Cardiovascular diseases (CVDs), which includes diseases that affect the heart and blood vessels, has been one of the world’s most significant problem - causing the death of an estimated 17.9 million lives each year, accounting 32% of the total global deaths. Treatments of cardiovascular diseases have been progressing significantly throughout the past decade, yet CVDs are still prevalent in the lives of many individuals today, such as the 244.1 million people living with ischemic heart diseases (IHD) in 2020. A recent unpublished In vitro finding by the Department of Biology of Universitas Pelita Harapan has proven the ability of Actinidin, an enzyme derived from Actinidia Chinensis (Kiwifruit) in breaking down blood clots that can possibly result in life-threatening CVDs. This in-silico research aims to prove in more depth the fibrinolytic activity between the enzyme Actinidin and the substrate Fibrin by computer 3D simulations to present the interaction down to its molecular structure and to support the claim made in the in-vitro experiment findings in recognizing of actinidin as a potent fibrinolytic agent for cardiovascular treatment.

Actinidin and Its Previous Studies

Actinidin is a cysteine protease enzyme derived from Actinidia chinesis (Kiwifruit) which is the dominant enzyme in kiwifruit, particularly in the bulk flesh of the fruit (Figure 1B) [5]. The enzyme was first discovered by Arcus in 1959 [6], yet further findings about the enzyme itself are still limited, especially in terms of its fibrinolytic activity. Currently, Actinidin has been better known as a substitute for meat tenderizers due to its proteolytic characteristics that are able to cause molecular changes to beef muscle proteins [7]. Moreover, Actinidin has been said to be able to enhance the gastric digestion of some foods including sodium caseinate and beef muscle protein due to the Actinidin’s proteolytic characteristic [8]. The current understanding about the fibrinolytic activity of Actinidin extends to the knowledge that the fibrinase of Kiwifruit has shown a significant relationship towards thrombolysis, as shown through the in-vitro simulation done by Habib and Saad.

Analysis Methods

• Actinidin Sequence Alignment: using CLUSTAL-Omega Web Server.

• Structural Modelling: The 3D structure of Actinidin was obtained from Protein Data Bank (PDB ID: 2ACT). The structure then validated through the Phi/Psi Ramachandran Plot in SWISS-MODEL Workspace to evaluate the accuracy of the 3D structure of Actinidin

• Structural Preparation: The active residues of Actinidin were predicted using CPORT.

• Protein-Protein Docking: Using the HADDOCK Web Server, the enzyme Actinidin and substrate Fibrin were processed and simulated to examine the existing interaction between the two proteins . The method used for this protein-protein docking is biased docking, where only the catalytic triad from the experimental data of Varughese et al. (1992) (Cys25, His162, Asn182) were inputted as the active residues. CPORT prediction was not included as it does not include the catalytic triad and due to the amount, will increase the competition of interaction between amino acids. Other parameters were left at default. Then, the energy of binding affinity was determined using PRODIGY, followed by 2D visualization using LigPlot+

• Protein-Ligand Docking: The preparation for docking consists of the substrate Fibrin being fragmented using Random Peptide Generator using Papain as the reference. These fragments were then modeled into ligands using PHENIX eLBOW. Then, Actinidin were docked together with the ligands and thus analyzed and visualized using the method used in Protein-Protein Docking.

Result and Conlusion

Overall, the results of the protein-protein and protein-ligand docking have shown the existence of fibrinolytic activity between enzyme Actinidin and substrate Fibrin. The protein-protein docking results which show two existing hydrogen bonds of Cys25 were able to prove that the thiolate of Cysteine-25 does attack the carbonyl carbon of the peptide bond in a nucleophilic addition. Moreover, the Protein-Ligand results of Chain Alpha also concludes Actinidin’s ability to degrade Fibrin, even in a smaller scope. Due to computational limitations, there are still a few elements to work on, including: the misprediction by two atoms of the Protein-Ligand result, and the hydrophilic bonds of Cys25 in Protein-Protein docking that should interact with the oxygen of the main chain of the Fibrin. Discovering different parameters would be a way to gain a more refined result in the future.