Secondary Screening Assays

Independent confirmation of primary hits is performed by a variety of reliable secondary assays. We use a number of direct binding assays such as biolayer interferometry (BLI) or (for strongly binding inhibitors) ITC as well as indirect binding assays such as thermal shift assays for hit validation. 

---

FIGURE 12.5 Partial view of a bioassay summary for a confirmatory (secondary screen) assay for ubiquitin-specific protease USP2a (AID 927). 

The layout of samples and controls configured on a plate during an assay. For example, for a primary screen in 384-well plates, columns 1, 2, 23, and 24 are controls, and columns 3-22 are for individual test compounds, whereas for secondary screening, each row will contain a single compound at varying concentrations. 

A screen applied to confirm independently actives from the primary screen. A secondary screen may employ an assay that differs in type from the primary screen, e.g., biochemical assay vs cell based assay, or it may be of the same type with different readout. 

After primary screenings, which are less quantitative than biological assays giving a positive result or HIT , another more precise secondary screening is conducted, by calculations of IC50 values. 

Extracts that exhibited significant inhibitory activity, defined as >50% inhibition of CPE at 100 (xg/mL, were advanced into secondary screening, which includes confirmation of activity observed during the primary screen using an expanded range of concentrations (dose response), plaque inhibition assay, and a one-step growth inhibition and testing of additional influenza viruses. As shown in Fig. 1.2a, the quantitative dose response assay was used to assess the potency of the most two... 

Additional assays included a selectivity evaluation and cytotoxicity assay where both good selectivity and low cytotoxicity were documented. The extract was also tested on unrelated viruses and other influenza viruses. Extract A4 was the only one to maintain the activity through secondary screening, and with that as a target, purification of the active extract commenced. 

The kitchen of a fast food restaurant is characterized by islands of automation, with well defined subprocesses focused on producing a certain kind of output, coordinated by a crew chief The principal advantage of a fast food restaurant is consistency and fast delivery. The dedicated subunits are designed to perform a certain type of process (assay) at a high rate with very little room for change. Economically, this model is difficult to sustain unless each assay type has sufficient demand to justify the existence of dedicated space, equipment and personnel. It is also not as efficient as a secondary screening model. For assays that are routinely, but not always, requested then this model is very appropriate (e.g., CACO-2 permeability, microsomal stability). However, for the more costly and complex assays that are requested less often, the cost of dedicated people and equipment is hard to justify and as a result the assay has to come off the menu. This is why most fast food restaurants have a relatively limited menu, including mostly foods that are simple to prepare. 

At present, the use of primary cells in the pharmaceutical industry is not as common as the use of recombinant cell-based assays. A rough estimate shows about 20% of all initial screening is carried out using primary cells and the rate is about the same for secondary screening. The use of primary cells is expected to increase in the next few years as emerging technologies start appearing on the market. 

---