In this article, we review a subset of affinity techniques that has been shown to be valuable for lead discovery ( Table 1 Nevertheless, the advantages of affinity techniques have extended their use into follow-up or secondary screens, especially in the field of ATP-dependent enzymes. Thus, affinity hits require biochemical, cellular or in vivo validation. On the downside, affinity-based technologies do not deliver a functional readout. Last, this approach eliminates many assay artifacts that arise from nonspecific aggregation, fluorescence absorption or quenching. Third, it is possible to carry out the assays at small-molecule concentrations much higher or lower than that of the corresponding biochemical assays. Second, it has the ability to investigate multiple forms of macromolecules, such as various ternary complexes, activated or inactivated forms and forms with or without cofactor. First, it has the capability to sample mixtures (including natural products) rather than discrete entities, thus enabling the exploration of larger chemical spaces without a concomitant increase in the number of samples. This approach has several advantages over biochemical assays for both drug-like and fragment-like methods (see Glossary). For affinity-based techniques, the readout is typically a qualitative or quantitative signal based on the physical interaction between macromolecule (RNA, DNA or protein) and the small-molecule partner. ![]() To address these issues, affinity or bio-affinity screening approaches have emerged as orthogonal methods for early lead discovery. Challenges facing HTS technologies include high false-positive and false-negative rates, the need for reporter assays, and the limitation in throughput imposed by testing compounds individually. Second, and most importantly for novel targets, sampling of drug-like chemistry space by a few million compounds is inadequate to consistently deliver several diverse lead classes. First, most compound decks in the industry are skewed in compound distribution favoring specific, well-studied protein subfamilies. ![]() Two key factors are likely to be responsible for this. Hit rates for targets tend to be extremely low or very high. The HTS era has thus far delivered mixed results. Typically, targets are interrogated with 1–3 million discrete compounds in parallel. Over the past decade, high-throughput screening (HTS) of corporate compound decks has emerged as the primary paradigm for hit or lead discovery. Lead generation is a critical first step in the drug discovery process.
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