Biomedical Engineering Reference
In-Depth Information
of detection approaches have specific sets of compound properties that interfere with
the assay; moreover, no approach is immune to compound interference artifacts. This
detection-aligned specificity of artifacts provides a basis for their identification and
elimination, either through screening of an unrelated target using the same detection
technique or through retesting hits against the target of interest using an orthogonal
assay with a different detection mode. Both of these approaches are heavily utilized
in follow-up hit confirmation and validation studies.
12.3.2 The Great (Biological) Divide
The HTS assays are designed to query and provide information on the status of bio-
logical targets in the presence of varied “perturbagens” such as interference RNA or
small molecules. The assays could employ intact cells or purified biological materi-
als and measure various functional characteristics of this biological matter, ranging
from enzymatic activity to phenotypic changes in the appearance of cells. All screen-
ing assays could be divided into two major groups: cell-based and cell-free assays.
As the names imply, cell-based assays employ intact native or engineered cells. In
contrast, cell-free assays employ cellular lysates and purified macromolecules: most
commonly, proteins. The advantages and disadvantages of both of these approaches
are outlined below.
One of the main advantages of the cell-based assays is that the target of interest
is studied as an integral part of interconnected cellular pathways of other macro-
molecules. This integration is greatly beneficial for identification of modulators of
multiple targets in a single assay, yet could also give rise to off-target compound
effects. In the light of the integral approach, selection of an appropriate cellular back-
ground is probably the most important step in the development of a cell-based assay.
Incorrect cellular background compounded with overexpression of the target of inter-
est would lead to misbalance of natural interactions and signaling, and would result
in generation of uninterpretable screening data. If available, primary patient-derived
cells offer a very attractive and relevant biological model. Continued progress in the
field of induced pluripotent stem (iPS) cells promises to overcome the current limi-
tations of the primary cells, their limited supply and inability to proliferate, making
iPS cells extremely attractive for future HTS.
Another advantage of cell-based assays is that they help with identification of
physiologically relevant molecules. Indeed, cellular membranes provide a set of nat-
ural barriers, driving off compounds with suboptimal membrane permeability. On
the other hand, the barrier properties of cellular membranes could also be disad-
vantageous, especially during early stages of a lead identification project. Certainly,
compound membrane permeability is easily altered later in the project, providing that
good starting-point compounds were not omitted simply on the basis of their polarity.
Another “inevitable evil” of cell-based assays is the presence of serum in cell culture
media that is, on the one hand, required to keep the cells alive, yet, on the other hand,
is characterized by a high capacity for nonspecific binding of compounds. To address
this problem, an increasing number of low-serum or serum-free media formulations
have been developed for varying cell types in recent years.
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