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Enhancing HTS Assays
High-throughput screening assays based on NMR
Companies and research institutes often generate thousands of compounds using combinatorial chemistry, in the hope of finding one lead compound. Whether the compounds are just off-the-shelf, purchased, or produced by synthetic means, all of these potential candidates must be screened. The development of high-throughput screening assays (HTS) has given scientists the ability to test large numbers of molecules for a desired biochemical activity, including binding and enzymatic catalysis. Designing such a specific assay isn’t always trivial; the assays can be complicated and may require several components that can interfere in the interaction of the drug candidate and the target protein. These assays also lend themselves to other problems such as low sensitivity, in the case of a weak signal or high background, and false positives.
The simplistic nature of Structure-activity relationship (SAR) by NMR makes it ideal for implementing as an assay - requiring only the target protein and the test compound - and the assay detects differences in the ligand-binding sites of the target protein.
Increasing the number of compounds screened in a given assay, can lower the required concentration of the small molecule and the protein and maximize the data collection from one assay. Longer acquisition times typically associated with increasing the mixture sizes of low protein concentrations can be overcome using a CryoProbe, which dramatically increases sensitivity, allowing larger libraries to be rapidly screened using smaller amounts of protein.
Cryogenic NMR technology, the helium-cooling of the preamplifier and radio frequency coils of the probe to about 20 K, enhances high-resolution NMR and increases the signal-to-noise ratio about fourfold. With cryoprobes, screening 100 different compounds at concentrations as low as 50 µM results in a satisfactory concentration of 5 mM and increases the data output of the assay by 10-fold when compared to the usual SAR by NMR assay.