RESUMO
Native nanoelectrospray ionization mass spectrometry is an underutilized technique for fragment screening. In this study, the first demonstration is provided of the use of native mass spectrometry for screening fragments against a protein-DNA interaction. EthR is a transcriptional repressor of EthA expression in Mycobacterium tuberculosis (Mtb) that reduces the efficacy of ethionamide, a second-line antitubercular drug used to combat multidrug-resistant Mtb strains. A small-scale fragment screening campaign was conducted against the EthR-DNA interaction using native mass spectrometry, and the results were compared with those from differential scanning fluorimetry, a commonly used primary screening technique. Hits were validated by surface plasmon resonance and X-ray crystallography. The screening campaign identified two new fragments that disrupt the EthR-DNA interaction inâ vitro (IC50 =460-610â µm) and bind to the hydrophobic channel of the EthR dimer.
Assuntos
DNA/química , Espectrometria de Massas/métodos , Mycobacterium tuberculosis/química , Proteínas Repressoras/química , Cristalografia por Raios X , Fluorometria/métodos , Interações Hidrofóbicas e Hidrofílicas , Conformação Proteica , Proteínas/química , Ressonância de Plasmônio de SuperfícieRESUMO
EthR is a transcriptional repressor that increases Mycobacterium tuberculosis resistance to ethionamide. In this study, the EthR-DNA interaction has been investigated by native electrospray-ionization mass spectrometry for the first time. The results show that up to six subunits of EthR are able to bind to its operator.
