Aurone as promising human pancreatic lipase inhibitors through in silico study

In this study, 82 aurone compounds, a subclass of flavonoids were investigated towards to human pancreatic lipase inhibitory activity. Molecular docking of the aurones was done successfully into the catalytic position of lipase (Pdb: 1LPB) using AutoDock Vina software 1.5.7.rc1. The results showed that 62 compounds interacted well with residues in the catalytic trial Ser152-Asp176-His263 and Phe77 of protein 1LPB. In particular, A32 was selected as the best binding compound (docking score: -10.6 kcal.mol-1) and suitable for oral drug following the 5-Lipinski rule. Combining the results of docking and molecular dynamics simulation of A32protein complex during 10 ns, this study performed that the A32 compound bound well and formed a stable complex with 1LPB protein. Therefore, the A32 compound was considered as the lead compound which could be synthesized and tested for pancreatic lipase inhibitor.

Aurone ( Figure 1) (IUPAC name is 2-benzylidene-1-benzofuran-3(2H)-on) is a potential subclass of flavonoid. Aurone plays an important role in the pigmentation of some type of flowers and fruits, which is typically bright yellow. They are also found in bark, wood, and leaves. Aurone is considered being phytoalexin, which is used by plants as a defense mechanism against infections.
Aurone is often found in some species of the Scrophulariaceae and Compositae families [8,9]. Many of the biological effects of aurone derivatives have been studied and published as anticancer, anti-inflammatory, antifungal, antibacterial, anti-malarial, anti-hepatitis B, anti-oxidant, etc [10].
However, the inhibition HPL ability of aurone compounds has not been studied, therefore this study conducted molecular docking of aurone derivatives to human pancreatic lipase protein (PDB ID: 1LPB) and investigated the molecular dynamics simulation of the best binding derivative, that contributes to expanding on the HPL potential inhibition of aurone compounds. R, R': OH, OMe, CH3, halogen, etc

Protein
The 3D structure of the lipase-colipase complex has a crystallined ligand in the catalytic cavity (PDB ID: 1LPB) which was retrieved from Protein Data Bank (https://www.rcsb.org). The lipase chain has 449 amino acids, including N-terminal from amino acids 1 to 335 and C-terminal from amino acids 336 to 449, which were attached to colipase. The protein is a crystalline structure with its ligand methoxy undecyl phosphonic acid (MUP). MUP binds and creates bonds with the catalytic cavity of HPL including the hydrogen bonds (H-bonds) with Phe77, Leu153, His263 and the covalent bonds with Ser152 [3,[11][12][13][14].
From the retrieved protein structure, protein chains binding with co-crystallization ligands were identified and extracted, and also added polarized hydrogens by AutoDock Tools 1.5.7rc1.

Ligand
The test compounds consisted of 82 aurone derivatives (symbols A1 to A82) collected from scientific papers investigating the biological effects of aurone derivatives [10,[15][16][17][18][19][20]. The 2D structures of these compounds were drawn by ISIS Draw 2.5 program and formatted in the MOL file. All 2D structures were converted to 3D structures and they were minimized energies with the YASARA Energy Minimization server (https://www.yasara.org).

Molecular docking
Molecular docking is a method to predict the structure and spatial orientation of one molecule when attached to another molecule to make the most stable complex. There are three common types of docking: rigid docking, flexible docking, and full flexible docking. A process that simulates ligand and protein binding will involve two basic steps: sampling and scoring [21,22].
The molecular docking process was implemented by AutoDock Vina software version 1.1.2 [21]. The binding site parameters (x: 8,431; y: 24,417; z: 52,623) and the docking box dimensions (18x18x18 Å) were determined through the re-docking of co-crystallization ligand in the catalytic cavity. The results of molecular docking were evaluated through the criteria of binding structure, binding energy, possible interactions between ligand and residues of the protein.

Five-lipinski rule
The five-Lipinski rule evaluates a chemical compound, which has a defined chemical and physical property, capable of being researched into an oral drug when it violates no more than one of the following criteria:

Molecular docking
The 82 aurone derivatives were successfully bond to the catalytic cavity with the docking score ranging from -10.6 kcal.mol -1 to -7.4 kcal.mol -1 . Of these, 62 compounds were classified into group A.
Group A created hydrogen bond with residues in the catalyst trial Ser152-Asp176-His263 and Phe77, which played an important role in the fat hydrolysis activity of pancreatic lipase enzyme. The remaining 20 compounds did not interact with the important residues, which were classified into group B.
62 compounds of group A were classified into 7 subgroups according to their similar structure and position ( Table 1, 2).  In general, there are some comments for the compounds of group A: -6'-hydroxy-aurone derivatives (subgroup 2A) and 4,6-dihydroxy-aurone (subgroup 3A-a) were more able to interact with the catalytic cavity than other derivatives. Because the hydroxyl substituent (OH) polarized and stayed in a favorable position for creating hydrogen bonding. In the same position, the methoxy substituent (OMe) gained the results not as good as the results of the OH substituents.
-The aromatic ring with multi-function group substituents in position 4 of the benzylidene ring (subgroup 1A) both increased the ability to create bonds and increased the length of the structure but did not hinder the compound binding to the catalytic cavity.
-The compounds had the small substituents (OH, CH3, OMe, halogen) in the benzylidene ring (subgroup 6A), whose the results of docking were not much different than the results of docking aurone frame.
The OH substituent at position 6' of the benzylidene ring created an additional hydrogen bond with Ser152.
Subgroup 3A (Table 1): Subgroup 3A-a had an additional OH substituent at position 4, which could make hydrogen bonds with important residues such as Ser152, Phe77, His263 to increase the interaction with the catalyst cavity. Therefore, the compounds with OH substituent at position 4,6 had better docking scores than those with OMe substituents.
The substituents and their positions of subgroups 4A, 5A, 6A, and 7A (Table 2) did not change the structure of the aurone frame much. Therefore, the compounds of these groups insided the catalytic cavity and their bonds with important residues (Ser152-Asp176-His 263 and Phe77) were made up of the aurone frame.
Subgroup B: Subgroup B consisted of 20 compounds, which did not interact with the important residues (Ser152, His263, Phe77), having structural frame types such as (Table 3)  Table 3 showed that the compounds of subgroup B had many adjacent methoxy substituents or the branching substituents, which made the compounds more bulky, difficult to penetrate the catalytic cavity, and did not interact and create bonds with the important residues (Ser152, His163, Phe77).

A32 compound as the potential compound for the treatment of obesity
In the 82 compounds after carrying out molecular docking, the A32 compound ((Z) -5-chloro-2-(4-

(2-(4-methoxyphenyl)-2-oxoethoxy) benzylidene) benzofuran-3(2H) -on) had the best docking result
with protein (PDB ID: 1LPB) (docking score: -10.6 kcal.mol -1 ) and created the bonds with the catalytic trial Ser152-Asp176-His263 and Phe77. According to 5-Lipinski rule, the structure of A32 compound fitted the criteria Table 4), so A32 was suitable for oral drug research. A complex of protein 1LPB-A32 was selected to run molecular dynamics simulation, which investigated the stability of protein-ligand complex under the influence of the environment.

Results of molecular dynamics simulation (MDs)
A complex of protein 1LPB-A32 was run molecular dynamics simulation in 10 ns. At the same condition, the protein without ligand A32 (apoprotein) was also conducted MDs to compare with the results of protein-ligand complex. The results were analyze following the parameters: 3.3.1. Stability of protein during dynamics simulation (RMSD: Root-mean-square deviation) The protein structure of the complex with the A32 ligand was more stable than apoprotein in 10 ns of MDs. When starting to 7ns, the RMSD value of the protein in the complex ranged from 1.0 Å to 2.5 Å. The amino acids of apoprotein fluctuated more than the residues of proteins in the complex with A32. Amino acids in the binding region, which created interaction with A32 ligand, had relatively stable with the RMSF value less than 2.0 Å. Especially, residues Ser152, His263, and Phe77 all had RMSF less than 1.0 Å (Figure 4)

Occupancies of hydrogen bonds between protein 1LPB and the A32 ligand
The occupancies of hydrogen bonds between the A32 ligand and protein 1LPB, which were determined by VMD software (d ≤ 3.5 Å and α ≤ 120o *26+). The hydrogen bonds with residues His263 and Phe77 had high expression occupancy, respectively 98.60% and 88.72%. Especially, the occupancy of hydrogen bonding with Ser152 was up to 100.00%. The data proved that the A32 ligand bound well and stable in the catalytic cavity of the HPL during 10 ns of MDs and always interacted with Ser152. The results showed that the compound A32 had potential inhibiting HPL.

Radius of gyration (Rg)
During 10 ns of the process dynamics simulation, the Rg value of the protein in the complex with A32 and apoprotein were average about 2.6 nm, which presented that the protein retained a stable structure during MDs ( Figure 6).
A B   Especially, the hydrogen bond with Ser152 was stabilized during the 10 ns MDs, indicating that A32 was a potential compound for resistance to HPL. Therefore A32 compounds could be synthesized and tested in vitro as new inhibitors for the anti-HPL effect. Funding: This research was funded by the NAFOSTED, Viet Nam with the research title "Sàng lọc một số dẫn chất flavonoid tổng hợp có tiềm năng ức chế lipase tụy hướng phát triển thuốc điều trị béo phì".