In this in silico study aimed for screening macroalgae compounds potential as ACE2 inhibitor. The Macroalgae derivative compounds such as carotenoids and sulphated polysaccharides. Macroalgae compounds were collected from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). There were 27 compounds collected (Table 1), then an initial screening study was conducted by Lipinski rule of five web server (http://www.scfbio-iitd.res.in/software/drugdesign/lipinski.jsp), the result is 25 compounds was approved by Lipinski rule of five and then 25 compounds carried out by PASS online prediction (http://www.way2drug.com/passonline/) web server. The result showed that 6 compounds were approved that have the potential as antiviral. The 3D structure of 6 compounds was approved shown in Fig. 1.

Table (1):
The ligand compounds were collected.

Table (2):
The result of Lipinski rule of five.

Note:MW: Molecular Weight, HBD; Hydrogen Bond Donors, HBA; Hydrogen Bond Acceptors, LOGP; High Lipophilicity, MR; Molar Refractivity.

Table 2 present the results of Lipinski’s five rule which generally predicts a compound as a drug like molecule. Druglikeness explained as the complex balance of diverse molecular properties and structural features that determine whether a particular molecule categorized as a drug or not. That properties include molecular weight, characteristics of hydrogen bonding lipophilicity, and existence of various pharmacophoric features in that compounds.¹⁹ The result is molecular weight value of alpha-carrageenan, galactan, fucoxanthin, and siphonaxanthin under 500.000 dalton, it is predicted that alpha-carrageenan, galactan, fucoxanthin, and siphonaxanthin can enter the cell membrane. While the molecular weight of lambda-carrageenan and (1->4)-beta-galactan have a molecular weight more than 500.000 dalton, so that both of the compound estimated unable to pass membrane cells. Advanced study be required for making molecular weight smaller under 500.000 dalton. Log P value indicates the coefficient of solvability in water or fat, the coefficient range is -0.4 to 5. The larger log P number represents that the more hydrophobic molecules characteristics would be. The over hydrophobic molecules cause the increased level of toxicity effect because of molecules longer disconnected in the lipid bilayer membrane will be spread broadly in the body. Thus, the selective effect of the compound inhibit target protein is reduced. Meanwhile, the more negative the log P number, the more non-permeable the molecule will be.^20,21

Table (3):
The result of PASS online prediction.

Table 3 shows the result of PASS online prediction webserver for prediction the ability of the bioactive compound from macroalgae as antiviral for activation or inhibition by human cells. The principle is that the Pa (potential activation) value must be higher than Pi (potential inhibition) value. In this in silico study Pa>0.3. The results showed macroalgae Pa value larger than its Pi value. It is indicates when a macroalgae compound penetrate human body, it have potential to inhibit ACE2 protein.

Table (4):
The result of molecular docking simulation.

Table 4 shows the result of macroalgae compound molecular docking simulation. Molecular docking simulation was done by PyRx 0.8 version and the followed grid docking result are center X: 166.4430 Y: 223.6339 Z: 301.4075 and dimensions (Å) X: 67.1115 Y: 74.6594 Z: 67.3432. Due to the target protein functional domain is unknown, so in this study aimed blind docking method.²²

Table (5):
The result of molecular docking simulation from drugs as control.

Based on the result of the binding energy value from the macroalgae compound selected, siphonaxanthin has the lowest binding energy to bind ACE2 receptor with binding energy value -8.8 kcal/mol. While, from sulfated polysaccharides derivate compound (1->4)-beta-galactan has the lowest binding energy with -8.3 kcal/mol. Ligands with the lowest binding energy are predicted to have a target protein’s biological activity. It refers to the aim of this study the compound is predicted to have the ability to inhibit ACE2 receptor. The lowest binding energy interaction ligand-target protein allows molecular complex formation constant temperature and pressure.²³ This study also using common antiviral drug such as remdesivir, molnupiravir, baricitinib, lopinavir, oseltamivir, and favipiravir (avigan).^5,24-26 These antiviral common drugs used as positive control for comparing the binding energy value to target protein ACE2 receptor. Based on the binding energy result, siphonaxanthin, fucoxanthin, (1->4)-beta-galactan, and (1->4)-beta-galactan has the lowest binding energy than antiviral common drug.

The docking simulation is finished, then molecular complex visualization was conducted by PyMOL v.2.4.1 showed in Fig. 2. Macroalgae compounds can bind to the ACE2 receptor. Fig. 3 present the complex ligands-protein visualization 2D diagram was carried out by DS BIOVIA Discovery Studio 2016 v16.1.0 x64. There are various bond types of macroalgae compounds with ACE2 receptor. Macroalgae carotenoids such as fucoxanthin and siphonaxanthin showed that Van der Waals, Alkyl, and Pi-Alkyl formed the dominant type bond (Fig. 4 and Table 6). While in Macroalgae sulfated polysaccharides, the dominant type bond formed is Van der Waals and Conventional Hydrogen Bond. The higher hydrogen bonds total between protein-ligand complex, the more stable the interaction of protein-ligand.²¹

Table (6):
The summary of amino acid residues from macroalgae.

There are various types of amino acid residues that form an interaction between ligand and target protein ACE2 receptor, shown in Table 5. Binding to these amino acid residues could potentially contribute to structural alterations of the ACE2 receptor, resulting in functional changes. Therefore, futher research related to in vitro and in vivo study will be interest to know the mechanism of macroalgae compounds as ACE2 receptor and against SARS-CoV-2.

Nowadays, in silico studies is important for drug development against SARS-CoV-2. Recent studies promising macroalgae compounds as SARS-CoV-2 drug candidates such as sulfated polysaccharides can inhibit SARS-CoV-2 by binding RBD spike protein. There are 17 seaweed sulphated polysaccharides that were screened as SARS-CoV-2 antiviral. The docking results lowest binding energy is -8.2 kcal/mol from xylan sulphate with positive control using heparin tetrasaccharide N-sulfated and heparin.²⁷ In comparison, the other in silico study used 9 investigated compounds marine sulphated polysaccharides promising antiviral agents and using heparin as positive control.²⁸ In both studies used heparin as the positive control. Meanwhile, other studies recommend remdesivir, molnupiravir, baricitinib, lopinavir, oseltamivir, and favipiravir as drugs against SARS-CoV-2. Hence, it is needed for extensive research to develop SARS-CoV-2 antiviral drugs using another positive drug control. Both of those studies do not explain the drug-likeness analysis Lipinski rule of five and predict the bioactive compounds as antiviral using PASS online prediction webserver. Hence, this research is important to conduct with initial screening studies that were done by Lipinski rule of five and PASS online prediction webserver to screen sulfated polysaccharides as antiviral drugs. Other than that, macroalgae is rich with antioxidant compounds that have the potential as SARS-CoV-2 antiviral. Therefore, this study also conducted molecular docking to screen macroalgae antioxidant compounds as SARS-CoV-2 antiviral.