Regardless of a large number of false hits, HTS has the advantage of testing large libraries rapidly. Unfortunately, these methods often select for hundreds or even thousands of misleading compounds, including nonspecific hits, promiscuous aggregators, or other assay-related artifacts, that render follow-up optimizations time-consuming, tedious, and often unproductive and unsuccessful ( Böcker et al., 2011 Feng et al., 2005, 2007 Shoichet, 2006a, 2006b). Generally, plate-based spectrophotometric assays are used in HTS. Hence, HTS libraries, which usually contain more than 10 5 compounds, cannot be screened by using NMR or other biophysical approaches, as these methods have a limited throughput. Compounds are usually tested in mixtures of 10–20, but higher throughput is unlikely to be possible given the limitations of sample consumption and the relatively long measurement times required. Using protein-based NMR approaches, fragment libraries of up to 10,000 compounds are routinely screened in a relatively short time (from several hours to several days). Binding information is usually obtained by using chemical shift mapping techniques with 15N and/or uniformly or selectively 13C labeled protein, provided that resonance assignments for the target and its three-dimensional structure are known. NMR spectroscopy has been the most widely applied method in FBDD given its unique advantages of (1) detecting fragment hits of weak binding affinity (K d values up to mM level) with little ambiguity (when spectra of the target are obtained in the presence and absence of a test compound), and (2) providing crude but insightful information on the binding sites of hit compounds ( Pellecchia et al., 2002, 2008). Compared to HTS libraries, fragments libraries contain lower molecular weight compounds (MW < 300 Da), and the resulting hits are consequently of weak binding affinity (with dissociation constants in the micromolar to millimolar range). Subsequently, these fragment hits are matured into more potent binders by a variety of approaches, most often guided by structural studies using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy ( Congreve et al., 2008 Dalvit, 2009 Hubbard, 2008 Murray and Blundell, 2010 Pellecchia et al., 2002, 2004, 2008). The basic idea behind FBDD approaches is to initially identify, usually by screening small focused libraries of low molecular weight compounds (fragments) via biophysical methods, key chemical substructures or pharmacophores sufficient to confer a minimal yet specific interaction with the given target. In recent years, fragment-based ligand discovery (FBLD) approaches, also known as fragment-based drug discovery (FBDD), have become popular alternative strategies to conventional high-throughput screening (HTS) campaigns in both academic and industrial drug discovery projects ( Congreve et al., 2008 Fischer and Hubbard, 2009 Hajduk and Greer, 2007 Murray and Rees, 2009).
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