Virtual Screening Studies on Bone Healing Phytochemicals Isolated from Cissus quadrangularis (round variant) and Cissus rotundifolia

Sabeerali Ansarali¹, Ganapathy Murugan Alagu Lakshmanan²*, Selvarasuvasuki Manikandan³, Shagufta Rashid⁴

¹Assistant Professor, PG Department of Botany, Sri Vidya Mandir Arts and Science College,

Uthangarai 636 902, Krishnagiri, Tamil Nadu, India.

²Department of Botany, Thiru Kolanjiappar Government Arts College,

Vridhachalam - 606001, Tamil Nadu, India.

³Guest Lecturer in Botany, Government Arts and Science College, Thittakudi - 606106, Tamil Nadu, India.

⁴Department of Botany, Annamalai University, Annamalai Nagar - 608002, Chidambaram, Tamil Nadu, India.

*Corresponding Author E-mail: gmalakshmanan@gmail.com

ABSTRACT:

Bone fractures are a prevalent orthopaedic issue that many people experience. While the body can naturally repair a broken bone, the duration of the healing process can range from weeks to years, depending on the injury's severity and potential complications. Different techniques are used in the process of bone healing, including both experimental and computational modeling methods. Computational modeling and simulation have been used to address the limitations of experimental methods. This study focuses on screening and investigating bone healing compounds found in the leaves of Cissus rotundifolia and the stem of Cissus quadrangularis (round-stemmed variant) using molecular docking studies to identify potent compounds for bone healing treatment. GC-MS analysis revealed two active compounds: alpha-methylglucofuranoside and tetradecanoic acid. These compounds were tested against the bone healing protein ABMP2 (ID: 4MID) sourced from the Protein Data Bank (PDB) and showed good docking scores and reasonable stability. In vivo and in-vitro approaches are recommended to further elucidate the molecular mechanisms of these compounds and develop them into potent drugs for treating bone fractures. The present study clearly observed with the screened compounds could possibly develop into potent drugs for treating bone fractures.

KEYWORDS: GC-MS, Bone healing, ABMP2 Chimera, Molecular docking, PDB.

INTRODUCTION:

Bone fractures are a common injury often referred to as "the silent disease" because they occur without early symptoms but result from gradual bone loss due to conditions like osteoporosis. The bone healing process is physiologically intricate, including tissue differentiation, tissue synthesis, encompassing cell migration, and the release of cytokines and growth factors, all governed by the mechanical environment, Although the body can naturally repair a broken bone, the healing process can span weeks, months, or even years, based on the injury's severity and potential complications, depending on the injury's severity and complications.

Elderly patients, smokers, and those with metabolic diseases such as osteoporosis or diabetes experience even longer healing times and a higher risk of non-union (non-healing of the bone)^1,2.

Studying bone healing through experimental methods requires advanced equipment, precise accuracy, and controlled conditions, and is often costly. These methods are further complicated by factors like unknown subject backgrounds, comorbidities, and genetic variations^3,4. To address these issues, computational models and simulations of the bone healing process have been developed. However, these approaches also have limitations in clinical applications and need further refinement and validation to produce clinically relevant results^5,6. Computational models deepen our comprehension of the mechanisms of bone healing by demonstrating how the mechanical environment influences the healing process⁷. These models also predict how mechanical environments and drug treatment strategies affect the biological and cellular processes essential for the healing of bones^8,9. Furthermore, these models offer critical insights for the optimization, design, and outcome predictions of future treatment strategies. To reach clinically relevant results, continued development and refinement are necessary^10,11.

Cissus rotundifolia and Cissus quadrangularis, both belonging to the grape family (Vitaceae), are plants used in traditional African and Ayurveda medicine. C. rotundifolia leaves and the fleshy stem of C. quadrangularis are primarily used to treat bone fractures, gout, rheumatoid arthritis, osteoporosis, cancer, upset stomachs, and haemorrhoids. This study aims to delve deeper into the bone healing constituents present in C. rotundifolia leaves and C. quadrangularis stem (round variant) using molecular docking studies to identify potent compounds that can be developed into drugs for bone healing treatment.

METHODS:

Plant Source Materials:

The plants were meticulously gathered from their natural habitats, and herbarium specimens were created and stored at the Department of Botany, Annamalai University. The accession numbers assigned were AUBOT360 for C. quadrangularis and AUBOT362 for C. rotundifolia. For experimental use plants were thoroughly washed with running tap water to eliminate soil particles, after which the plant parts were separated and dried in the shade. The shade-dried and powdered stem of C. quadrangularis (round-stemmed variant) and leaves of C. rotundifolia were extracted with methanol, then filtered and concentrated by vacuum distillation. GC-MS analysis results were compared and used for molecular docking studies to achieve the study's objective.

Molecular Docking:

Target Protein Identification and Ligand Structure Preparation:

For docking studies, the X-ray crystal and NMR structures of the bone-inducing protein ABMP2 (PDB ID: 4MID) were obtained from the Protein Data Bank (PDB). These structures served as the foundation for detailed analysis and interactions with potential ligands. Missing loops in the protein molecule were filled by adding hydrogen atoms and removing water molecules from the structure. The RMSD value was minimized to 0.30 Å. Using the bioactive compound structures retrieved from PubChem and ChemSketch, ligand molecules were generated and saved in SDF format¹².

High-throughput virtual screening:

Major plant-derived compounds found in C. quadrangularis (round stemmed variant) and C. rotundifolia were selected as ligands for target-based virtual screening using PyRx. Prior to screening, the compounds were assessed for drug-likeness according to Lipinski's Rule of Five¹³. PyRx, incorporating the Open Babel program for protein and ligand visualization, was employed in this process. Both the ligands and the protein target underwent energy minimization and charge assignment. Subsequently, they were converted to pdbqt format to enable docking with the Auto Dock Vina program¹⁴.

In-silico Pharmacokinetics:

Undesirable properties of pharmacokinetic can lead to the failure of most drugs during development. Addressing these issues early in the drug discovery process is advantageous. The pharmacokinetic properties of the identified compounds, such as absorption, distribution, metabolism, and excretion (ADME) toxicity, were determined using the ADMET tool in BIOVIA Accelrys Discovery Studio v4.5 (2016) under the "Calculate Molecular Properties" section and visualized accordingly¹⁵.

RESULTS:

High-throughput virtual screening:

The PyRx virtual screening software was utilized for high throughput virtual screening. Ten major compounds identified through GC-MS analysis were carefully chosen for computational screening analyses. The 2D and 3D structures of these compounds were obtained by using online tool PubChem. The high throughput virtual screening of C. quadrangularis (round variant) revealed pharmacological activities in three compounds: stigmast-5-en-3-ol, alpha-methylglucofuranoside, and stigmasterol. In C. rotundifolia, tetradecanoic acid and 7-tetradecenal were found. Overall, five biological compounds showed drug-likeness. The binding affinities observed from the virtual screening were -5.2, -6.0, -8.2, -6.2, and -7.2 for the respective compounds (Table 1).

Table 1. Virtual screening results of C. quadrangularis (round variant) and C. rotundifolia derived compounds

Name of the Species	Name of the Compound	Hepato toxicity Binding Affinity
Cissus quadrangularis (round stem variant)	Stigmast-5-en-3-ol, (3.beta.)	-5.2
	Alpha-Methylglucofuranoside	-6.0
	Stigmasterol	-8.2
	Ergost-5-en-3-ol	-5.3
	Palmitic acid	-9.1
Cissus rotundifolia	Tetradecanoic acid	-6.2
	7-Tetradecenal	-7.2
	Beta.-Amyrin	-8.4
	Olean-18-En-3-ol	-5.4
	Squalene	-8.1

Screening of Compounds through Pharmacokinetic Properties:

Pharmacokinetics uses mathematical principles to study how drugs enter the body, spread through it, are broken down, and are removed. This analysis helps determine the drug's duration and impact. (ADMET). Understanding these parameters is crucial for designing an appropriate drug regimen. Based on the virtual screening results, five compounds were filtered from the initial ten and tested for their ADMET properties (Table 2).

The ADMET properties of all ten compounds were evaluated using the pkCSM software, which can be accessed at http://biosig.unimelb.edu.au/pkcsm. Among these five compounds, two exhibited drug-likeness. Alpha-Methylglucofuranoside showed a Log P value of -3.2214, and tetradecanoic acid showed a Log P value of -2.7247, both with very loss toxicity levels and adherence to Lipinski's Rule of Five (Figures 1 and 2). Alpha-methylglucofuranoside from C. quadrangularis (round variant) and tetradecanoic acid identified in C. rotundifolia were selected for molecular docking analysis using the bone healing target protein (Figure 3). The compounds exhibited different interactions, including conventional hydrogen bonds and alkyl-shaped interactions with the target protein.

Table 2. ADMET Properties of GC-MS derived compounds from the Cissus quadrangularis (round stem variant) and Cissus rotundifolia

S. No	Compound Name	Absorption
S. No	Compound Name	Water Solubility	Permeability of CaCO₂	Human Intestinal absorption Percentage	Permeability of Skin	P.glyco protein substrate	P.glyco protein I inhibitor	P.glyco protein II inhibitor
1	Stigmast-5-en-3-ol	-6.773	1.201	94.464	-2.783	No	Yes	Yes
2	Alpha-Methylglucofuranoside	-0.581	-0.153	45.236	-3.276	No	No	No
3	Stigmasterol	-6.682	1.213	94.97	-2.783	No	Yes	Yes
4	Tetradecanoic acid	-4.952	1.56	92.691	-2.705	No	No	No
5	7-Tetradecenal, (Z)-	-1.119	-0.258	32.274	-2.737	No	No	No

			Distribution						Metabolism				Excretion
			Human VD_SS	Unbound Fraction	Permeability of BBB		Permeability of CNS		CYP3A4 substrate		CYP1A2 inhibitor		Clearance Total
1		Stigmast-5-en-3-ol	0.193	0	0.781		-1.705		Yes		No		-0.628
2		Alpha-Methylglucofuranoside	-0.07	0.915	-0.756		-4.001		No		No		0.696
3		Stigmasterol	0.178	0	0.771		-1.652		Yes		No		0.618
4		Tetradecanoic acid	-0.578	0.171	-0.027		-1.925		No		No		1.693
5		7-Tetradecenal, (Z)-	-0.217	0.821	-0.894		-3.667		No		No		0.639
			Toxicity
			Toxicity of AMES	Max. tolerated dose in human	Inhibitor of hERG II	Oral rat acute toxicity (LD50)		Oral rat chronic toxicity (LDAEL)		Toxicity of T.pyriformis		Minnow toxicity		Log P value
1	Stigmast-5-en-3-ol		No	-0.621	Yes	2.552		0.855		0.43		-1.802		8.0248
2	Alpha-Methylglucofuranoside		No	1.951	No	1.288		3.113		0.285		4.272		-3.2214
3	Stigmasterol		No	-0.664	Yes	2.54		0.872		0.433		-1.675		7.8008
4	Tetradecanoic acid		No	-0.559	No	1.477		3.034		0.978		-0.601		-2.7247
5	7-Tetradecenal, (Z)-		No	1.626	No	1.128		3.529		0.285		4.869		-2.3214

Table 3. Molecular docking of Alpha-Methylglucofuranoside interact with a target protein

Diseases/

Protein

PDB – ID

Docking energy

(kcal/mol)

Interactions

Amino acid

Binding site of amino acid

Bone healing

4MID

-8.04418

Conventional Hydrogen Bond

Cysteine

111

Unfavorable Acceptor- Acceptor

Table 4. Molecular docking of Tetradecanoic acid interact with a target protein

Diseases/ Protein	PDB - ID	Docking energy (kcal/mol)	Interactions	Amino acid	Binding site of amino acid
Bone healing	4MID	-11.5209	Unfavorable Bump	Phenylalanine	23
			Conventional Hydrogen Bond	Tryptophan	28
			Alkyl	Methionine	89, 106
			Alkyl	Isoleucine	103

DISCUSSION:

Drug development and discovery is a lengthy, complex, costly, in addition risky process. Computer-aided drug design (CADD) methodologies are extensively employed in the industry pharmaceutical to expedite this process. Applying computational tools in hit-to-lead optimization expands the exploration of chemical space while minimizing the number of compounds needing synthesis and in vitro testing¹⁶. The ADME profiles of the selected five compounds from C. quadrangularis (round variant) and C. rotundifolia showed good bioavailability. The two active components identified in this study, alpha-methylglucofuranoside and tetradecanoic acid, were subjected to further in-silico studies to analyse their effectiveness against the target protein. Commonly, rhBMP2 (recombinant bone morphogenetic protein) is used in clinical treatment for bone fracture patients but can cause side effects. The novel activin ABMP2 chimera promotes bone healing more effectively than rhBMP2¹⁷. The structures of the target protein, ABMP2 chimera (ID: 4MID), were sourced from PDB (Protein Data Bank). These two compounds showed good docking scores and reasonable stability. The ADMET profile and Log P values support the bioavailability of these compounds compared to others. This investigation demonstrates significant bone healing activity in two Cissus species.

CONCLUSION:

In this study, phytocompounds from C. quadrangularis (round stem variant) and C. rotundifolia leaves were docked with the bone healing protein (ABMP2 chimera). Alpha-methylglucofuranoside and tetradecanoic acid were identified as key compounds. Based on molecular docking analysis, tetradecanoic acid showed promising target formation against bone healing. Both in-vivo and in-vitro approaches were recommended to elucidate the molecular mechanisms of this compound, aiming to develop it into a potent drug for treating bone fractures.

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Received on 11.06.2022 Revised on 13.11.2023

Accepted on 23.09.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5724-5728.

DOI: 10.52711/0974-360X.2024.00871