Protein NMR in biomedical research

Abstract

Nuclear magnetic resonance (NMR) spectroscopy is a versatile biophysical technique with wide applicability in drug discovery research, particularly for the detection and characterization of molecular interactions. This review highlights in a comprehensive manner the aspects of biomolecular NMR which are most beneficial for pharmaceutical research and presents them as contributions to the different stages of a drug discovery program: target selection, assay development, lead generation and lead optimization. Emphasis is put on the concept of the particular NMR application, rather than on technical details, and on recent examples. Finally, an appendix of frequently asked questions is given.

Determining the optimal size of small molecule mixtures for high throughput NMR screening

“…Random libraries usually have hit rates on the order of 0.1-0.5% (Dove and Marshall, 1999), but focused chemical libraries, such as SHAPES used for NMR assays, may exhibit significantly higher hit rates of 0.7-20% or higher (Lepre et al, 2002;Jahnke and Widmer, 2004). The hit rate, thus, plays a significant factor in determining the optimum size of a mixture.…”

“…The absolute hit rate is an unknown quantity prior to executing a screen, but an expected value can be predicted based on the composition of the screening library. For directed libraries that are routinely used in NMR higher hit rates are anticipated (0.7-20% or higher) (Lepre et al, 2002;Jahnke and Widmer, 2004). Our analysis of OMS would predict that a mixture size of 5 compounds would be a reasonable choice for the high hit rates predicted for NMR screens of directed compound libraries.…”

“…Second, NMR screens tend to be more sensitive than traditional HTS where observing K D 's in the 100 μM to mM range are common (Hajduk and Burns, 2002). Finally, NMR-based assays are also increasingly being used as secondary assays to confirm hits identified from HTS (Hajduk and Burns, 2002;Jahnke and Widmer, 2004). In this context, the expected hit rate would be very high when using these enriched compound libraries where it is theoretically possible to approach 100%.…”

“…A comparable approach to reduce the size of screening libraries is to use NMRbased assays as a secondary screen to validate hits from HTS assays. In this manner, the HTS assays reduce a large corporate library to a small, focused list composed of a few hundred to a few thousands compounds that is more applicable to an NMR screen (Hajduk and Burns, 2002;Jahnke and Widmer, 2004).…”

Mapeamento das interações proteína-ligante através de técnicas de RMN de ¹H utilizando detecção do ligante

“…A vantagem desta metodologia é identificar um ligante em uma grande mistura de compostos e fornecer um K d aproximado em um experimentos simples. O epitopo pode ser evidenciado a seguir, aplicando-se técnicas como STD e WaterLOGSY 17,18 . Dessa forma, para implementar e interpretar experimentos de triagem por RMN é necessário revisar alguns conceitos básicos Figueiredo e Marsaioli sobre o equilíbrio de ligação, de forma a permitir uma melhor distinção entre princípios biológicos e físico-químicos a partir de novos desenvolvimentos na área da RMN.…”