ThermoFMN - A Thermofluor Assay Developed for Ligand-Screening as an Alternative Strategy for Drug Discovery

A tecnica de Termofluor constitui uma importante ferramenta na identificacao de moleculas prototipos a farmacos. No presente trabalho, foi desenvolvido um metodo alternativo para a tecnica de Termofluor, chamado ThermoFMN, que explora o grupo prostetico flavina mononucleotideo (FMN) como sonda fluorescente. A validacao do metodo foi feita atraves do monitoramento da fluorescencia do FMN para diferentes alvos macromoleculares na presenca de uma biblioteca aleatoria de ligantes. Alem disso, farmacos com eficacia comprovada tiveram seus perfis de inibicao seletiva avaliado. Alem de demonstrar que o rendimento quântico do FMN fornece intensidade adequada para deteccao, nossos resultados revelam que o metodo de ThermoFMN utiliza-se de baixas concentracoes de proteina e e compativel com uma vasta quantidade de tampoes e aditivos quimicos. A metodologia apresentada nesse trabalho propoe uma estrategia alternativa na busca por ligantes para proteinas dependentes de FMN, como uma importante ferramenta no desenvolvimento de novas terapias contra doencas negligenciadas. 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.