Biomedical Engineering Reference
In-Depth Information
number of close analogs within each scaffold. This property of chemical collections
is greatly beneficial for nascent identification of structure-activity relationship (SAR)
in scaffolds. It could also serve for cross-confirmation of screening data and recovery
of false negatives. Since most representatives of any given scaffold share signifi-
cant similarity, they are expected to demonstrate a similar sign of activity within the
screen. Therefore, false negatives would appear as outliers in comparison with their
analogs; these compounds would be included for a retest in a hit-confirmation stage
and thus could potentially be recovered.
Lead identification and optimization is a highly collaborative process. Aforemen-
tioned HTS data safeguards exemplify close interdependence of disciplines adjacent
to HTS. The primary function of biologists in this process is to design and opti-
mize the sensitive and robust assays, providing a reliable platform for screening.
Automation/HTS specialists ensure that the instruments and robotics systems are
in working condition for the reliable execution of assays in HTS. In addition, they
perform global HTS data analysis aimed at identification and correction of plate sig-
nal patterns. Cheminformatics specialists perform HTS scaffold analysis using data
generated and feed back into the assembling of a list for hit confirmation, helping to
eliminate frequent hitters and recover potential false negatives.
12.2.5 Stages of Lead Identification Projects
HTS is an integral part of most lead identification and optimization projects
(Figure 12.3). HTS could be viewed as an initial enrichment process, aimed at siev-
ing out inactive compounds, preferably without discarding the active compounds.
Other project stages that follow the HTS stage are generally responsible for confir-
mation and characterization of initial hits as well as further optimization of the most
attractive scaffolds.
Typically, a screening assay is designed and optimized during the assay develop-
ment stage. The design and optimization of the assay usually takes into account all
aspects of the project, especially the properties of the screening target, the properties
of the chemical library, and the desired properties of the probe compounds. Primary
HTS is commonly performed using the single-concentration single-replicate mode.
Active compounds are identified using a distribution curve of all compounds or, alter-
natively, could be selected using the percent response with respect to positive and
negative controls, as was described above.
The primary hit list is compiled from HTS data through hit analysis. At this
stage, the hit list is compared to all other assays previously utilized to screen the
same chemical library. Promiscuous compounds are normally removed from the
list, while the rest of the hits proceed into reconfirmation. Initial reconfirmation is
performed using single-concentration replicate wells for each hit. Confirmed hits
undergo further reconfirmation in dose-response mode using replicate serial-dilution
curves for each compound. The same hits are normally also tested in additional
(secondary) assays aimed either at confirmation of the hits (orthogonal assays) or
identification of compounds interfering with the assay (interference assays).
