Screening cascade

Each compound passing these initial filters will be considered to enter the secondary screening cascade. 

There are some general pitfalls for the medicinal chemist to consider when running and evaluating a cellular GPCR agonist single-point screen. 

The cutoff criteria are biased and mainly designed to avoid too many felse positives. 

Effect data do not necessarily reflect the potency of the compounds. It is tempting for the chemist to interpret high-effect values as high potency and vice versa. There is no linear correlation between effect data in a singlepoint screen and the % effect values generated in a full curve concentration response assay. 

Compounds having an EC50 around 3-30 pM are probed in the steep part of their EC50 curves. In a single-point experiment, small concentration shifts inside the well due to titration can lead to big changes in the observed effect in a retest. 

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Bioavailability is an important parameter in drug screening cascades. It gives a good indication of the efficiency of the delivery of the compound to the systemic circulation by the chosen route. It can only be measured in vivo but, as will be described below, it can be predicted for man using a number of methods. 

There are several approaches to estimating absorption using in vitro methods, notably Caco-2 and MDCK cell-based methods or using methods that assess passive permeability, for example the parallel artificial membrane permeation assay (PAMPA) method. These are reviewed elsewhere in this book. The assays are very useful, and usually have an important role in the screening cascades for drug discovery projects. However, as discussed below, the cell-based assays are not without their drawbacks, and it is often appropriate to use ex vivo and/or in vivo absorption assays. 

Figure 17.6 Hematotoxicity screening cascade. Assays used during drug discover with tier 1 as the early discovery frontloading assay.

The second approach necessitates a mechanism to identify compounds or chemical series from a project that enter the screening cascade. These triggers may include target-related information, previous experience and in silica analysis of compounds. 

Mayer et al. (19) reported the use of FCG to identify compounds that affect the cellular mitotic process through a screening cascade reported in Fig. 9.2. A commercially available, diverse collection of 16,320 compounds was screened on a whole cell primary cytoblot assay (20) measuring the phosphorylation level of a nucleolar protein called nucleolin (step a). This protein is phosphorylated when cells enter mitosis, and inhibitors of the mitotic process are expected to increase the level of phosphonucleolin. 139 positive compounds were able both to penetrate the cell and to increase the level of phosphorylated nucleolin. 

The positive compounds from the primary screening are further profiled to check their usefulness. This characterization includes prehminary physicochemical property determination, toxicity data, and specificity/selectivity data when possible. The secondary screening cascade will restrict the screening outcome to a smaU number of... 

In developing screens for KCAs, the primary objective is to identify the general KCA mechanism of action, and to distinguish this from other mechanisms of action. This is because any programme of synthesis of potential KCAs could produce compounds which activate channels and also have (an) additional mechanism(s) of action as part of their inhibitory effect. Thus, it is important that a screening cascade can distinguish between... 

Fig. 15 Screening cascade taken to the virtual screen for ChK-1 inhibitory activity.

The screening cascade in SPR based fragment screening contains a series of assays that enable the application of different filter criteria for the selection of true positive binders. An overview on the most commonly used filters is given in Table 2. 

Perez-Pineiro, R., Burgos, A., Jones, D.C., Andrew, L.C., Rodriguez, H., Suarez, M., Fairlamb, A.H., and Wishart, D.S. (2009) Development of a novel virtual screening cascade protocol to identify potential trypanothione reductase inhibitors. Journal of Medicinal Chemistry, 52 (6), 1670-1680. 

Very commonly the first tests in a screening cascade will be biochemical tests performed in vitro (i.e. in the test tube) involving pure enzymes, cell cultures or cell membranes. These are commonly cheap, capable of testing... 

The first step in a screening cascade is normally the determination of the minimal inhibition concentrations against bacteria, fungi or algae. 

Molecular-based biochemical assays utilizing purified cellular fraction or proteins, albeit attractive in their simplicity and streamlining ability, do not allow for the observation of a phenotypic response and so are not suitable starting points for a primary assay, yet they have utility as follow-up assays later in the screening cascade. 

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