Steroidal Plant Growth Promoters vs. Phytopathogens, via Enzymatic Regulation; An in Silico Approach

Steroidal plant growth promoters (SPGP) have been continuously studied due to their high activity increasing biomass and resistance to diverse stress fac-tors. In our hands, a new SPGP family of 22-oxocholestanic compounds stands out at a comparative level to brassinosteroids (BSs). The potential activity of new SPGP against phytopathogens was studied through in silico molecular docking, these assays were performed with relevant ensymes of phytopato-gens Chitinase B and 1,3-β-Glucanase. Nine Chitinase B inhibitors and two 1,3-β-Glucanase inhibitors were proposed. The launched study analyzed the interactional and spatial level, determining the presence of interactions with key amino acids in receptors in comparison to reference inhibitors. Even more, the AVR4 and ECP6 effectors were also examined. No compound that blocks ECP6 was found; due to, probably, the influence of its highly hydrophilic environment. In the case of AVR4, two SPGP showed a better docking score (DS) than a chitin fragment (endogenous ligand); this fact demonstrates the latent potential of the 22-oxocholestanic derivatives against phytopathogens, with a specific regulation via proliferation inhibition. Moreover, this SPGP does not affect the symbiotic fungi that are beneficial for the natural plant system.


Introduction
Plant growth promoters are compounds commonly called phytohormones, which are responsible for important roles, as regulation processes for increasing biomass, flowering, resistance to stress conditions, among others. A highly active group of growth promoters are Brassinosteroids; for example, brassinolide (1) has shown a huge promoter effect than other kinds of phytohormones and resistance for abiotic stresses, this discovery has led to an increased interest in the synthesis of diverse BSs analogues, but in most cases low yields have been reported [1]- [8]. Compounds of the novel family of 22-oxocholestanes (SPGP1, SPGP3, SPGP5), (Figure 1) have been positioned as SPGP alternative due to its comparable or even more potent activity to that of BSs and moreover, can be generated through higher reaction yields. Some 22-oxocholestanes have been successfully tested in rice, red-beans, and maize [9] [10] [11]. Although these compounds have demonstrated an exceptional plant growth promoting effect in in vitro and greenhouse assays, for field tests and future applications it is necessary to predict synergistic effects on beneficial/non-beneficial phytopathogens and to study their effect on the intrinsic defense system of vegetal models.
Plants have developed several mechanisms to recognize microbial infections and respond appropriately by activating defense responses [12]. Therefore, the knowledge of the relationship between plants and the pathogenic hosts is crucial for their control [13]. It has been found that the interaction that takes place between guest and host is carried out by means of special proteins, which have effects on both cells and phenotypes of hosts [14] (Table 1). Fortunately, all plant cells possess a sophisticated surveillance system that can register and distinguish many signals of different origins, which manages to induce a more efficient and  Cladosporium. fulvum [26] effective defense response at the infection site [15].
There is a special class of proteins of low molecular mass, diverse chemical composition having a different hydrophilic character that is directly related to the pathogenic fungi activity, known as Pathogenesis-Related (PR or pathogenicity proteins), such as chitinases and 1,3-β-glucanases [16]. Another unique kind of proteins that acts as a defense mechanism in plants is named effectors, like AVR4 and EPC6 [17]. When these molecules are recognized by the plant, an unfavorable interaction between the plant and pathogens, therefore, the effectors' function has not been clearly clarified [18].
In the present work, fifteen 22-oxocholestanic SPGP, which previously showed a positive experimental results as growth promoters [9] [10] (Figure 2), were in silico studied on the effect in the two principal enzymes involved in fungal proliferation (Chitinase B and 1,3-β-Glucanase). Also, the influence against two effectors of infection in plant systems (AVR4 and ECP6) was studied, to prove the potential of SPGP in fungal pests.

Protein and Ligands Preparation
The enzymes used were obtained from the protein data bank (PDB) with codes

Results and Discussion
The synthesized bioactive SPGP, SPGP1-SPGP15 (22-oxocholestane compounds) through an acetolysis opening of the spiroketal framework of spirostan steroids  [11]. Various modifications at A, B and C rings and at C-26 were introduced ( Figure 2): at C-3 an acetoxy, or an α,β-unsaturated ketone group is found. At ring B, a double bond at C-5 or a ketone group at C-6 is present. At ring C, the presence of a carbonyl group at C-12 was studied. Ring D was derivatized by the introduction of an acetoxy or a ketone group at C-16. The 22-oxocholestane side chain was modified introducing oxygenated groups at C-26 or introducing a trans diol at C-22 and C-23; similar function present in natural brassinosteroids [37].
Two enzymes associated with fungal proliferation were studied: chitinase B, which is responsible for the regulation of chitin in the fungus, and 1,3-β-glucanase [38]. Both enzymes are associated with the hydrolysis of 1,3-and 1,6-β-glucan ( Figure 2). For the case of chitinase B, the chitin fragment has a better coupling energy than most plant growth promoters: SPGP13, SPGP7 and SPGP1 have better energy than the reference inhibitor (FO0), while SPGP12 presents the same level. For Glucanase, the SPGP15 compound has a greater affinity for the enzyme than 1,3-Glucane and Apegin, while SPGP7 has the same energy the substrate ( Figure 3).
The AVR4 and ECP6 effectors are responsible for the intramolecular regulation and protection of chitin, so the bound to the active site of this is essential for fungal non-proliferation. For AVR4, SPGP1 and SPGP6 compounds have a higher energy than the Chitin fragment that binds at the site, while in the case of  ECP6, no steroidal derivative has the potential to bound to the active site of this protein. This study is at the energy level, which in the cases of the enzyme has previously established a direct relationship between the coupling energy and the enzyme inhibition constant [39], but they should be studied accordingly to the amino acid residues interact.

Chitinase B
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 fun-   (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.

1,3-β-Glucanase
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.

ECP6 and AVR4
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  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.

Conclusion
SPGPs compounds have a huge plant growth promoting effect in various biological systems, and an effect against phytopathogens (specifically for Chitinase B and 1,3-β-Glucanase). Some SPGPs were studied in silico finding 5 competitive inhibitors better than Chitin and 4 preferred than FO0 (reference inhibitor).
While for 1,3-β-Glucanase, 2 potential inhibitors were found (SPGP7 at the level of 1,3-β-glucane and SPGP15) having a better activity than Apegin (reference inhibitor). For the blockage of chitin effectors (AVR4 and ECP6), only an allosteric blockade against AVR4 was achieved, so the 22-oxocholestan studied compounds have a latent potential as inhibitors of fungal proliferation at the enzymatic level. In conclusion, SPGPs have a potential dual action, as promoters for plant growth and as antifungal against phytopagens.