RESUMO
Bacterial Topoisomerase I is a potential target for the identification of novel topoisomerase poison inhibitors that could provide leads for a new class of antibacterial compounds. Here we describe in detail a fluorescence-based cleavage assay that is successfully used in HTS for the discovery of bacterial topoisomerase Ι poisons.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Escherichia coli/enzimologia , Inibidores da Topoisomerase I/síntese química , Yersinia pestis/enzimologia , DNA Bacteriano/química , Descoberta de Drogas , Escherichia coli/efeitos dos fármacos , Fluorescência , Conformação de Ácido Nucleico , Relação Estrutura-Atividade , Inibidores da Topoisomerase I/química , Inibidores da Topoisomerase I/farmacologia , Yersinia pestis/efeitos dos fármacosRESUMO
The thermodynamics of interaction of neomycin and lincomycin with bovine serum albumin (BSA) and human serum albumin (HSA) has been studied using isothermal titration calorimetry (ITC), in combination with UV-visible, steady state and time resolved fluorescence spectroscopic measurements. Neomycin is observed to bind weakly to BSA and HSA whereas lincomycin did not show any evidence for binding with the native state of these proteins, rather it interacts in the presence of surfactants. The ITC results suggest 1 : 1 binding stoichiometry for neomycin in the studied temperature range. The values of the van't Hoff enthalpy do not agree with the calorimetric enthalpy in the case of neomycin, suggesting conformational changes in the protein upon ligand binding, as well as with the rise in the temperature. Experiments at different ionic strengths, and in the presence of tetrabutyl ammonium bromide and surfactants suggest the predominant involvement of electrostatic interactions in the complexation process of neomycin with BSA and HSA, and non-specific interaction behaviour of lincomycin with these proteins.
