and stabilization [15,16]. Similarly, the IKKβ alias IKK2 kinase has been accorded to activate the NF-κB via phosphorylation for aggravating hepatic inflammation and HSCs activation cum apoptosis inhibition via TGF-β1 and IL-6 stimulation [17,18]. This study aimed to explore novel P. niruri phytochemicals-based inhibitors of LOXL2, HSP47, BRD4, and IKKβ which have been speculated to impact some of the key features of liver fibrosis like activation of HSCs, stabilization of collagen fibrils and induction of hepatic inflammation using in silico analysis. By conducting in silico ADME analysis and molecular docking studies, a comprehensive assessment of the binding interactions and druggability of many P. niruri compounds against liver fibrosis has been explored in this research. The schematic workflow adopted for this research is shown in Figure 1.

2. Materials and Methods

2.1. Initial Compounds Selection and ADME Studies

The phytoconstituents of P. niruri were obtained through the manual literature search and from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/ (accessed on 6 September 2024)). The active phyto-compounds were examined for molecular weight (Mol wt), oral bioavailability (OB), drug-likeness (DL), compliance to Lipinski’s rule, and the additional literature evidence for hepato-protective potential using online webservers Swiss ADME and Molsoft [19]. The selection criteria for Mol wt (150–500 Da), OB (>0.17), DL (>0.18), and non-violation of Lipinski’s rule were considered as parameters for selecting potentially pharmacological ingredients, and additionally, phytocompounds with the literature evidence for hepato-protective potential but having violations to the examined ADME parameters were also included in the further analysis.

2.2. Preparation of Ligands, Protein Structures

The MGLTools and AutoDock 1.5.7 software were utilized to prepare the ligand and protein structures. The reference inhibitor compounds and the P. niruri phytocompounds that were selected based on initial ADME studies were individually downloaded.SDF files were further prepared and saved.PDBQT files. The protein structures were retrieved as.PDB files from the RCSB database (https://www.rcsb.org (accessed on 8 September 2024)) and saved as.PDBQT files post-preparation include the removal of HOH molecules and the addition of Gasteiger charges. The prediction of grid box parameters to facilitate docking was also performed [20]. The list of details about the PDB files and the corresponding inhibitor compounds used as reference for docking analysis is listed in

Table 1. A list of details about protein PDBs and inhibitor references was used in this study.

Target Protein	PDB Id	Inhibitor Compound	Reference for Inhibitor Compound
BRD4	6C7Q	Compound CE277 (BRD4 inhibitor)	Used in the PDB 6C7Q
HSP47	3ZHA	Hsp47 inhibitor Col003 (HSP47 inhibitor III)	[21]
LOXL2	5ZE3	Levoleucovorin	[22]
IKKβ	3RZF	Ligand K-252A	[23]

PDB: protein data bank.

.

2.3. In Silico Molecular Docking Analysis

The AutoDock Vina virtual screening algorithm was utilized to predict the binding. Top 15 compounds exhibiting docking scores ≤ −7.0 kcal/mol were chosen for further analysis. The binding interactions of the docked ligand and protein results were visualized using Pymol version 3.1.1 and Discovery Studio visualizer tools.

2.4. In Silico Prediction of Toxicity Parameters

The Absorption, distribution, metabolism, excretion, and toxicity (ADMET) toxicity parameters like predicted LD50, predicted toxicity class, hepato-toxicity, immunogenicity, carcinogenicity, and activity status of Tox21 nuclear receptor parameters like the PPAR-γ, MMP, and p53 were explored using the Protox II web server [24].

3. Results and Discussion

3.1. ADME/T Profiles of the Best

Around 35 compounds have been listed in

Table 2. List of selected P. niruri compounds based on initial ADMET profiles.

Compound Name	Molecular Weight (150–500 Da)	Bio Availability (>0.17)	Lipinski’s Rule Violation	Drug Likeliness (>0.18)
Phyltetralin	416.51	0.55	0	0.70
PubChem CID 13872461	434.39	0.55	1	1.08
(+/−)-Hypophyllanthin	430.49	0.55	0	0.81
PubChem CID 24832108	430.49	0.55	0	0.81
5-((1S-2R-3R)-6-7-dimethoxy-2-3-bis(methoxymethyl)-1-2-3-4-tetrahydronaphthalen-1-yl)-1-3-benzodioxole	400.46	0.55	0	0.37
7-Hydroxysecoisolariciresinol	378.42	0.55	0	0.41
Amariin	968.64	0.17	3	0.49
Astragalin	448.38	0.17	2	0.67
Beta-Glucogallin	332.26	0.55	1	0.81
Beta-Sitosterol	414.71	0.55	1	0.78
Captopril	217.29	0.56	0	−0.20
Cianidanol	290.27	0.55	0	0.64
Corilagin	634.45	0.17	3	0.64
Ellagitannin	992.71	0.17	3	0.29
Epicatechin	290.27	0.55	0	0.64
Formononetin-7-O-Glucuronide	444.39	0.11	0	0.10
Gallocatechin	306.27	0.55	1	0.57
Geraniin	268.48	0.55	1	0.30
Hinokinin	354.35	0.55	0	−0.74
Hypophyllanthin	430.49	0.55	0	0.81
Isocorilagin	634.45	0.17	3	0.64
Isolintetralin	400.46	0.55	0	0.60
Isoquercetin	464.38	0.17	2	0.68
Lintetralin	400.46	0.55	0	0.37
Niranthin	432.51	0.55	0	−0.19
Nirtetralin	430.49	0.55	0	0.84
Niruriflavone	364.33	0.56	0	0
Nirurinetin	356.37	0.55	0	0.77
Niruriside	770.73	0.17	2	−0.63
Pectolinaroside	622.57	0.17	3	0.87
Phyllanthin	418.52	0.55	0	−0.56
Quercetin	302.24	0.55	0	0.52
Quercitrin	448.38	0.17	2	0.82
Rutin	610.52	0.17	3	0.91

as potential P. niruri phytochemicals based on ideal molecular weight, oral bioavailability (OB), drug likeliness score (DL), and non-violation of Lipinski’s rule. Additionally, phytocompounds with the literature evidence for hepato-protective potential but having violations of the examined ADME parameters are also included for further analysis.

3.2. Molecular Docking Analysis

The docked interactions of selected 8 P. niruri compounds like astragalin, quercitrin, niruriflavone, amariin, corilagin, hinokinin, pectolinaroside, and isocorilagin were analyzed for each of the four target proteins taken for the current study.

When comparing the interaction of BRD4 inhibitor against the P. niruri compounds, it was identified that astragalin, quercitrin, niruriflavone, corilagin, and hinokinin had been found to interact with many of the crucial sites that the BRD4 inhibitor has been identified to bind within the BRD4 protein via our docking analysis and also to sites closer to the desirable residues (Figure 2A). With respect to binding energy scores, hinokinin with −8.1 kcal/mol and quercitrin with −8.8 kcal/mol binding energy are nearer to the BRD4 inhibitor score of −9.7 kcal/mol. Similarly, the eight P. niruri compounds were also reported to interact with HSP47 (Figure 2B), LOLX2 (Figure 2C), and IKKB (Figure 2D) target proteins. Among the compounds, quercitrin and hinokinin have been identified to show interactions with many of the desirable residues of all four target proteins.

The cross-binding interactions studies of each of the inhibitor compounds against the other target proteins for example, docking of BRD4 inhibitor against other target proteins, namely HSP47, LOXL2, and IKKB, were also carried out, and we identified that certain inhibitors like BRD4 inhibitor and antibiotic K255 an (IKKB inhibitor) have been identified to even show high interaction to other proteins like IKKB and BRD4, respectively (Figure 2E).

With respect to the molecular docking binding energy (BE) scores, compounds interacting with BE scores lower than −7 kcal/mol are accorded as hit compounds, as shown in the heat map profile (Figure 2E). The potentially suitable P. niruri hit compounds, which also show interactions against target proteins at key residues and satisfy the BE scores requirement, are identified as desirable anti-liver fibrosis inhibitor compounds. In this study, P. niruri compounds like astragalin, quercitrin, corilagin, and hinokinin have been identified to interact with most of the key residues of BRD4 target protein, and amongst them, hinokinin exhibits higher BE of −8.8 kcal/mol as shown in Figure 2A,E. Similarly, pectolinaroside, hinokinin, and quercitrin show binding to some of the key residue sites where HSP47 inhibitor III binds along with lower BE scores, as in Figure 2B,E. Compounds isocorilagin and amariin have been identified to interact with sufficient desirable residues of LOXL2 protein shown in Figure 2C,E. Compounds like quercitrin, niruriflavone, and hinokinin also promisingly interact with the IKKB target, as shown in Figure 2D,E.

Overall, the molecular docking results of these eight P. niruri compounds demonstrate promising interactions with BRD4, HSP47, LOXL2, and IKKB, suggesting that they may exhibit therapeutic efficacy against the prime features of liver fibrosis. Compounds quercitrin and hinokinin show preferential interaction cum desirably lower BE scores with all four target proteins.

3.3. In Silico Toxicity Prediction

The in silico toxicity activeness for hepato-toxicity, carcinogenicity, and immunogenicity of P. niruri compounds and the reference inhibitor compounds explored in this current research was examined using the ProTox-III webserver. Amongst the inhibitor compounds, only levoleucovorin (LOXL2 inhibitor reference) showed inactivity for the toxicity potential

Table 3. In silico toxicity results for P. niruri compounds.

Compounds	Predicted LD50 (mg/kg)	Predicted Toxicity Class	Hepato-Toxicity	Carcino-Genicity	Immunogenicity	Tox21-Nuclear Receptor Signaling Pathways
						PPAR-γ	MMP	p53
BRD4 inhibitor	3000	5
HSP47 inhibitor III	1600	4
Levoleucovorin	135	3
Antibiotic K 252a	11	2
Amariin	620	4
Astragalin	5000	5
Corilagin	2260	5
Hinokinin	1500	4
Isocorilagin	2260	5
Niruriflavone	3919	5
Pectolinaroside	5000	5
Quercitrin	5000	5

Color palate: , .

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The predicted toxicity class and LD50 results show that P. niruri compounds administration could potentially be harmful only beyond high dosages as per the predicted LD50 values, and also, these compounds have been predicted to belong to mostly class IV and V, unlike the reference inhibitor compounds. The Tox21 nuclear receptor signaling activeness results shown in Table 3 imply that almost all the analyzed inhibitor compounds and P. niruri compounds, except the IKKB inhibitor and flavonoid astragalin, show inactivity towards triggering the PPAR-γ, MMP, and p53 cascades to induce cytotoxic effects at concentration beyond the predicted LD50 threshold.

4. Discussion

The selected eight P. niruri compounds have been identified to be non-hepatotoxic, though most compounds show active immune-toxic potential, which could possibly emphasize the anti-oxidant, radical scavenging potency of phytochemicals, and their applicability as anti-cancer agents [25]. Hinokinin has earlier been reported to mediate beneficial health effects like neuroprotective and anti-cancer abilities [26,27]. The anti-myocardial fibrotic prowess of hinokinin [28] substantially advocates the predictive inference of this current research for a similar anti-liver fibrosis effect. Quercitrin has been identified to prevent mitochondrial damage in liver cells [29], reduce hepatic steatosis [30], and suppress HCC metastasis [31], and its potential therapeutic effect against liver fibrosis is attempted for exploration in this current research. Previous studies on P. niruri extracts report reduced hepatic and nephrotic injury cum lowered fibrotic scores in animal model studies [32,33], which also supports the computational modeling outcomes of the current research work.

P. niruri has many substantial previous studies emphasizing its therapeutic potential, like anti-cancer and hepato-protective efficacy [34,35]. For instance, one of the major P. niruri phytochemical phyllanthin has been identified to protect from hepatic injuries cum toxicity [36] and impart anti-fibrosis effect via downregulation of TGF-β1 cascade [37] and TNF-α/NF-κB levels in vitro [38]. Quercetin, a prominent flavonoid, has also been proficient against pulmonary liver fibrosis and NAFLD [39,40,41,42]. Likewise, methanolic extracts of P. niruri have been identified to mitigate liver fibrosis progression and NAFLD complexities concerned with atherosclerosis [43].

5. Conclusions

This computational analysis provides comprehensive insights into the anti-liver fibrosis potential of Phyllanthus niruri compounds against some of the prominent therapeutic targets influencing key features of liver fibrosis. The in silico molecular docking and in silico ADMET studies provide valuable insights into hit compounds of P. niruri, like quercitrin and hinokinin, to have good docking scores below −7 kcal/mol threshold and molecular interactions with many key residues of all the four liver fibrosis targets explored in this research namely the BRD4, HSP47, LOLX2, and IKKB proteins. Both quercitrin and hinokinin compounds have been previously accorded for their health benefits. This present study holds promising computational predictive findings of both quercitrin and hinokinin as anti-liver fibrosis therapeutic druggable compounds. Further in vitro and animal model based in vivo experimental analysis needs to be explored for future validations.