Biofísica da interação entre urease com metabólitos secundários de líquens e da albumina do soro humano com cocaína e cocaetileno

Abstract

The nature and dynamics of the binding between small molecules and proteins represent an area of intense study, due to the interaction processes are related to the availability and the transport of these compounds. The interaction between secondary lichen metabolites, as the usnic acid isomers, (+)-USN and (-)-USN) and fumarprotocetraric acid (FUM) as urease inhibitors as well as cocaine (COC) and cocaethylene metabolite (CE) interacting with human serum albumin (HSA) were evaluated simulating physiological conditions. In the interaction studies between secondary metabolites of lichen and urease, the Kb (binding constant) values obtained indicated intermediate to strong binding strength between the enzyme and inhibitors, varying from 0.25 to 35.5 105 M-1 at 30ºC. The (+)-USN was the compound with higher interaction, while the FUM presented lower affinity. The quenching mechanism of the process is preferably static and the electrostatic forces (ΔH < 0 and ΔS > 0) shown the main contribution. Through synchronous fluorescence and 3D it was possible to infer that there were changes in the secondary structure of the native urease. Finally, the influence of the substrate and some classical inhibitors (thiourea, hydroxyurea and omeprazole) on the interaction between urease and lichen compounds, which interact in the active site of the enzyme, was evaluated. Finally, an inverse relationship was observed regarding Kb values and minimum inhibitory concentration (MIC) for resistant strains of H. pyroli in in vitro assays, which credential the compounds evaluated to possible candidates for the treatment of this bacteria. In the interaction studies between the alkaloids COC and CE with HSA, the binding constants obtained for the CE varied from 2.92 to 8.33×104 M-1, being greater than COC (1.15 to 3.77×104 M-1) at different temperatures. These results agreement with the resonant energy transference (FRET) study, since the distance between donor (HSA) and receptors (ligands) for CE (1.76 nm) was lower than for COC (2.25 nm). Considering the thermodynamic parameters, the COC presented preferential interactions by van der Waals forces and hydrogen bonds, whereas the CE were hydrophobic interactions. The employ of the ANS probe, which binds to hydrophobic sites of the HSA, confirmed the hydrophobic characteristics of the CE. However, the Kb value for both compounds decreased with increasing ionic strength (NaCl 25-200 mM), indicating electrostatic component in the interaction with the protein, probably due to nitrogen protonation (pKa = 8.6). Synchronized fluorescence shown that both compounds interact preferentially in the microenvironment near the Trp214 residue. The 3D fluorescence, UV-vis and circular dichroism studies indicated that there were changes in the secondary structure relative to the native protein. Besides, the HSA binding sites were evaluated using warfarin (site I), diazepam (site II) and digitoxin (subdomain IIIB) as markers, COC and CE preferentially interact at site I of protein. The evaluation of the competitors influence indicated that only Ca(II) ions there was an increase in the interaction constant, being more accentuated for the CE. Finally, based on 1H Nuclear Magnetic Resonance studies was possible to propose the epitope of the molecules. Therefore, based on results was concluded that EC has the highest affinity for HSA, justifying its longer plasma life when compared with COC, and thus prolongs the effects.