Biomedical Engineering Reference
In-Depth Information
confines of high-throughput screening (HTS), a discipline of the biomolecular sci-
ences. HTS relies on a specialized, constantly expanding group of biological tests that
are amenable to miniaturization and screening in high-density formats. The design
and optimization of these tests, known as screening assays , is usually the domain of
assay development , a sister discipline within biomolecular sciences.
12.2 BASIC PRINCIPLES OF HTS
The main goal of a high throughput screening project is the identification of lead
compounds with a desired specific set of properties. These properties are defined
by the requirements of the future use of the lead compounds. Nevertheless, to be
considered useful, all lead compounds have to possess some general properties. First,
the compounds identified have to be genuine modulators of the target of interest, not
of the assay utilized for screening. Second, the lead compounds need to be sufficiently
potent and efficacious in modulating the target of interest. Finally, they need to be
sufficiently specific and selective, so their modulation of the target of interest could
be distinguished from any potential off-target effects. These expectations define the
screening process and its components.
12.2.1 Specifics of HTS Assays
HTS provides an environment in which biological processes are queried through
screening assays that establish a link between cellular and macromolecular phe-
nomena/functions and a quantifiable signal (Figure 12.1). HTS became the central
methodology within drug discovery for testing small-molecule compound collections
and for the generation and optimization of chemical leads. It emerged and matured
in response to a demand of the drug discovery industry in screening large-scale
compound libraries and was supported by a concomitant development of screening
instrumentation and methodologies.
Most HTS assays are performed in microplates, in which the wells represent
separate compartments of nanoliter to microliter volume. Diversity of plate formats
exist; nonetheless, the great majority of HTS plates adhere to Society for Biomolecular
Screening plate format, established to ensure interchangeability and compatibility of
instrumentation utilized for screening. Most frequently used plates come in 96-, 384-
and 1536-well densities (Figure 12.2). Variations in plate materials, color additives,
and surface treatment make them suitable for different screening applications.
Screening plates could be seen as a unit of an HTS-based experiment. As such,
they contain internal controls that allow for judging the success of the experiment.
These control wells are used to assess the overall data quality and to serve as the
reference markers of efficacy for screened compounds. Working reagent solutions are
dispensed in the wells with the help of microchannel pipetters or automated liquid
handlers.
There are three main types of samples within HTS plates. First, wells containing
all assay components but lacking the tested compounds are called negative control
wells ; they represent unaffected biological process corresponding to a zero-efficacy
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