A Molecular Docking of New 9β-Halogenated Prostaglandin Analogs with an Ester Group at C-6 Atom of the α-Side Chain

Prostaglandins with cytoprotective activity were studied for a long time and a few PGE1 and PGE2 stable analogs were promoted as drugs: arbaprostil, enprostil, misoprostol and rioptostol. Nocloprost, a 9β-chlorine prostaglandin analog, has been also promoted as a cytoprotective drug; the succes with this compound stimulated the reserches, and many 9βor 11β-substituted prostaglandins were synthesized and studied for their biological activity. In the same dirrection we previously synthesized new 9β-halogenated prostaglandins having also an ester group at the carbon atom 6. These compounds were now used in a molecular docking study to predict their potential cytoprotective (anti-ulcer) activity. The study has been done with CLC Drug Discovery Workbench 2.4. software and an oxidoreductase enzyme receptor, chosen from the Protein Data Bank, ID: 4KEW. Two recognized drugs, omeprazole (co-crystallized with the enzyme) and nocloprost were used as standard in the study. The 9β-halogenated prostaglandin analogs were finally docked. Nocloprost and all 9βhalogenated compounds had docking score greater than that of omeprazole. The majority of the 9βhalogenated analogs have a docking score even greater than that of nocloprost, indicating that these compounds could have potential cytoprotective activity. Correlations between docking score and substituents on the prostaglandin skeleton have been done.

9-Halogenated prostaglandins mimic the 9-keto group and acts at the receptors of PGE2 (Nocloprost is in this group of prostaglandin analogs) and 11-halogenated prostaglandins have great affinity for receptors of PGD2, like for example compound ZK 110841. In the same dirrection we previously synthesized 9β-halogetaned prostaglandins and the results were published in a paper [15] and a patent request [16]. Brossing the literature, a molecular docking study of the 9-halogenated prostaglandins had not been found for predict their cytoprotective (anti-ulcer) activity.

Materials and methods
The docking studies have been realized according to the docking protocol [18]: importation of the protein/enzyme receptor, preparation of the protein receptor, setup the binding site and setup the binding pocket, introduction of the ligands in the "Molecule Project", docking the ligands in the active binding site, extraction of the docked ligands in to a "Molecule Table", calculation of the molecular properties of the ligands, screening the docking results. The validation of the method and of the docking parameters acquired from the molecular docking studies, have been carried out by redocking the co-crystallized docked in the active binding site of the protein receptor. The docking score and the hydrogen bonds established with the amino acids from the group of interaction are used to predict the binding modes, the binding affinities and the orientation of the docked compounds in the active site of the protein/enzyme receptor. The molecular properties of the small molecules, such as parameters of the Lipinski's rule of five: the molecular weight, number of hydrogen bond donors, number of hydrogen bond and Log P (octanol-water partition coefficient), have been calculated using the "Calculate Molecular Properties" tool [22].

3.Results and discussions
The paper was taken in the study a molecular docking to predict the cytoprotective (anti-ulcer) activity of the 9β-halogenated prostaglandins of type 2 and 3. The compounds were obtained by oppening the δ-lactone group of compounds 1 with diols (n = 0 to 4), or 2-butyn-1,4-diol, catalyzed by toluenesulfonic acid (Scheme 1), with the formation of an ester having an alcohol group at C-1 (prostaglandin numbering) spaced to carboxylic group by two to six methylene group or a 2-butyn group. The compounds have not only a 9β-halogen (Cl, Br and F) to act at the PGE2 receptors, but also an ester group at the carbon atom 6. Such an ester group was presented in the literature and the PGE2 type compounds showed cytoprotective activity [17] and the synthesized compounds presented in Scheme 1, having both modifications in the molecule, are waiting to have also cytoprotective activity. 2a, Hlg = Cl, X = Cl, n = 0 2b, Hlg = Cl, X = Cl, n = 1 2c, Hlg = Br, X = Cl, n = 1 2d, Hlg = F, X = Cl, n = 1 2e, Hlg = Cl, X = Cl, n = 2 2f, Hlg = Cl, X = CF 3 , n = 2 2g, Hlg = Br, X = Cl, n = 2 2h, Hlg = F, X = Cl, n = 2 2i, Hlg = Cl, X = Cl, n = 3 2j, Hlg = Cl, X = Cl, n = 4 2k, Hlg = Cl, X = Cl, n = 2, R 1 = OH, R 2 = H 2l, Hlg = Cl, X = CF 3  To put in evidence their predicted cytoprotective activity, we have done a molecular docking study, using CLC Drug Discovery Workbench 2.4. software [18]. It is to be mentioned that in the literature we didn't found a similar molecular docking for Nocloprost or for other 9 or 11-halogenated prostaglandins.
The affinity of a compound to an identified protein or enzyme target is consider a relevant parameter in the process for development of a new drug. The prediction of the mode of binding of the ligand (generally, compounds in study) to the target (protein/enzyme) by molecular simulation could allow the restricting of the organic synthesis to the most promising chemical compounds.
We chose the 9β-halogenated prostaglandin analogues 2a-2l and 3a-3d, presented in Scheme 1, for the molecular docking study. The computational molecular simulation was performed to determine the affinity and orientation of the compounds and their mode of binding to an oxidoreductase enzyme receptor, chosen from the Protein Data Bank, ID: 4KEW. (www.rcsb.org) [19]. In the study we used as standard two recognized drugs, omeprazole (co-crystallized with the enzyme) and nocloprost (F2α prostaglandin analogue with a 9β-chlorine atom instead of 9α-OH group, Figure 1).
As usual, the binding site and binding pockets, used in the molecular docking of the ligands, were well established, and the search was carried out inside the binding site volume ( Figure S1a, green sphere). The protein receptor, ID: 4KEW was loaded from Protein Data Bank, water molecules were removed, omeprazole co-crystallized was extracted, the binding site and also the binding pocket (which play an important role in orientation during molecular docking) were defined. The cocrystallized omeprazole was reposed in the protein pocket, docking validation and hydrogen bonds between co-crystallized omeprazole and amino acid residues of receptor were done. The prostaglandin analog drug nocloprost, used as standard in the study, then has been docked and the results are presented in Table 1 (entry 2); docking pose of the interactions between nocloprost and the amino acid residues are presented in Figure 2. The docking score (PLANTPLP score) is a function described in Korb et al. [20]. "For a strong binding, the score has a negative value; for weak or non-existing binding, the score has a less negative or even positive value" [21].  Table 1.
-by reduction of 15-keto group to 15α-OH group, the docking score increase: -74.67 docking score of 2e increase to -75.03 for 2k and -74.92 docking score for 2f increase to -77.57 for 2l; the same is for 2-butyn synthon of compounds 3a-3d: -75.09 for 3a increase to -82.27 for 3b and -77.47 for 3c increase to -84.91 for 3d.
An expressive presentation of the docking score is presented in Figure 3: Besides the parameters mentioned in Table 1, group interaction, hydrogen bonds of ligands with amino acid residues were determined and hydrogen bond length was calculated. These are presented in the Table 2.
The docking poses of the ligands with the best score interacting with the amino acid residues of the protein are presented as follows: 3d in Figure 4, 3b in Figure 5, 3c in Figure 6 and 2l in Figure 7.  In fact, majority of 9β-halogenated ligands were found to have the same orientation with that of cocrystallized omeprazole and nocloprost, as can be observed in the Table 2. The compounds 2b, 2d, 2g, 2h and 2i adopted a different orientation than that of the co-crystallized and nocloprost.

Conclusions
Prostaglandin analogues substituted with a 9β-halogen and with an ester group with a diol at the C-6 carbon atom, previously synthesized, were used in a molecular docking study to determine their potential cytoprotective (anti-ulcer) activity. The study has been done with CLC Drug Discovery Workbench 2.4. software and an oxidoreductase enzyme receptor, chosen from the Protein Data Bank, ID: 4KEW. (www.rcsb.org). In the study we used as standard two recognized drugs, omeprazole (cocrystallized with the enzyme) and nocloprost. The 9β-halogenated prostaglandin analogs 2a-2l and 3a-3d were finally docked. Nocloprost and all 9β-halogenated compounds had docking score greater than that of omeprazole. The majority of 9β-halogenated analogs have a docking score greater than that of nocloprost, with the exception of the compounds 2a, 2c, 2g and 2j. The compounds 2b, 2d-2f, 2h-2i, 2k-2l, 3a-3d had a greater docking score than that of nocloprost; this indicate that the compounds could have potential cytoprotective (anti-ulcer) activity. A few correlations between docking score and substituents on the prostaglandin skeleton have been done.