In-silico screening of phytochemicals

to find a potential neutralizer

against Phospholipase A2s
in Hypnale hypnale venom
SS Edirisinghe (D/ PMY/ 20/ 0015)
SNA Karunathilake (D/ PMY/ 20/ 0026)
Introduction
• One of the six highly venomous snakes in Sri Lanka. Accounts for the highest
envenomation in the country (Maduwage et al., 2013).
• No specific anti-venom is available. Only symptomatic treatment is offered to the
patients (Sri Lanka Medical Association).
• The need for alternatives to vaccines is evident due to many reasons: early and
late reactions to anti-venom, cost associated with anti-venom treatment in
resource-constrained settings, lack of clinical efficacy for irreversible local tissue
damage and VICC.
• Heavy reliance of people on traditional treatments for snakebites also underscores
the importance of scientific validation of traditional treatment options (Ediriweera
et al. 2017).
Objectives
General Objective: To evaluate the neutralizing effect of phytochemicals against
Phospholipase A2 (PLA2) to treat Hypnale hypnale envenomation

Specific Objectives:
• To prepare a phytochemical library from selected ayurvedic plants with anti-venom
properties.
• To Predict the ADMET parameters and molecular properties of the phytochemicals.
• To predict the anti-venom ability of phytochemicals by structure-based virtual
screening/ molecular docking approaches against the PLA2 of Hypnale hypnale.
• To validate the selected phytochemicals and target protein interactions by
performing Molecular Dynamic (MD) simulations.
Methodology
1. Protein selection and
preparation for virtual
screening
• Snake Venom Phospholipase
A2
• Approximately 40% of the
venom content
• Responsible for the local
tissue damage
• Found across all venomous Adapted from Tan, C.H., Tan, N.H., Sim, S.M., Fung, S.Y. and Gnanathasan, C.A.
(2015) ‘Proteomic investigation of Sri Lankan hump-nosed pit viper (Hypnale
snake families hypnale) venom’
Methodology
2. Compound library preparation
• 11 plants (Dharmadasa et al. 2015 ) –
966 molecules
• IMPPAT – Indian Medical Plants, and the
Photochemistry and Therapeutics
database (Mohanraj et al., 2018 and
Vivek-Ananth et al., 2023)
• Aristolochia bracteolata – Sassanda
• Asparagus racemosus – Hatawariya
• Terminalia chebula – Aralu
• Terminalia bellirica – Bulu
• Piper betle – Bulath
• Citrus aurantiifolia – Dehi
• Citrus aurantium – Dodam
• Murraya koenigii – Karapincha
• Citrus limon – Naran
• Solanum melongena – Elabatu
• Zingiber officinale – Inguru
Methodology
3. Structure-based virtual screening
• AMBER ff14SB force field
• UCSF Chimera (Pettersen et al., 2004)
• PyRx
• AutoDock tools

4. Prediction of ADME parameters – Swiss ADME (Daina et al., 2017)

5. Molecular Dynamics (MD) simulation – Schrodinger suite (Bowers et
al., 2006)
• TIP3P • 100ns
• OPLS 2005 • 1ATM

• 310K
Results
Virtual Screening:
• Binding energy < -6.5
kcal/mol
Results
Interaction Analysis:
• Aristololactam and Ellagic acid
formed hydrogen bonds with all
four targets, whereas
Pabularinone, and Heraclenin
established hydrogen bonds
with three out of four targets.
• Those molecules were Q9PVF2 – Ellagic acid complex
ultimately chosen for further
analysis.
Results

4RFP – Ellagic acid complex Q9PVF4 – Ellagic acid complex

Results
ADMET Analysis:

• The selected
candidates
displayed drug-like
characteristics and
adhered to the "rule
of five" without
violation.
Results
MD simulation:
• Ellagic acid emerged as the lead
molecule, demonstrating
successful binding to three of the
four protein targets.
• None of the four ligands
demonstrated binding affinity to
the P81479 protein.
Results of MD simulation

RMSD Fluctuation of Empty Protein vs. Protein-Ligand Complex

Results of MD simulation

Q9PVF2 - Ellagic acid L-P contact

Q9PVF2 - Ellagic acid P-L contact

Results of MD simulation

Q9PVF4 - Ellagic acid L-P contact

Q9PVF4 - Ellagic acid P-L contact

Results of MD simulation

4RFP - Ellagic acid L-P contact

4RFP- Ellagic acid P-L contact

Conclusion
• The research has identified potential hit molecule Ellagic acid which is
found in Terminalia chebula and Terminalia bellirica; could inhibit the
svPLA2 of Hypnale hypnale.
• Ellagic acid maintained stable Hydrogen bonds with 4RFP, Q9PVF2 and
Q9PVF4 with 55%, 99% and 51% respectively of the simulation period
indicating a strong ligand binding and therefore a high specificity for
the target.
• RMSD of 4RFP fluctuated within an acceptable range of 1-3 Å
throughout the simulation period, both without and with the ligand
Ellagic acid indicating the stability of the formed complex.
Future Aspects
• Findings should be validated in-vivo and in clinical trials.
• Future research is important to experimentally obtain the protein's
complete sequence and validate the accuracy and completeness. This
can be achieved through techniques such as Edman degradation or
tandem mass spectrometry.
• Homology modelling can be used to predict the structure of target
proteins after obtaining the complete sequence.
• X-ray crystallography is a standard method for experimental structure
determination and should be used to validate the predicted protein
structures.
Thank You!