RESUMO
This work aimed to evaluate the inhibition of Candida rugosa lipase by five guanylhydrazone derivatives through biological, biophysical and theoretical studies simulating physiologic conditions. The compound LQM11 (IC50â¯=â¯14.70⯵M) presented the highest inhibition against the enzyme. Therefore, for a better understanding of the interaction process, spectroscopic and theoretical studies were performed. Fluorescence and UV-vis assays indicate a static quenching mechanism with non-fluorescent supramolecular complex formation and changing the native protein structure. The binding process was spontaneous (ΔGâ¯<â¯0) and electrostatic forces (ΔHâ¯<â¯0 and ΔSâ¯>â¯0) played a preferential role in stabilizing the complex ligand-lipase. The compounds were classified as non-competitive inhibitors using orlistat as a reference in competition studies. Based on the 1H NMR assays it was possible to propose the sites of ligand (epitope) that bind preferentially to the enzyme and the theoretical studies were consistent with the experimental results. Finally, LQM11 was efficient as a lipase inhibitor of the crude intestinal extract of larvae of Rhynchophorus palmarum, an important agricultural plague, showing potential for control of this pest. Within this context, the real potential of this biotechnological application deserves further studies.
Assuntos
Candida/enzimologia , Inibidores Enzimáticos/farmacologia , Hidrazonas/farmacologia , Lipase/antagonistas & inibidores , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Animais , Biotecnologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Hidrazonas/química , Hidrazonas/isolamento & purificação , Lipase/metabolismo , Estrutura Molecular , Termodinâmica , Gorgulhos/químicaRESUMO
The interaction between bovine serum albumin (BSA) and thimerosal (TM), an organomercury compound widely employed as a preservative in vaccines, was investigated simulating physiological conditions and using different spectroscopic techniques. The results, employing molecular fluorescence showed the interaction occurs by static quenching through electrostatic forces (ΔHâ¯<â¯0 and ΔSâ¯>â¯0), spontaneously (ΔGâ¯=â¯-4.40â¯kJâ¯mol-1) and with a binding constant of 3.24â¯×â¯103â¯M-1. Three-dimensional fluorescence studies indicated that TM causes structural changes in the polypeptide chain of the BSA, confirmed by circular dichroism that showed an increase in α-helix (from 43.9 to 47.8%) content after interaction process. Through synchronized fluorescence and employing bilirubin as a protein site marker, it was confirmed the preferential interaction of TM in the subdomain IB of BSA. The interaction mechanism proposed in this work is based on the reaction of TM with BSA through of free Cys34 residue, forming the adduct BSA-HgEt with the thiosalicylic acid release, which possibly interacts electrostatically with positive side chain amino acids of the modified protein. Finally, it was proven that both TM and EtHgCl accelerate the protein fibrillation kinetics in 42 and 122%, respectively, indicating the toxicity of these compounds in biological systems.