Identificação in silico dos alvos proteicos de novos nematicidas: tioxazafeno e fluazaindolizina

Abstract

Plant-parasitic nematodes (PPNs) are microscopic organisms that inhabit soil and plant tissues. Among these organisms, those of the genera Meloidogyne and Heterodera stand out because they affect more than 2,500 different plant species, with economic losses exceeding US$ 100 billion per year. In Brazil, these nematodes constitute a notable threat to the country's progress and food security. The most popular methods for the control of PPNs are based on the use of chemical nematicides. However, animal and environmental safety laws have limited their use. As a result, new nematicides such as tioxazafen (NemaStrikeTM) and fluazaindolizine (SalibroTM) have emerged. Although they are considered safe, their mechanisms of action are not yet known. Consequently, the main objective of this work was to identify the possible enzymatic targets of these nematicides. For this, an in silico approach was employed, which included: pharmacophore search, protein molecular modeling and molecular docking. In the search for proteins inhibitors pharmacophorically similar to

tioxazafen and fluazaindolizine, two compounds were selected: BXZ (4-bromo-6-(6-hydroxy- 1,2-benzoxazol-3-yl)benzene-1,3-diol) and EW0 (7-chloro-4-(3-methoxyanilino)-N-(4-

methoxyphenyl)sulfonyl-1-methylindol-2-carboxamide) with Tanimoto scores of 0.52 and 0.40, respectively. BXZ is a potent inhibitor (IC50 = 190 nmol/L) of Hsp90-type chaperones; whereas EW0 is a strong inhibitor of the key enzyme of gluconeogenesis, fructose 1,6- bisphosphatase (FBPase; IC50 = 29 nmol/L). On Hsp90 not produced by PPNs, molecular docking results showed that tioxazafen binds to such enzymes with the same affinity energy as BXZ (-7.5 kcal/mol), suggesting that tioxazafen may act as an inhibitor of Hsp90-type chaperone enzymes. On the other hand, in Hsp90 of H. glycines the affinity results depended on the conformational state of the N-terminal domain (NTD) of Hsp90 modeled from the selected sequences. In Hsp90 models with the NTD region in closed state there was no statistically significant difference when comparing the affinity energy with that obtained in mammalian Hsp90; however, in Hsp90 models with the NTD region in open state the affinity energy was statistically different (-6.5 kcal/mol). Fluazaindolizine, on the other hand, bound to FBPase enzymes not produced by PPNs with a half affinity energy of -8.0 kcal/mol. In FBPases produced by Meloidogyne graminicola and Meloidogyne enterolobii, and which were modelled until their quaternary structures were obtained, the calculated affinity energies were not statistically different from those obtained in FBPases not originating from PPNs. Compared with potent FBPase inhibitors, fluazaindolizine was among the three best affinity results recorded in both docking software employed. In conclusion, the methodology employed here shows that the results obtained are in agreement with the pharmacophore characteristics of the compounds BXZ and EW0. The tioxazafen, similarly to other benzisoxazoles has the necessary structural characteristics to act as competitive antagonist of ATP in Hsp90 enzymes. On the other hand, fluazaindolizine, like other sulfonylcarbaxamides, has the necessary qualities to be a potent inhibitor of FBPases enzymes.