AN IN-SILICO STUDY OF SOME NATURAL AND SYNTHETIC COMPOUNDS AS POTENTIAL INHIBITORS FOR FACTOR XA

Recent research has demonstrated the significance of factor Xa and its inhibitors as a possible treatment approach. Since currently approved drugs have a wide variety of side effects, new anticoagulants must be developed. So, the goal of this in silico research is to find molecules that may act as factor Xa inhibitors. The structure of factor Xa (2w26) was obtained from the Protein Data Bank database, while the structures of the organic compounds were obtained from the ZINC database or via other means. In order to verify the anti-factor Xa action of these chemical compounds, iGemDock program was used to perform molecular docking. The compound (1) [5-hydroxy-2-(3-hydroxy-4-phenylmethoxyphenyl)-3,7bis(phenylmethoxy)chromen-4-one] showed the best interaction value against the 2w26 enzyme, and the binding energy was (–167.702 kcal/ mol); whereas the reference rivaroxaban was (– 149.661 kcal/mol). These results lead to suggest new organic compounds as factor Xa inhibitors and further in vitro studies are required to confirm.


INTRODUCTION
Several recent studies have demonstrated the importance of factor Xa as a promising anticoagulant target, in addition to, the usage of many factor Xa inhibitors to prevent and treat a lot of thrombotic diseases [1][2][3][4][5][6] . The coagulation process involves proteins in coagulation reactions, which are called blood coagulation factors. Generally, they are present in the plasma in an inactive state but can be activated. Therefore, the coagulation process is a complex cascade of enzymatic reactions that can be activated via both intrinsic and extrinsic pathways 7 . The final step in coagulation is the formation of the fibrin clot from fibrinogen, by the action of thrombin, which acts as a serine protease, which is generated by prothrombin through factor Xa 8 . Therefore, factor X is considered the connecting point between the coagulation cascades. Recent investigations have established the relationship between thrombosis and Covid-19 9 where Covid-19 causes vast inflammation-promoting cytokine. The Cytokines in turn increase the liver's production of clotting factors, as explained by Beverley Hunt, medical director of Thrombosis UK and a practicing clinician. Besides, there is a new study suggesting that there is a similarity among SARS-CoV-2 Mpro and coagulation factors thrombin and Factor Xa, which gives more importance to Factor Xa inhibitors 10,11 . Direct Factor Xa (FXa) inhibitors have anticoagulant, anti-inflammatory, and antiviral activities, and they probably hold a considerable promise in treating COVID-19 12 .
FXa structurally belongs to the serine proteases of the trypsin-like family. The crystal structure of human factor Xa was deposited in May 1993 for the first time. The active site of factor Xa is divided into four sub-pockets as S1, S2, S3, and S4. The S1 sub-pocket determines the major segment of selectivity and binding. Factor Xa inhibitors usually bind with the active pocket in an L-shaped conformation 13 .
The in-silico study is a technique that predicts the preferred location of small molecules (ligands) within the active site of the FXa (ID:2W26) in this research. This method applies a cavity detection algorithm for distinguishing invaginations in the protein as candidate active site regions 14 .
This research which is part of a master's degree thesis attempts to survey some organic compounds (more than 1000 compounds) that have been included in some recent studies in recent years. The interaction of Factor Xa was investigated with derivatives of phenolic compounds 15 or phenolic acids compounds 16 including benzoic acid, cinnamic acid, naphthoic acid, salicylic acid, gallic acid, and chromen 17 , in addition to some flavonoids with a various type. In this part of the research, the focus was on the compounds that fulfill Lipinski's rules. The chemical structures of the investigated compounds, along with some proven drugs, are compared with rivaroxaban (Figure 1 illustrates its structure) as a reference in Table 1. These compounds and their inhibitory effect on FXa were investigated by molecular docking for visualizing the efficiency of their potential effect as factor Xa inhibitors, theoretically.

Protein Preparation
The receptor protein related to human Factor Xa, and required for the docking study has been retrieved from the Protein Data Bank (PDB) 18,19 which is the main source in sites of structural biology, and it is a major repository for 3D structure data of giant molecules. The protein (PDB ID:2w26) had a resolution factor of 2.08 A°. The enzyme was downloaded, then saved in PDB file format. Figure 2 shows the active site of (2w26) and binding ligand Rivaroxaban (RIV).

Ligands preparation
The organic compounds that we reported here had been chosen after a study of the literature. Indeed, more than 1000 compounds that are derivatives of phenolic compounds or Phenolic acid were chosen. Some of which were drawn in the two-dimensional (2D) structures using ACD chem sketch software 20 . Some other chemical structures were reclaimed from the Pubchem database [21][22][23] or were retrieved from the ZINC database 24 . Afterward, the compounds were saved in Sdf or mol format and converted to mol2 format using the OPEN BABEL software 25 .
Afterward, a total of 100 high binding energy compounds resulting from the in-silico study were made to follow the previous rules. Data of the compounds were obtained from the SwissADME website 26 .
However, despite the importance of the aforementioned rules, there have been critical opinions that relied on the fact that 6% of oral medications don't fulfill the Lipinski's rule, such as azithromycin, cyclosporine, digoxin, and many anti-tumors and viruses, in addition to the fact that several drugs for oral use have been licensed by the Food and Drug Administration in recent years although they didn't fulfill to previous bioavailability rules. Some recent studies have also shown the biological effectiveness of compounds of natural origin, despite exceeding the limits of these rules [27][28][29][30][31][32] .

Protein-ligand docking
After a literature review to collect compounds 33 , iGemdock v2.1 was used to perform the docking study for the enzyme (2w26) that interacts with the selected phenolic or Phenolic acid compounds, natural compounds such as aglycones and their derivatives, vitamins, and some approved drugs by the FDA. It is available for free and was used in previous research [34][35][36][37][38] .

iGemdock v2. 1
IGemdock is an integrated virtual screening (VS) environment for preparations through post-screening analysis with pharmacological interactions. The docking software iGemdock was used as a molecular docking tool to dock the protein of the enzyme (2w26) with our selected compound. iGemdock provides interactive interfaces to prepare the screening compounds' library and the binding site of the targeted enzyme. Then, using the in-house docking tool iGemdock, each compound in the library was docked into the binding site. Afterward, iGemdock inferred protein-compound interaction and then clustered the screening compounds for the postscreening analysis based on profiles of hydrogen-bonding (H), electrostatic (E), and Van der Waal's (V) interactions. Furthermore, based on compound structures, iGemdock inferred the pharmacological interactions. Finally, iGemdock ranks and visualizes the screening compounds by combining the pharmacological interactions and energy-based scoring functions made available by iGemdock. the protocol -accurate docking‖ was used by setting a population size of 800 with 80 generations and 10 solutions.
After the docking, the software performed the post-docking analysis to find the docking pose and its energy values. The empirical scoring function of iGemdock was estimated using: Energy = vdW + H-bond + Elect…. (1) whereas the vdW term is van der Waal energy, H-bond term is hydrogen bonding energy, and Elect term is electro statistic energy.

Results
In silico docking is the best method to predict the usefulness of any chemical as a remedy before continuing with any in vivo or in vitro study to shorten the experiments and ensure cost savings. A literature review was done for the organic compounds, and the chosen compounds were docked later against factor Xa by using iGemdock to evaluate the theoretical effect as a factor Xa inhibitor and conclude its interactive analysis before the compounds are introduced to investigate the in vitro anticoagulant activity.
Several studies have emphasized the importance of using Lipinski's rules when developing and researching new drugs. This rule helps to determine if a biologically active chemical is likely to have the chemical and physical properties to be orally bioavailable. Noting that Lipinski's rules allow for one transgression for each molecule 39-41 .

Lipinski's Rule
The "Rule of Five" was formulated by Christopher A. Lipinski in 1997, based on the observation that most orally administered drugs are relatively small and moderately lipophilic molecules. The "Rule of Five" describes molecular properties and is used to predict drug pharmacokinetics in the human body, which consists of four important stages that are Absorption, Distribution, Metabolism, and Excretion (ADME). The compounds are more likely to be membrane-permeable and easily absorbed by the body if it matches the following criteria 42 : 1. Molecular weights not more than 500 Da. 2. Calculated octanol-water coefficients (CLogP) not more than 5. 3. Less than 5 hydrogen bond donors 4. Less than 10 hydrogen bond acceptors. From this point of view, some FDAproved drugs and organic compounds like those mentioned in table 1 were selected for docking and were compared to (RIV).
Rivaroxaban which is a proven known factor Xa inhibitor was employed as a positive standard, then the docking investigations were performed using iGemdock v2. 1. The kcal/mol results demonstrated that the best 20 compounds had high binding energy ranging from (-167,702 kcal/ mol) to (-146/,294kcal/mol). This is demonstrated in Table 2 compared with the standard (-149,661kcal/mol). Therefore, these molecular docking analyses could express the most potent 2w26 inhibitors for the prevention and treatment of thrombosis. Table 2 summarizes the results of the docking study based on binding energies.        figure 4 illustrates the predicted interaction of compound (1) with amino acid residues. It was synthesized as a derivative of quercetin, and then its antibacterial and antioxidant effects were measured. However, the results showed that this derivative is less effective than Quercetin as an antibacterial and antioxidant 43 , however, the anti-coagulant effect hasn't been investigated yet. Whereas, quercetin has many biological effects as a powerful antioxidant, antibacterial, antiinflammatory, and anticoagulant, especially as an inhibitor of activated factor X 44-47 .    (1) with amino acid residues in the active pocket of (PDB ID:2w26) using iGEMDOCK. The pink color represents the corresponding ligand molecule and the green color represents the corresponding reference. The Green and grey color represent the amino acids involved in hydrogen bonding and van der Waals interactions respectively.

Conclusions
The current research has provided an insight into the search for new phenolic compounds or phenolic acids compounds as a suggested Factor Xa inhibitor. Various in silico tools like Lipinski's rule. Molecular docking has been applied to select the best compounds as anticoagulant inhibitors whereas the derivative [5-hydroxy-2-(3-hydroxy-4phenylmethoxyphenyl) -3, 7-bis (phenylmethoxy) chromen-4-one] showed great anticoagulant activity theoretically. Later, further in vitro investigations should be applied to determine the biological effect. Therefore, the in-silico investigation has been essential in predicting molecules enabling the minimization of time spent searching for compounds, and can be recognized as an appropriate technique for screening novel compounds to target other enzymes.