Biological activity, molecular docking, and ADME predictions of amphibine analogues of Ziziphus spina-christi towards SARS-CoV-2 M pro

. The main protease of the SARS-CoV-2 virus, SARS-CoV-2 M pro , can be discovered as a promising target to treat the COVID-19 pandemic. The peptide-based inhibitors may present better options than small molecules to inhibit SARS-CoV-2 M pro . Ziziphus spina-christi species reported have a peptide-based of alkaloids group, i.e., amphibine whose analogues can be identified the potential as an inhibitor of SARS-CoV-2 M pro . The compound structure was drawn and optimized using semi-empirical AM-1 method using Quantum ESPRESSO v.6.6, while the biological activity using PASS. Prediction server and molecular docking simulation using MGLTools 1.5.6 with AutoDock 4.2 were performed. Afterward, the ADME profiles were predicted using the SWISS-ADME server. PASS server was predicting amphibine B-F and H showed potency both as antiviral and as a protease inhibitor. The molecular docking simulation of amphibine analogues showed lower binding energy than the native ligand. The binding energy of the native ligand was −7.69 Kcal/mol compared to the lowest binding energy of amphibine analogues was −10.10 Kcal/mol (amphibine-F). The ADME prediction showed that amphibine-F has the best bioavailability as an oral drug, amphibine-B, C, and D have good bioavailability, and amphibian-E and H have poor bioavailability. Concluded, amphibine B-F and H of amphibine analogues showed potency as COVID-19 treatment targeting SARS-CoV-2 M pro .


INTRODUCTION
The Coronavirus disease 2019 has spread worldwide and still become a health problem that needs attention (Thompson, 2020;Zhu et al., 2020). Released on March 2021 in the present situational report from WHO, 3.8 million COVID-19 new cases, and 64000 recent deaths were reported globally. (WHO, 2021). Clinically, COVID-19 can lead to severe respiratory complications and death with fever and respiratory symptoms (Calcagno et al., 2020).
The natural product compounds based on peptide-like from medicinal plants become our orientation research. Hence, they have not been explored intensively in drug discovery, especially those that can inhibit COVID-19 (Dias et al., 2012;Harvey et al., 2015;Benarba & Pandiella, 2020;Lakshmi et al., 2020). Z. spina-christi as important medicinal plant (El Maaiden et al., 2019) is a deciduous tree that generally comes from warm and subtropical climates, such as North Africa, South Europe, Mediterranean, tropical America, South and East of Asia, and others, including Indonesia (Kwape et al., 2013;Moossavi et al., 2017). There are many names for Z. spina-christi, known as Christ's thorn jujube, belongs to the Rhamnaceae family with large shade tree (Baghazadeh-Daryaii et al., 2017;Gorai et al., 2019).
The previous studies have reported that Z. spina-christi provided a variety of pharmacological activities, including antibacterial, antifungal, antioxidant, antihyperglycemic, and anti-diabetic (Kalayou et al., 2012;Ads et al., 2017;Al-Ghamdi et al., 2017;Alotibi et al., 2020). According to our previous studies, the main phytochemicals were discovered in this plant include alkaloids, flavonoids, and saponins . Tuenter et al. (2017) and Sakna et al. (2019) stated cyclopeptide alkaloids can be found in their stem-bark.
To date, the need for a vaccine and antiviral development is increasing, especially those targeting SARS-CoV-2 M pro . Computational approaches were demonstrated to predict the affinity and molecular behavior of amphibine analogues from the plant compound. We are interested in investigating the potency of the amphibine analogues (cyclopeptide alkaloids) from Z. spina-christi, as a promising future treatment for COVID-19 targeting SARS-CoV-2 M pro .

MATERIALS AND METHODS
Ligand preparation. The ligands chosen for this research were peptide alkaloids, amphibine analogues in Ziziphus spina-christi, i.e., amphibine A-H compounds. The 3D ligand structures were drawn and optimized based semi-empirical AM-1 method using Quantum ESPRESSO v.6.6 (Giannozzi et al., 2020). The research protocols were following our previous studies .
Receptor preparation. The 3D structure of SARS-CoV-2 M pro was obtained from the Protein Data Bank (PDB) (http://www.rcsb.org/pdb/). The high resolution of the SARS-CoV-2 M pro receptor (2.15 Å) with PDB ID: 6WTT was chosen (Ma et al., 2020). The receptor was complexed with boceprevir, an HCV protease inhibitor as a native ligand. Afterward, all the unique ligands and water molecules were removed from the receptor, and then the polar hydrogen and a charge (Kollman charge) were added to the protein structure. The protein preparation procedures were executed using MGLTools 1.5.6 with AutoDock 4.2 (Tanbin et al., 2021).
Biological activity prediction. The biological activity spectra of amphibine analogues were assessed using the PASS prediction web server (http://www.way2drug.com/PASSOnline/predi ct.php) (Lagunin et al., 2000). The predicted spectrum was estimated as probable activity (Pa) and probable inactivity (Pi), based on structure-activity relationship analysis of the training set containing more than 205000 compounds exhibiting more than 3750 kinds of biological activities. Pa and Pi values vary from 0.000 to 1.000 since they are probabilities. The PASS prediction was interpreted and used flexibly, according to Anzali et al. (2001).
Molecular docking simulation. The molecular docking simulation method was validated using RMSD calculation by redocked the crystallographic native ligand. The best conformation of docked native ligand was taken and superimposed with the native ligand before docked, and the Root-Mean-Square Deviation (RMSD) was calculated. The acceptable RMSD value must be less than 2.0 Å (Bell & Zhang, 2019). Afterward, the amphibine analogues were docked into the binding pocket of the SARS-CoV-2 M pro . The grid box was set with coordinates 5. 499, 27.197, and −11.76 (x, y, and z, respectively), and the dimensions of the grid box were 64, 60, and 60 (x, y, and z), and numbers of GA run was 100 (Atilgan & Hu, 2011). ADME prediction. We analyzed the adsorption, distribution, metabolism, and excretion (ADME) profile of the amphibine analogues, which could be used as a drug. We used the SWISS-ADME web server to predict the ADME profile (https://www.swissadme.ch), which allows the user to draw or input their molecules data and Vol 9(1), June 2021 Biogenesis: Jurnal Ilmiah Biologi 111 provides the parameters such as lipophilicity, water-solubility, pharmacokinetics, druglikeness rules, and medicinal chemistry (Mahanthesh et al., 2020).

RESULTS AND DISCUSSION
Biological activity prediction. The PASS prediction web server's biological activity was carried out on amphibine analogues (A-H) compounds to see the level as a COVID-19 main protease inhibitor ( Table 1).
The PASS web server predicts various biological activities of Amphibine analogues, but the focus of the research here is on the prediction of antiviral and protease inhibitor agents. All amphibine A-H were predicted to have an activity as antiviral agents. As protease enzyme inhibitors, amphibine B, C, D, E, F, and H showed activity, whereas amphibine A and G showed no activity. Amphibine-C showed the best-predicted activity spectrum of 0.  Molecular docking simulation. Molecular docking simulation study was performed on the crystal structure of SARS CoV-2 M pro to assess the binding affinity potency of amphibine analogues (amphibine B, C, D, E, F, and H) that were previously predicted using the PASS website. The docking methods were validated to see the strength of binding mode prediction through re-docking the native ligand. The RMSD value of the native ligand obtained was 1.30 Å, which shows that the molecular docking method was valid. The amphibine analogues were then docked into the binding site of the crystal structure of SARS-CoV-2 M pro . All of the docking results of amphibine A-H showed high binding energy and Ki compared to the native ligand ( Table 2). The negative sign or the lowest binding energy is considered to be a stable binding affinity to the receptor. The binding energy of the native ligand was −7.69 Kcal/mol. The amphibine analogues binding energy sort by lowest to highest were −10.  Molecular interactions. The ligandreceptor interactions of the best binding mode of the Amphibine analogues were analyzed and compared to reference native ligand binding mode toward the SARS-CoV-2 M pro . The tabulation data of the amino acid interactions were provided in Table 3, and the 2D interaction was provided in Fig. 2. Native ligand in its interactions, form hydrogen bonds with amino acids Glu166, His164, Phe140, Gln189, Cys145, and other types of interaction with residue Cys145 (unfavorable bump), His163 (unfavorable acceptor-acceptor), Pro168 (Pi-alkyl), Met165 (alkyl), His41 (alkyl & carbon-hydrogen bond), and His172 (carbonhydrogen bond). Amphibine-B showed hydrogen bond interaction with three amino acids, i.e., Glu166, Ser144, Asn142, and other types of interaction with amino acids Glu166 (Pi-Anion), Pro168 (Pi-sigma), His41 (alkyl), Cys145 (Pi-alkyl), and His163 (unfavorable acceptor-acceptor). Amphibine-C showed hydrogen bond interaction with two amino acids, including Glu166 and Gln189. Other types of interaction with amino acids Gln189 (carbon-hydrogen bond), Glu166 (carbonhydrogen bond), Pro168 (Pi-sigma), Met49 (Pisulfur), His41 (Pi-alkyl & Pi-pi stacked), Leu167 (Pi-alkyl), Leu141 (alkyl). Amphibine-D showed hydrogen bond interaction with one amino acid (Glu166) and other types of interaction with amino acids Met165 (alkyl & Pi-sulfur), His41 (carbon-hydrogen bond), Thr24 (carbon-hydrogen bond), Cys145 (Pihydrogen bond). Amphibine-E showed hydrogen bond interaction with three amino acids, including Glu166, Gln192, and Gln189, and other types of interaction with amino acids Ala191 (Pi-alkyl), Pro168 (Pi-alkyl), His41 (Pisigma). Amphibine-F showed hydrogen bond interaction with three amino acids, including His41, Glu166, and Gln189, and other types of interaction with amino acids His41 (Pi-sigma & unfavorable acceptor-Acceptor), Glu166 (Pianion), Gln189 (carbon-hydrogen bond), Arg188 (carbon-hydrogen bond), Met165 (alkyl). Amphibine-H showed hydrogen bond interaction with three amino acids, including Glu166, Gln192, and Thr190, and other types of interaction with amino acids Ala191 (Pialkyl), Met165 (alkyl), Glu166 (carbonhydrogen bond), and Phe140 (carbon-hydrogen bond).  The hydrogen bond is an attractive interaction between a hydrogen atom from fragment X-H, and enhance receptor-ligand interactions (Arunan et al., 2011;Chen et al., 2016). Native ligands binding mode still showed the highest intensity of hydrogen bonding (five hydrogen bonds), followed by amphibine-B, amphibine-E, amphibine-F, and amphibine-H with three numbers of the hydrogen bond, then amphibine-C (two hydrogen bond), and Amphibine-D (1 hydrogen bond). The similarity of amino acid interaction types between the native ligand as a reference and amphibine analogues showed in the amphibine-B, amphibine-C, amphibine-D, and amphibine-H provided one type of hydrogen bond interaction similar (Glu166). The amphibine-E and amphibine-F compounds showed two similar hydrogen bond interactions to the reference ligand (Glu166 & Gln189). The other interactions, i.e., Pi-sigma, Pi-alkyl, and Pi-sulphur, mostly involve charge transfer assisting in intercalating the drug at the receptor-binding site. The highest number of amino acid interactions that form those other interactions were dominated by amphibine-C, amphibine-B, amphibine-F, amphibine-H, amphibine-E, and amphibine-D, respectively. Amphibine-F Amphibine-H ADME prediction. The amphibine analogues predicted before (B, C, D, E, F, and H) have been analyzed by ADME profile using SWISS-ADME (Fig. 3). The ADME profile was provided with radar that shown six predicted ADME parameters that are closely related to the oral bioavailability of a compound, including LIPO (lipophilicity), SIZE (size), POLAR (polarity), INSOLU (insolubility), INSATU (instauration), and FLEX (flexibility). The colored zone was a physical chemistry area that is suitable for oral bioavailability. Analysis of ADME profiles performed by radar showed Amphibine-B, Amphibine-C, Amphibine-D, and Amphibine-F have suitable in polarity and insaturation following by oral drug bioavailability criteria, except for the lipophilicity, size, insolubility, and flexibility parameter. Amphibine-E compound radar is suitable for insaturation parameters. Amphibine-F compound radar shows a suitable in all parameters, i.e., lipophilicity, size, polarity, insolubility, insaturation, and flexibility. Amphibine-H compound radar shows a suitable in polarity, insolubility, insaturation, and lipophilicity criteria. Fig. 3. Administration, distribution, metabolism, and elimination (ADME) parameters for amphibine B, C, D, E, F, and H that were evaluated by SWISS-ADME. Furthermore, lipinski analyses were performed to look for drug similarities or determine whether a chemical compound has specific pharmacological or biological activity has chemical and physical properties that make it pharmacokinetically effective in the human body, including ADME. In Lipinski druglikeness analysis, amphibine-B provided one violation (MW >500), amphibine-C provided one violation (MW >500), amphibine-D provided one violation (MW >500), amphibine-E provided two violation (MW >500), and (NorO > 10), amphibine-F provided one violation (MW>500), Amphibine-H provided two violations (MW>500, NorO>10). The drug-likeness results showed amphibine-E and amphibine-H provided more than one violation, indicates poor bioavailability as oral drugs. Amphibine-B, C, D, and F showed one violation for drug-likeness criteria, indicates good bioavailability. In general, the amphibine-F compounds showed the best bioavailability as an oral drug, amphibine-B, C, and D showed good bioavailability as an oral drug, and amphibine-E and H showed poor bioavailability as oral drugs.

CONCLUSION
The amphibine analogues from Ziziphus spina-christi species analyzed by biological activity, molecular docking, and ADME predictions were showed as potentially inhibitor candidates for the SARS-CoV-2 M pro receptor. The biological activity prediction by PASS web server of amphibine analogues (A-H) showed amphibine-B, C, D, E, F, and H have potential as antiviral and protease inhibitor agents. The molecular docking results of the amphibine-B, C, D, E, F, and H showed better binding affinity against SARS-CoV-2 M pro compared to the native ligand as a reference inhibitor. These compounds also form interactions that are similar in some residues with the native ligand. The ADME prediction showed amphibine-F has the best bioavailability as an oral drug, amphibine-B, C, and D have good bioavailability as an oral drug from drug-likeness criteria, while amphibine-E and H show poor bioavailability as an oral drug. Concluded the Amphibine-B, C, D, E, F, and H have potential as a treatment of COVID-19 through inhibits the protease enzyme of SARS-CoV-2 M pro , and some compounds can be formulated as oral administration (amphibine-B, C, D, and F), and some in other administration (amphibine-E and H).