Investigation of Momordica charantia phytochemicals against PIM1 Kinase: A Computational Approach

: Prostate cancer is a significant contributor to male cancer-related mortality. PIM1 kinase has implications in the development and progression of various cancers, particularly prostate cancer. PIM1, a serine/threonine protein kinase plays a crucial role in cellular processes including survival, growth and differentiation. In prostate cancer increased PIM1 expression is associated with a more aggressive phenotype and poorer patient outcomes. It has emerged as a promising therapeutic target for prostate cancer treatment. The development of PIM1 kinase inhibitors has greatly enhanced and progressed significantly. Different stages of clinical trials demonstrating their potential as therapeutic agents. Momordica charantia , or bitter melon has a long history in traditional medicine for various health conditions, it is very rich in secondary metabolites like triterpenoids, glycosides, alkaloids, flavonoids and phenolic acids. Bitter melon is considered to have medicinal properties including potential anticancer phytochemicals. This study employs virtual screening, molecular dynamic simulation and ADME/T analysis to explore bitter melon's phytochemicals and their interaction with PIM1 kinase. The goal is to understand the molecular details and pharmacokinetics of bitter melon compounds evaluating their potential as therapeutic agents against prostate cancer. In our present study, it was found that out of all investigated phytochemicals catechin and gallic acid shows satisfactory result depending upon various parameters taken into consideration for conducting the study.


INTRODUCTION
PIM1 kinase is a serine/threonine protein kinase belonging to the PIM kinase family, plays a pivotal role in regulating various cellular processes like cell survival, growth and differentiation through the phosphorylation of target proteins [1].It is implicated in modulating signalling pathways activated by growth factors, cytokines and stress signals [2].In the context of prostate cancer elevated PIM1 expression is linked to more aggressive tumor characteristics, potentially leading to poorer prognoses for prostate cancer patients [3].Inhibitors of PIM1 kinase have been developed and are currently undergoing early-stage clinical trials for prostate cancer treatment.In preclinical studies, these inhibitors have demonstrated efficacy in impairing prostate cancer cell proliferation, survival, and angiogenesis leading to tumour regression [4].Clinical trials play a fundamental role in advancing lifeextending and curative interventions for cancer patients.They are essential for facilitating the transition of novel treatments, also generating essential data for regulatory approvals, enabling the integration of new drugs into widespread clinical practice.PIM1 kinase inhibitors exhibit anti-inflammatory and antioxidant properties further underscoring their potential as anticancer agents [5].Several promising PIM1 kinase inhibitors, including small molecules like SGI-1776 and peptide inhibitors such as PIM447, are currently in various stages of clinical development [6][7].However, it is crucial to emphasize that the use of PIM1 kinase inhibitors for prostate cancer therapy is still in the early stages necessitating further research to understand their mechanisms of action comprehensively and optimize their clinical applications.Momordica charantia, commonly known as bitter melon has long been employed in traditional medicine for centuries to address diverse health conditions including hepatitis [8] diabetes, obesity and various neurodegenerative disorders [9] [10].Bitter melon has recently garnered utmost attention for its potential source of natural compounds with anticancer properties.It contains several secondary metabolites such as triterpenoids, glycosides, alkaloids, flavonoids and phenolic acids which are essential in alleviating the broad range of diseases [11].Studies have revealed that the phytochemicals present in bitter melon extract have great potency to inhibit cell proliferation, induce apoptosis and modulate the immune system, critical factors in managing prostate cancer [12] [13].Additionally, these phytochemicals also demonstrate antiinflammatory, antioxidant and anti-angiogenic properties, further bolstering their potential as anticancer agent [14].In this study, various in-silico methods are employed to analyze the phytochemicals of bitter melon extract concerning their interaction with PIM1 kinase, specifically in the context of prostate cancer.

MATERIAL AND METHODS 2.1 Target protein preparation
The PDB website (https://www.rcsb.org/)was employed to obtain the pdb file of human Pim-1 kinase with the ID 6MT0.The Protein Data Bank (PDB) stores data on experimental protein and nucleic acid structures.To refine the protein for docking, water molecules were eliminated using PyMOL [15], an open-source molecular visualization software.

Ligand retrieval and preparation
Compound structures of bitter gourd were acquired in sdf file format from the PubChem database, which offers details on chemical compounds, including their structure, formula, and molecular weight (https://pubchem.ncbi.nlm.nih.gov/source/15751).Table 1 presents the compound information.Ligand preparation was conducted using the OpenBabel [16] tool from PyRx 0.8 [17], and the ligand energy was minimized using the mmff94 force field.The sdf file format of the ligands was converted to pdbqt format to render it executable.
Table1: Phytochemicals with their molecular weight and Pubchem ID.

Molecular Docking
A molecular docking research was performed utilising bitter gourd compounds as ligand groups and pim1 kinase as a macromolecule.The AutoDock Vina [18] tool from PyRx 0.8 was utilised to conduct the molecular docking investigation.

Visualization of Docking Results
After the docking simulation, the most favorable docked pose, determined by the best negative score (docking score), was selected as the optimal configuration for the respective chemical and protein.To analyze unbound interactions, Discovery Studio 4.5 [19] was employed for visualizing and presenting the top-docked position.

Analysis of protein-ligand interactions
The generation of Ligplot involved utilizing the Ligplot+ program to assess hydrophobic and hydrogen bond interactions between the ligand and the target protein [20].Ligplot provides a two-dimensional representation of the interactions between the compound and the protein.

Prediction of Physiochemical Properties
The assessment of the drug-like characteristics of the compounds was conducted using the DruLito program.This investigation determined the count of rotatable bonds and compliance with Lipinski's Rule of 5 [21], which outlines the criteria for orally active drugs to maintain their pharmacological integrity.Absorption, distribution, and metabolism prediction of the chosen compound were done using admetSAR [22] (http://lmmd.ecust.edu.cn/admetsar2/).

Prediction of toxicity
ProTox-II (https://tox-new.charite.de/protox_II/index.php?site=compound_input) has been employed for toxicity prediction of chosen compound [23].It is a web-based virtual toxicity laboratory for predicting several toxicological endpoints connected to a chemical structure.It is accessible to academic and non-commercial users.ProTox-II includes computer-based models trained on actual data (in vitro or in vivo) to forecast the hazardous potential of current and hypothetical substances.

Prediction of biological activity of the compound
To anticipate the biological activities of the selected molecules, the PASS web server (http://www.pharmaexpert.ru/passonline) was used [24].Using multilayer atom neighbour descriptors, the PASS analysis assists in analysing the effects of a drug entirely based on its molecular formula, meaning that its biological behaviour is exclusively governed by its chemical structure.

RESULT AND DISCUSSION
3.1 Docking score of the compounds 3D crystal structure of pim-1 kinase (PDB ID: 6MT0) was used for docking study.Autodock Vina from PyRx 0.8 was used for analysis.Protein was converted to macromolecule and all the selected compounds were first minimised with mmff94 forcefield and then finally converted to pdbqt format suing OpenBabel in PyRx.Blind docking was performed with grid box dimension (49.22 Å ×55.71 Å ×46.56 Å) and centre (-39.17,-13.23, -0.41).The exhaustiveness was set to 8 by default.Table 2 summarises the details of ligands or compounds with their docking score.Best docked poses and schematic 2D representation of their interaction with target protein is given in Figure 1 (a-f) .We observed that all of the chosen compound present in bitter gourd extract shown good docking score.

Pharmacokinetics and Toxicological Properties Analysis
To assess drug similarity, it is essential to determine the ADME/T characteristics of ligands.The DruLito program was employed to evaluate the pharmacological properties of compounds present in bitter gourd extract.Various drug similarity rules, including Lipinski's rule, MDDR-like rule, Ghose filter, BBB similarity, CMC-50-like rule, unweighted QED, Veber filter, and weighted QED, were applied for compound identification.The findings are consolidated in Table 4. Additionally, Table 5 presents the predicted absorption, distribution, and metabolism of selected compounds using the admetSAR server.All molecules exhibited positive human intestinal absorption, with Molecules 1, 3, and 5 demonstrating blood-brain permeability.Tables 4 and 5 provide a summary of the results.
Table 4: Pharmacological properties of the selected compounds evaluated using DruLito server.cancer.Current approaches to inhibit PIM in cancer treatment have predominantly centered on a monotherapeutic strategy.This typically involves the use of ATP (adenosine triphosphate)-competitive drugs targeting the kinase activity of the protein, preventing its phosphorylation of downstream effectors.These efforts utilize compounds like quinones or other classes of small molecule inhibitors.A paradigm change including computer-based simulations and data analysis provides vital insight into the complexities of proteins and ligands.The research concentrated on computational approaches for determining the therapeutic capabilities of phytochemicals found in M. charantia against PIM1 kinase.In this research, several in silico methodologies were used, and the results were reviewed.Based on the numerous parameters utilised in our research, we conclude that catechin and gallic acid has shown good biological activity of being PIM1 kinase inhibitor and also exhibited satisfactory docking scores, ADME, and toxicological measures.Further efficacy of these phytochemicals might be confirmed by using in vitro and in vivo methods.

CONFLICT OF INTEREST:
Authors declare no conflict of interest.

Table 2 :
Molecular details (MW and PUBCHEM ID) alongside docking scores (kcal/mol) for selected ligands.Lower scores, such as -8.7 for Momordicoside K, indicate strong binding affinity.

Table 3 :
Summary of hydrophobic and hydrogen bond amino acids interaction between ligand and protein

Table 5 :
Absorption, distribution, and metabolism of the chosen compound as per admetSAR online toolkit ISSN:

Table 7 :
Biological activity prediction of compounds (Pa = probability to be active; Pi = probability to be inactive 4. CONCLUSION M. charantia has long been utilised as an herbal treatment with strong pharmacological benefits.Research has explored its medicinal attributes like antibacterial, antiviral, antitumor, immunomodulatory, antioxidant, anthelmintic, antimutagenic, antilipolytic, antifertility, hepatoprotective, anti-inflammatory, anti-ulcerogenic, antioxidative and immune-modulatory properties.PIM1 has emerged as a key player in prostate cancer (PCa) carcinogenesis, where its overexpression enhances the tumorigenicity of prostate ISSN: