Thermofluor has become a well-known and widely practiced methodology for screening of ligands that enhance stability and solubility of proteins, and also a powerful tool for hit identification in early drug discovery. In the present work, we developed an alternative Thermoflour method, named ThermoFMN, which explores the endogenous prosthetic group flavin mononucleotide (FMN) of flavoproteins as the fluorescent probe. Validation of ThermoFMN method was achieved by monitoring fluorescence signal of FMN of several drug targets in the presence of an unbiased library of ligands. In addition, drugs with known efficacy had their selective inhibition profile evaluated. Besides demonstrating that FMN signal provides sufficient fluorescence intensity for detection, our results revealed that ThermoFMN assay requires low concentration of protein samples and is compatible with a wide range of chemical reagents. The methodology presented here proposes an alternative strategy in the search for ligands of FMN-binding drug targets, therefore an important tool for the development of new therapies against neglected diseases.
FMN; Thermofluor; drug targets; ThermoFMN; drug discovery; dihydroorotate dehydrogenase
