High-Throughput Nuclear Magnetic Resonance-Based Screening

Abstract

A high-throughput screening strategy is described that involves the acquisition of two-dimensional 15N/1H correlation spectra in less than 10 min on 50 microM protein samples using cryogenic NMR probe technology. By screening at these concentrations, small organic molecules can be tested in mixtures of 100, which dramatically increases the throughput of the NMR-based assay. Using this strategy, libraries of more than 200 000 compounds can be tested in less than 1 month. There are many advantages of high-throughput NMR-based screening compared to conventional assays, such as the ability to identify high-affinity ligands for protein targets with no known function. This suggests that the method will be extremely useful for screening the large number of targets derived from genomics research.

Structure–activity relationships by interligand NOE-based design and synthesis of antiapoptotic compounds targeting Bid

“…Recent years have witnessed the development of a number of chemical fragment-based approaches to inhibitor design and drug discovery that have been applied to even unconventional and challenging drug targets such as those involving protein-protein interactions (14)(15)(16)(17). Along these lines of research, we recently described a powerful NMR method that led us to the identification of high-affinity ligands for given targets by linking low-affinity fragments (18).…”

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

“…The inherently low-sensitivity and long acquisition times necessary to acquire even the simplest NMR experiment limits the feasibility of screening a corporate library using a one compound per sample approach. Thus, the advantage of mixtures is both apparent and paramount to the successful application of NMR in high-throughput assays that require screening thousands of compounds in a reasonable time-frame, where an order of magnitude improvement in throughput may be achieved compared to screening singletons (Meyer et al, 1997;Lin et al, 1997;Jacoby et al, 2003;Hajduk et al, 1999a;Dalvit et al, 2003;Chen and Shapiro, 1999). There are multiple acceptable paradigms that contribute to the proper design of individual mixtures in a library.…”

“…Additionally, NMR may be used to evaluate the physical properties of a chemical lead, measure K D 's (Fielding, 2003), identify ligand binding sites (Roberts, 2000), and determine a co-structure (Clore and Gronenborn, 1994;Cooke, 1997;Kay, 1997;Roberts, 2000). A diverse number of NMR screening approaches have been developed, which include SAR by NMR (Shuker et al, 1996;Hajduk et al, 1997a;Hajduk et al, 1997c;Hajduk et al, 1999b;Johnson et al, 2003), SHAPES (Moore et al, 2004;Lepre et al, 2002;Fejzo et al, 1999), and MS/NMR (Moy et al, 2001). NMR spectroscopy is a relatively insensitive technique requiring higher amounts of material and acquisition time compared to standard methods used in traditional HTS assays.…”

“…Screening mixtures of 5 to 100 compounds have been described where it may be feasible for an NMR assay to screen upwards of hundreds of thousands of compounds (Devlin et al, 1996;Glick et al, 2003;Hann et al, 1999;Jacoby et al, 2003;Pratt Steven et al, 2004;Hajduk et al, 1999a). Thus, the use of mixtures makes an NMR assay readily amenable for screening smaller, directed libraries with the potential to screen larger, random libraries comparable to standard HTS assays.…”

Parallel screening and optimization of protein constructs for structural studies

“…[2][3][4][5][6][7] This type of spectrum provides a ''fingerprint'' of the protein which informs on the suitability of a sample for structural studies and also on the degree of folding of the protein. [8][9][10] Taking advantage of these improvements, we have developed a protocol that allows rapid evaluation of the feasibility of structural studies for multiple protein targets expressed in parallel in E. coli. We have accelerated construct screening by reducing the common pipeline described above to a simple small-scale, fully automatable process.…”