RESUMEN
In-cell NMR spectroscopy is a powerful tool to study protein structures and interactions under near physiological conditions in both prokaryotic and eukaryotic living cells. The low sensitivity and resolution of in-cell NMR spectra and limited lifetime of cells over the course of an in-cell experiment have presented major hurdles to wide acceptance of the technique, limiting it to a few select systems. These issues are addressed by introducing the use of the CRINEPT pulse sequence to increase the sensitivity and resolution of in-cell NMR spectra and the use of a bioreactor to maintain cell viability for up to 24h. Application of advanced pulse sequences and bioreactor during in-cell NMR experiments will facilitate the exploration of a wide range of biological processes.
Asunto(s)
Reactores Biológicos , Resonancia Magnética Nuclear Biomolecular/instrumentación , Proteínas/química , Supervivencia Celular , Evaluación Preclínica de Medicamentos/instrumentación , Evaluación Preclínica de Medicamentos/métodos , Diseño de Equipo , Escherichia coli/química , Escherichia coli/citología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Células HeLa , Humanos , Viabilidad Microbiana , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular/métodos , Mapeo de Interacción de Proteínas/métodos , Proteínas/metabolismo , Programas InformáticosRESUMEN
In-cell NMR spectroscopy was used to screen for drugs that disrupt the interaction between prokaryotic ubiquitin like protein, Pup, and mycobacterial proteasome ATPase, Mpa. This interaction is critical for Mycobacterium tuberculosis resistance against nitric oxide (NO) stress; interruption of this process was proposed as a mechanism to control latent infection. Three compounds isolated from the NCI Diversity set III library rescued the physiological proteasome substrate from degradation suggesting that the proteasome degradation pathway was selectively targeted. Two of the compounds bind to Mpa with sub-micromolar to nanomolar affinity, and all three exhibit potency toward mycobacteria comparable to antibiotics currently available on the market, inhibiting growth in the low micromolar range.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Espectroscopía de Resonancia Magnética/métodos , Mycobacterium tuberculosis/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/crecimiento & desarrollo , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Ubiquitinas/metabolismoRESUMEN
We developed an in-cell NMR assay for screening small molecule interactor libraries (SMILI-NMR) for compounds capable of disrupting or enhancing specific interactions between two or more components of a biomolecular complex. The method relies on the formation of a well-defined biocomplex and utilizes in-cell NMR spectroscopy to identify the molecular surfaces involved in the interaction at atomic scale resolution. Changes in the interaction surface caused by a small molecule interfering with complex formation are used as a read-out of the assay. The in-cell nature of the experimental protocol insures that the small molecule is capable of penetrating the cell membrane and specifically engaging the target molecule(s). Utility of the method was demonstrated by screening a small dipeptide library against the FKBP-FRB protein complex involved in cell cycle arrest. The dipeptide identified by SMILI-NMR showed biological activity in a functional assay in yeast.