Mapeamento de interações entre compostos biologicamente ativos com urease: desenvolvimento de inibidores de urease com potencial aplicação clínica e agrícola

Abstract

The inhibition of urease activity can be explored in several areas, and mainly in the clinical and agricultural context. Thus, efforts have been made to develop effective inhibitors for this purpose. Thus, this work proposes to evaluate the activity of julolidine and amino acid derivatives (with thiourea nucleus) as in vitro urease inhibitors. Kinetic studies indicated that the RS12-J2 derivative of the julolidine class acted as a competitive inhibitor, while the other derivatives as mixed inhibitors. By molecular fluorescence it was verified that the RS12 derivatives presented higher Kb values (0.10 - 7.33×106 M-1 ), with the occurrence of static quenching and non-radiative energy transfer in the interaction process. Hydrophobic interactions were the predominant forces in complex stabilization for RS11 derivatives (ΔH > 0 and ΔS > 0), and van der Waals forces and hydrogen bonds (ΔH < 0 and ΔS < 0) for RS12 derivatives. Using the RS12-J2 derivative as a model, it was verified in the 3D fluorescence, UV-vis and synchronized fluorescence studies that the interaction process led to alteration in the enzyme structure and the microregion close to the Tyr residue (active site) was more affected, corroborating the study of interaction with Ni(II) cations by molecular absorption spectroscopy. Through competition studies, it was proved that the RS12-J2 derivative acts in the active site of the enzyme. The evaluation of the inhibitory potential of the RS12-J1 and RS12-J2 compounds on soil ureases showed that the RS12-J2 derivative showed equivalent efficiency to the NBPT (commercial inhibitor) for soils S1 and S4, which have a high content of organic matter. Finally, in studies using molecular docking, it was observed that the orientation of the dihydrofuran ring plays an essential role in the coordination involving the free electron pair of the oxygen atom of the dihydrofuran ring and the Ni(II) ions. For the amino acid derivatives, the values of the inhibitory concentration (IC50) were initially determined to evaluate the potential of these compounds to inhibit the activity of Jack bean urease (model), obtaining values between 0.37 - 0.74 µM, however, no influence of enantioselectivity was verified, since the values did not differ statistically at a confidence level of 95%, this tendency was also obtained in the interaction studies for Kb values. The possible intermolecular interactions involved in the interaction process for derivatives 110-L, 110-D, 110 ac-L, 110 ac-D and 116 were van der Waals forces and hydrogen bonds and for derivatives 106 ac-L and 106 ac-D were the hydrophobic interactions. Furthermore, the occurrence of static quenching was observed, and that there was no influence of ionic strength on the interaction process at a confidence level of 95%, with the exception of compound 110-L. The 3D fluorescence and UV-vis studies indicated changes in the secondary structure after the interaction process, in which there was non-radiative energy transfer. The study by synchronized fluorescence indicated that derivatives 110 ac-L, 110 ac-D and 116 bind to the microregion close to the Tyr residue. Derivatives 110-L and 110-D interact close to Trp residues. The competition study indicated that amino acid derivatives competed for the active site, in the presence of classical inhibitors, corroborating the study of interaction with Ni(II) cations by molecular absorption spectroscopy. In tests using soil samples, it was observed that for all soils, derivatives were more potent when compared to NBPT. Thus, the derivatives of julolidines and the derivatives of amino acids showed inhibition of urease activity in vitro, and in soil samples, being possible potent urease inhibitors.