Primary HTS

ReproducibiUty in a primary HTS even with a good a/ (quality metric) >0.5 is an issue [9]. The concordance in a tripUcate HTS with identical target, identical screening format and identical compounds seldom exceeds 65%. Problems persist post primary HTS. IC50 rankings and selectivity panels can be seriously flawed if nominal rather than actual concentration is used. AH competent drug discovery... 

A second approach to handling false negatives relies on a computational analysis of actives in the primary HTS. Were there analogs or similar compounds to actives that appeared inactive in the original HTS These inactives are retested perhaps in a more careful screen and some of the original inactives will now be found to be active. Most commonly these were truly active compounds that appeared inactive in the original screen because they were not in solution under the initial HTS assay conditions. 

In order to determine whether compounds identified in the primary HTS screen are specific, a counterscreen is required to identify and eliminate false positives that will arise in the primary screen. For protein—protein interaction screens, it is preferable to test an unrelated protein pair that uses the same mode of detection. For our purposes, we adapted a previously described TR-FRET assay that monitors the interaction between bacterial Staphylococcus aureus Dnal and phage protein 77ORF104 (Liu et al., 2004). 

Taken together, these classes of compounds can cause a significant enrichment of false positives among the most active compounds. This is illustrated by the sudden spike in the number of actives at the highest levels of inhibition (the most active 0.2%) in a recent primary HTS assay run here at Wyeth see Figure 6.1. In the study by SiUs... 

Fig. 6.1 Activity histogram (left Y axis) and cumulative frequency curve (right Y axis) versus binned percent inhibition (X axis) for a recent primary HTS run at Wyeth. Note the sudden spike in the cumulative frequency curve for compounds with >100% inhibition, corresponding to the 0.2% most active compounds.

Fig. 6.2 Plot of percent inhibition in the primary HTS assay versus IC50 ( J.M) in a secondary in vitro assay for 118 compounds of interest identified from a recent Wyeth HTS. Note the relatively flat correlation such that compounds with low percent inhibition (e.g., 50%) have IC50S, on average, only one order of magnitude less than compounds with maximal ( 100%) inhibition.

So, if other factors are to be considered in hit selection, what should they be We suggest that chemical structure is at least as important as potency in the primary HTS. In the following paragraphs, we describe ways in which the limitahons of the Top X approach can be addressed, often involving the use of chemical structure. In the remainder of the chapter, we describe a method that combines these techniques and offers substantial improvements in several regards. 

In many projects, the throughput limit for the confirmation assay is significantly lower than the number of actives from the primary HTS, and in these cases, a Top X approach based on a preselected threshold has the obvious disadvantage of failing to consider a potentially large number of true actives, leading to an artificially high... 

Even if the concepts are good, the use of primary cells in screening laboratories is not yet widespread. Primary cells are mainly employed in secondary assays to validate effects of compounds identified in initial screens conducted with recombinant cells. Secondary assays using primary cells to validate positive hits found via primary HTS should aid in the selection of meaningful leads with enhanced probability of identifying successful new compounds (Gebrin-Cezar, 2007). 

Many commercial suppliers (Table 10.3) have established protocols and procedures that address many of these issues. Primary human and animal cells of a variety of types are often available, sometimes as ready-to-use frozen stock aliquots or already added to 96-or 384-well plates. Specific culturing medium and detailed protocols for the correct maintenance of the cell line in culture are usually provided as well. This should facilitate the use of primary cells for primary HTS and compound validation. 

In practice, an HTS collection of microclusters around diverse scaffolds provides a certain redundancy in noisy primary HTS data and facilitates the data analysis methods described above to identify active series. Recent library design efforts have shifted away from the optimal diversity approach and focus more on scaffold representation, high quality lead-like (Hann and Oprea, 2004) compounds, synthetic feasibility, and smart focused libraries to provide the best starting points for medicinal chemistry (Davies, Glick, and Jenkins 2006 Schnur, 2008). 

Primary HTS data are captured in PubChem [9], which has author-defined labels for active and inactive chemical probes. However, most of the other databases listed above do not capture biological endpoints in a simple searchable manner There are no fields that one can query in a quantitative manner to identify what is the target-related activity of a particular compound, or what other measured properties it has. Such information is important if... 

Fig. 32.5. Changes in the usage of the total screening capacity from primary HTS to in-depth hit characterization. HTS high throughput screening. Confirmation repetition of assay for hits with duplicates. Validation IC50 determination. Control tests tests for interference with the assay system. Profiling tests to characterize the properties of the inhibitors. 2006 data predicted.

One prerequisite to get precise affinity values in ITC is related to the shape of the binding isotherm and thus the affinity of the compoimd. A typical experimental setup using 20 pM of a target protein would enable to determine accurate affinity values in the range of Ap = 5 pM to about 5 nM. If affinities are either lower (e.g., fragments or primary HTS hits) or higher (e.g., advanced compounds in lead identification and/or optimization), one has to use competition experiments, also referred to as displacement ITC [35]. In the case of weakly binding... 

---