This study is a continuation of our previous results published [1] . The same structures were used. The full details of the preparation of the structures are published in Cabrera et al. [1] .

Similar to the previous work [1] before docking of the halogenated structures of the Figure 1, the docking protocol was validated by re-docking of the co-crystal ligands in their corresponding crystallographic structure. As reference, Table 1 shows the ligands and their respective enzymes used in validation. For this validation were considered semi-flexible the three co-crystal ligands (R165481, R157208 and GS9137) and was done with the MMFF94x force field with the

default settings of MOE [16] (e.g., 500 iterations in total with 30 consecutive attempts to select the best result). The binding pocket was defined as the set of amino acids within of 4.5 Å of the co-crystal ligand.

The flexible alignment of the new quinolones structures was performed to explore if these new structures could adopt similar conformation as the co-crystalized pyridinone analogues. Similar to our previous work, we selected a sample of 10% (32 structures) and each was aligned flexibly to the co-crystal coordinates of R157208 and R165481. For this study, the structure of the co-crystal compound was kept rigid. The docking was conducted with MOE maintaining the default settings (500 iterations in total with 30 consecutive attempts to find the best result) with the MMFF94x force field.

In order to explore the potential oral bioavailability of the structures proposed, we calculated the pharmaceutical properties molecular weight (MW), the partition coefficient octanol/water (Log P) as a measure of lipophilicity, topological polar surface area (TPSA), number of hydrogen bond donors (HBD), number of hydrogen bond acceptors (HBA), and number of rotatable bonds (RB) [17] [18] .

The coordinates obtained in our previous work [1] of the structures aligned and superposed were used to run the docking using 320 structures of pyridinone derivates. The structures selected for this study were PDB ID: 2BAN and 2B5J [13] .

Before docking the newly structures proposed in this work (Figure 1), the docking protocol was validated as described in the Methods section. The root-mean square deviation (RMSD) values between the co-crustal and predicted positions were low: the RMSD values were 0.8222 and 0.7393 for 2BAN and 2B5J, respectively. The relative docking scores obtained were −9.1224 and −8.6151 for 2BAN and 2B5J, respectively. These results indicate that the settings used in the docking are correct to reproduce the binding modes shown in the crystal structure.

A sample of 10% of the data (32 structures) was taken of the 320 structures in a scheme of stratified random sampling. The structures were aligned flexibly with the position of crystallographic structures of R157208 and R165481 (Table 1). In the analysis of the results, the best values were obtained for the structures aligned with R165481. The results were not so different to those obtained in the previous work [1] . In this study, the values of alignment of the new structures with R157208 were less negative than the values of the alignment of the same structures but with R165481. The 32 structures with R157208 had an average of alignment energy of −80.05 Kcal/mol with a standard deviation of ±9.58 Kcal/mol. The average of alignment energy for R165481 was −83.40 Kcal/mol and standard deviation of ±11.14 Kcal/mol (Table of 3D alignment results in supplementary material as Table S1). As an example of the alignment, Figure 3 shows the 3D alignment of structures 180 and 270 with R157208 and R165481, respectively. The results of the alignments of structures 180 and 270 are close to the average values and are possible to observe the good overlap with reference structures.

After the docking assay performed with MOE for 320 new molecules using PDB ID: 2BAN, we select 72 structures maintaining interaction with important aminoacids for this investigation. The contacts of interest observed were between the substituent in C-4 of each molecule with Tyr188 and the conserved aminoacids Trp229; and the interaction of substituent in C-3 with conserved aminoacid Pro236 due to a 180˚ rotation of the molecule but always maintaining the characteristic interaction of pyridinones and other non-nucleoside RT inhibitors [19] [20] [21] between hydrogen in N-1 and the oxygen of Lys101 (Figure 4). Pro236 was the aminoacid with most contacts with the ligand. Another common interaction observed with the docked molecules was with the chlorine atom in C-8 and the oxygen of the carbonyl group in Pro236. The distances shown for the structures that have halogen bond are between 2.9 and 3.5 Å.

Also, is important to indicate that as result of docking we could identify some structures with double interaction with aminoacids of interest because at the same time show the possibility to have contacts with Pro236 and Tyr188 (structures 152 and 181); and contacts with Pro236 and Trp229 observed in the structures 123 and 158 (Figure 5). The structures mentioned have good results of score as we can see in the Table 2.

In general, Table 2 highlights seven structures with the best results and 10 structures with less favorable results. This distinction is based on the relation of average and standard deviation in the docking assay with PDB ID: 2BAN.

Structures with multiple interactions with aminoacids of interest have better possibilities as inhibitors according with the docking with 2BAN.

The results of docking of 320 structures with PDB ID: 2B5J helped us to identify

64 structures with contact with aminoacids of interest as Trp229, Pro236, Tyr181 and Tyr188. The average of score as result of the docking was −7.0418 with a standard deviation of 0.897 in this study. Taking in count the average and the standard deviation, there are 10 outstanding molecules and seven molecules with lower priority (Table 3). In general, the most common contact of the ligands is with Trp229.

Docking with the structure PDB ID: 2B5J revealed that compounds 7 and 33 have the possibility to interact with two aminoacids simultaneously with the substituent in C-4 that can confer to the ligands maintain activity against RT. Figure 6 shows that the substituent in C-4 of compound 7 interacts with Trp229 and Tyr188 at same time while a related substituent at the equivalent position of compound 33 interacts with Tyr181 and Tyr 188.

Another interesting interaction that we observed in docking with quinolones 266 and 270 was a halogen interaction between Iodine and Pro236 as is shown in Figure 7 for quinolone 270. The conformation adopted by the ligand enables to contact with Lys101 that is the characteristic interaction of pyridinone.

In the docking performed with PDB ID: 2B5J we could observe less halogen interactions than the docking with PDB ID: 2BAN. The contacts with chlorine in position C-8 are with Lys103. This kind of contact helps to fix the quinolone to

the allosteric site and the flexibility of the substituent in C-4 let the ligand to make contact with Tyr181 or Trp229 (Figure 8). As example of this, we show docking of 58.

Another interesting result was observed with the docking of 143 (Figure 9),

which at same time show interaction with Tyr188 and pro236 with C-4 and C-3 substituents respectively. So, if the interaction with Tyr188 is lost because a mutation the compound could maintain activity against RT because Pro236, which is an aminoacid, conserved.

We found 21 structures that coincide in Table 2 and Table 3; this could mean that they have a better probability to be RT inhibitors. Some structures have contact with conserved aminoacids, as Trp229 and Pro236 mentioned by Li et al. [5] which is favorable because this evidence give more probability to think that this contacts could help in the activity of the compounds. In the Table 4 are

shown the 21 structures and the aminoacid of contact depending of PDB structure of RT.

In order to validate the docking with IN, we re-docked the co-crystal ligand in PDB ID: 3L2U (Figure 10), Elvitegravir. The binding mode was reproducible by MOE with a RMSD value of 1.3 Å (similar to our previous work [1] ).

We docked 73 selected quinolone structures that showed key interactions in the docking models with PDB ID: 2BAN and 2B5J, respectively. The docking with IN was done with the structure of IN PDB ID: 3L2U (Structures of quinolones in supplementary material as Figure S1). The quinolones have ester or carboxylic acid groups at C-3 and carbonyl group in C-2. Similar to the quinolones, Elvitegravir has a carboxylic acid at C-3 and a carbonyl group at C-4. In Table 5 is

summarized the results of 14 structures with potential to be dual inhibitors of RT and IN.

In Table 5 we can see how quinolone 138 have the best result of score docking (−6.686) with PDB ID: 3L2U and have similar interaction to Elvitegravir with the two Mg⁺⁺ ions (Figure 11). In the Figure 11, we can see in yellow color the quinolone overlapped on Elvitegravir (green color). The distances and the space IN-DNA between the Mg⁺⁺ ions and the oxygens of quinolone has an average of 2.5 Å according with MOE. The distances between Mg⁺⁺ ions and Elvitegravir in MOE are near to 2 Å.

The less favorable result in docking with 3L2U is for quinolone 82 because have a score of −4.031. The distances between oxygens in quinolone and the Mg⁺⁺ ions have an average of 2.6 Å. The Figure 12 show the interactions of quinolone and Mg⁺⁺ in the space between IN and DNA.

As result of the entire analysis of docking with RT and IN we got 14 quinolones that could act like dual inhibitors. In Figure 13 is showing the five best structures that could be dual inhibitors. The first five structures are recommended to be synthetized according with the score results and the contact with Mg⁺⁺ ions and aminoacids of interest in the space between IN-DNA and the allosteric space in RT, respectively.

The structures identified as potential compounds with activity against RT and IN were evaluated in base to the rules of Lipinski and Veber, which comprise six pharmacological properties of interest. The pharmacological properties calculated to 73 structures were MW, Log P, HBD and HBA, RB and TPSA. The results are included in supplementary material (Table S2).