The enzymes used were obtained from the protein data bank (PDB) with codes 7CB1 (Chitinase B), 3N9K (1,3-β-Glucanase), 6BN0 (AVR4) and 4B8V (ECP6) [27] [28] [29] [30]. They were processed with the Protein Preparation Assistant [31] [32] using the Schrödinger Suite. Structural integrity was reviewed and adjusted; missing recurrences and loop segments near the active site were added by Prime. Hydrogen atoms were added to each protein to returned it to its original state. The protonation and tautomeric states of Asp, Glu, Arg, Lys and His were adjusted to a pH of 7.4. The water molecules were removed within a 5 Å sphere at the active site. The orientation of the hydrogen bonds was adjusted around the active site using PROPKA at a pH of 7.4. Finally, the protein-ligand complex was minimized using the OPLS4 force field [33], with a convergence of heavy atoms with an RMSD of 0.3 Å.

Reference ligands (substrates, inhibitors, endogenous ligands) were obtained from crystals. 22-oxocholestane steroidal derivatives were designed according to the 2D Sketcher program of the Master program in Schrödinger Suite, and converted to their most stable 3D conformer. For molecular docking, ligands were prepared using LigPrep [34] from Schrödinger Suite. After of 3D structures were generated, the OPLS4 force field and the loads were prepared in all stages. All possible protonated centers and ionization states were calculated for the scaffold using ionizer at pH 7.4. The stereoisomers were retained according to their original structures limited to 32 isomers for each ligand. Tautomeric states were generated for each group. The conformers with the best energy were selected for each ligand.

Molecular docking between catalytic sites and substrates was performed using the Glide module [35] [36] from Maestro 12.2 and the receptor grid for each target was prepared using OPLS4. Each grid was built based on co-crystallized reference inhibitors or ligands. The softening of the non-polar parts of the receivers was carried out by scaling the van der Waals radii by a 0.8 factor. Atoms were considered as nonpolar if their absolute partial atomic charge was determined to be <0.25. In flexibility, additional ligand rotations were allowed for the hydroxyl groups at Ser, Thr, and Tyr, and the thiol group at the Cys residues. Furthermore, the lowest binding position of each ligand was maintained. Slip coupling scores were performed in three high-throughput virtual detection modes (HTVS), standard precision (SP), and additional precision (XP). Before SPGP were coupling, the coupling with reference molecules of the respective target proteins was performed to validate the coupling protocol by limiting RMSE as heat cutoff < 2.0 Å in all proteins in the validation process. The XP mode to dock was applied.

Chitinase B is a chitinolytic enzyme responsible for the regulation of chitin levels in the invading fungus. Chitin is an essential protein (natural substrate) for fungus proliferation. Herein, the first objective is to evaluate a DS between SPGP and Chitin, compared to the reference inhibitor, to delimit its potential. It was found that both Chitin and SPGP can be placed in the catalytic site; this fact validates the docking and the inhibitory potential of the steroid derivatives. The steroidal nuclei are hydrophobic compared to Chitin fragment one, as confirmed in Figure 4. The potential inhibitory effect can be observed with interacting amino acids, showing the formation of characteristic hydrogen bridges with TRP97, TRP145 and ASP215. These interactions can be observed in both the chitin fragment and the reference inhibitor. Most of the steroidal derivatives present hydrogen bridges with TRP97, even that the compounds with the best DS also present with TRP145, and in some cases with others such as GLH144 (Figure 4).

The van der Waals and π-alkyl interactions are due to the acetyl groups of the substrate; this fact reveals importance highlighting the lipophilic character of the SPGP derivatives. The hydrophobic caracter of the rings let to observe interactions with with PHE191, TYR145, TRP97 and PHE190 in the case of SPGP13, SPGP7 and SPGP1 (Table 2). SPGP7 there is a hydrogen bridge with TRP220, as well as SPGP12, which has better DS than chitin but not than the reference inhibitor, demonstrating the importance of this residue.

The main function of 1,3-β-Glucanase lies in the hydrolysis of these glucans, necessary for the adaptation-proliferation process of phytpathogen. It is a so specific enzyme that there are only a few selective inhibitors, such as Apegin. The formation of a hydrogen bridge between the residue TYR29 and GLU192 is crucial in the catalytic process of the breakdown of b-glucan into monosaccharide units.

The hydrophobic nature of the steroid nuclei hinders the interaction with the catalytic site of the enzyme (Figure 5). SPGP7 and SPGP15 interact in a polar way with the amino acids GLH 27 and ASP 145 due to its polyoxygenated functions. The latter is important to be underlined in the glucanase hydrolysis process, having a mechanism like Apegin, with the advantage that these SPGP interact with PHE258 and ASN191. SPGP15 also interacts with PHE144. For the case of phytopathogen glucanase-dependent SPGP7 and SPG15, demonstrated the need for a high polarity to bind to this enzyme. Chitinase B, that Chitin, by having acetyl groups, the residues in the protein have a hydrophobic character.

The rest of SPGP (1 - 6, 8 - 14) have docking scores between −6.0 kcal/mol to −4.0 kcal/mol (Table 3). These values are not comparable to glucans or commercial inhibitors. This is not necessarily negative, since 1,3-β-Glucanase is present not only in phytopathogens, but also in beneficial fungi for plant development, which allows the use of these SPGPs with a plant growth promoting effect without altering the symbiosis with the kingdom fungi.

Given that the regulation observed is via Chitinase B, it was interesting to study the effectors by the fungus necessary for fungal proliferation, with ECP6 and AVR4 being critical for the transport and proliferative process via Chitin, an indirect regulation option is by allosterically blocking the site. of transport that is located between two chains both in the ECP6 and the AVR4 (Figure 6(a) and Figure 6(d)). At the energy level, we can observe that only 2 compounds have a higher energy than the chitin fragment in the case of AVR4, but in ECP6 no compound has a competitive energy. At the spatial level we can see that the Chitin fragment in both cases is in the middle of the two chains, the SPGP in all cases bind at that site (Figure 6(b) and Figure 6(e)), but particularly in the case of ECP6 this region inter-chains are highly hydrophilic, so the steroidal nucleus being lipophilic does not allow a strong interaction with the site, although acetates and hydroxyls form hydrogen bonds is not enough to compare with chitin. In the case of AVR4, although chitin is hydrophilic, the site is not completely hydrophilic, presenting interaction with the nucleus and with a carbonyl or a enol at C-6 for SPGP1 and SPGP6 (Figure 6(c) and Figure 6(f)).

At the specific level for AVR4, the compounds interact at the particular site and are supported with an increase in van der Walls and π-alkyl interactions, as can be seen in Table 4, the amino acids for the chitin fraction are repeated in

interaction for derivatives SPGP, but in the formation of hydrogen bridges is where it is key for SPGP1 and SPGP6 since they interact with TYR D: 67, as well as annexing TYR C: 67, ASP C: 102 and LYS C: 98, which its increases the coupling energy allowing it to compete in the case of SPGP1 at the same energy level and for SPGP6 with a better DS than the endogenous ligand, thus allowing the AVR4 effector to be blocked.