Introduction

Cardiovascular disease, which affects both the heart and blood vessels  often stems from impaired blood flow. Blood plays a vital role in oxygen  transport, nutrient delivery, and waste removal within the body.  However, blood coagulation, necessary for injury response, can pose  risks when thrombus formation obstructs blood flow. Thrombi,  comprising platelets, fibrin, and blood cells, can cause blockages in key organs, leading to strokes and heart disease. While medications  like aspirin have been developed to manage cardiovascular diseases,  they carry the risk of adverse effects such as elevated blood levels.  This study aims to explore alternative, biologically derived treatments for cardiovascular ailments, utilizing fibrinolytic enzymes like Droserasin-4 found in the Sundew plant (Drosera Capensis) as healthier therapeutics for cardiovascular conditions.

Droserasins and its previous study.

Droserasin4, known for its antispasmodic, diuretic, and expectorant properties, serves as a natural remedy in villages and holds potential in  fibrinolytic activities. Droserasin is an aspartic protease enzyme found  in certain carnivorous plants like Drosera Capensis, commonly known  as Sundew, native to regions including Australia, South Africa, and  parts of Asia. This enzyme is vital for breaking down proteins in prey,  catalyzing the hydrolysis of peptide bonds. It is secreted by glandular  cells on the plant's tentacles, where it aids in digesting insects trapped in the sticky secretions. By breaking down prey proteins into smaller peptides and amino acids, Droserasin enables the plant to absorb essential nutrients like nitrogen and phosphorus, crucial for growth in nutrient-poor environments. Specifically, Drosera Capensis encompasses around six types of Droserasin, with Droserasin-4 showing potential in containing bioactive compounds. These  compounds may include therapeutic enzymes with anti-inflammatory and antimicrobial properties, and recent research suggests potential applications in thrombolysis. Utilizing computational techniques (In Silico), researchers are exploring Droserasin-4's three-dimensional structure to identify potential medications for cardiovascular diseases.

Methodology

• Prodigy: Prodigy is for scientific data collection of various biological structures, such as domains,  active, and binding sites.

• Haddock 2.4: HADDOCK is used to visualize protein-protein, protein-nucleic acid, and protein-ligand.  LigPlot+: LigPlot+ is used to visualize 2d structures from aspartic protease (Droserasin4) enzyme.  

• Cluspro: Cluspro is used to evaluate the proteins.  

• PyMOL: PyMOL is used to visualize proteins.  

• Swissmodel: Swissmodel is used to validate the 3D Fibrin models. 

• Rapid Peptide Generator (RPG): RPG is used to fragment the substrate.  

• Droserasin-4 PDB (enzyme) & 2HLO (Fibrin 3D structures).

Structure Obtainment and Structural Validity

Figure 3C at the image section > Carter T. Butts, Jan C. Bierma, and Rachel W. Martin  utilized Rosseta Prediction and deep neural networks to  analyze Drosera Capensis's structure, revealing insights  into computational methods for understanding protein structures. Performing computational analysis for  Droserasin4 for its catalytic residues of D18 and D182.

Figure 4D at the image section > 2HLO Fibrin (insoluble protein formed from fibrinogen during the  clotting of blood) came from RCSB  (database of 3d structure, peptide,  etc), with the PDB ID of 2HLO.

Figure 5E and 5E.1 > The Ramachandran plots diagram  yielded high-quality results,  indicating a strength of 93.17 for  Droserasin4 in potential fibrinolytic activities against blood clotting in cardiovascular diseases (CVDs). This success paves the way for further analysis of Droserasin4 as a prospective cure for blood clotting in  the future

Protein-Protein Docking

Using HADDOCK Web Server, the enzyme Droserasin4, aspartic protease and substrate Fibrin were processed and simulated to examine the existing interaction between the two proteins. This protein-protein docking approach predicts the three-dimensional structure of a protein complex to  mimic the binding process and find the most energetically advantageous configurations of the protein  complex. Results: Using the Prodigy Web Server, ∆G predicts the impact on protein interaction stability in the Droserasin4 model (0.3). With a ∆G value of -13.3, it exhibits moderate stability, falling between  weaker (∆G ≥ -4.3) and stronger (∆G ≥ -18.6) stability thresholds. Furthermore, the Kd value is 1.7e 10, indicating moderate binding affinity.The binding affinity (∆G) and Kd values (M) reflect the strength  of interaction, revealing moderate values in Droserasin4 model 0.3. Visualized through LigPlot+ [7], the interaction between Droserasin4 and Fibrin (2HLO) reveals catalytic residue D182 (Asp182) forming  hydrogen bonds with Asn160, indicating a significant molecular interaction at a specific binding site.

Protein-Peptide Docking

Protein-peptide docking preparation involved exploring the interaction between the aspartic protease,  Droserasin4 enzyme and the  fibrin (2HLO) substrate spanning Domains A-F. Droserasin4 showed significant interaction within its Domain B structure, as indicated  by ∆G and Kd values and LigPlot+ visualization. 

Results:  The ∆G value of -9.7 is obtained through the Prodigy Web Server, indicating the  interaction stability between Droserasin4 and Fibrin’s Domain B. Based on the  ∆G value, the results indicate an intermediate level of interaction stability in the  protein structure. Furthermore, the Kd value of 7.8e-0.8, falls under the protein peptide interaction’s category. Visualized through LigPlot+, the interaction  between Droserasin4 and fibrin domain B reveals catalytic residues D18 and  D182 forming hydrogen bonds with Arg194, indicating a correlation and  molecular interaction at a specific binding site.

Protein-Ligand Docking

The preparation for docking involves the fragmentation of the substrate Fibrin using RPG. The rules to fragment this fibrin are derived  from Pepsine (pH 2.0), justified with its 3D structure is the same as the Droserasin4 after being superimposed, including the catalytic  dyads. These fragmented segments are subsequently modeled into ligands, which serve as binding molecules. Then, the enzyme  aspartic protease Droserain4 is docked together with the ligands, allowing for analysis and visualization of their interaction. This  process utilizes the Protein-ligand docking method to examine the potential binding interactions between the enzyme and the ligands. 

Results:  The protein-ligand docking visualization using LigPlot+ revealed  that the interaction occurred within the ligand at Tyrosine4 (Tyr4).  Furthermore, the Droserain4 peopling ligand B1 displayed  interactions with both catalytic residues D182 and D18 in the  middle substrate, favoring the left side due to the nature of these  interactions, as research has suggested [9]. Both catalytic  residues of Droserasin4 interact with the Tyrosine4, D182  interacts using the hydrogen bonds, and D18 interacts using the  hydrophobic bond. The two catalytic residues of Droserasin4  exhibited hydrogen and hydrophobic interactions with the amino  acid of the ligand fragments from fibrin Chain Beta. Furthermore,  the prediction shows the interaction between the Droserasin4  catalytic dyad and the oxygen atom from Tyrosine4, whereas it  should be from the oxygen of the main chain in Tyrosine4. This  occurrence happens because of the computational limitation to  predict atomic precision due to this semi-flexible method. Despite  this, the docking results indicate high-quality interaction, with  moderate binding affinity observed for protein-ligand docking.  Notably, the best representation of the interaction stemmed from  the beta chain (B1), where both Asp18 and Asp182, forming part  of the catalytic dyad, interacted with the same amino acid.

Conclusion and Future Outlook

In summary, the outcomes from protein-protein,  protein-peptide, and protein-ligand docking analyses  confirm the possibility of fibrinolytic activity between the enzyme aspartic protease Droserasin4 and the substrate Fibrin, especially at the chain beta. The protein-ligand docking results reveal the interaction of  Droserasin4 catalytic residues D182 and D18 with  Tyr4, supporting the prediction of the RPG substrate fragmentation. Additionally, the protein-ligand results underscore the Aspartic Protease's capability to degrade Fibrin, albeit on a smaller scale. Despite  computational limitations, such as misprediction by  two atoms in the protein-ligand results, efforts to identify D18 as a hydrogen bond interacting with the oxygen of the main chain of Fibrin remain ongoing. Exploring different parameters represents a promising  avenue to achieve more refined results in future  investigations.