Biomedical Engineering Reference
In-Depth Information
number of primary hits could proceed directly to dose-response confirmation in the
primary and secondary assays. The arrows on the right side in Figure 12.3 represent
some advanced methodologies utilized for screening (see below).
12.2.6 Special HTS Modalities
An interesting approach was proposed by the NIH Chemical Genomics Center. The
approach,
quantitative high-throughput screening
(qHTS), is based on performing
primary HTS in a dose-response mode through, screening the same compound plate
at seven or more concentrations [5]. Understandably, the approach is costlier than
a single-concentration HTS, but it offers some great benefits. First, the approach
helps eliminating false negatives and false positives, as it relies on data curve
fitting and translates into embedded data confirmation. For example, the qHTS
approach allows identification of active compounds that would otherwise be missed
due to their precipitation at high concentrations. Second, qHTS allows determina-
tion of compound's potency and selectivity values, if also involves counter screen
assays. Thus, hits could be selected and prioritized based on their potency and
relative selectivity.
Another approach is based on testing compounds in the presence of varied concen-
trations of protein ligand, allowing to establish the MOA of primary hits, as described
in the next section. This approach, which could be called mechanistic HTS (mHTS,
by analogy with qHTS), has been utilized successfully by us [6,7] and by others [8].
An obvious drawback of the mHTS approach is the additional cost. On the other
hand, the benefit of this approach is that it allows prioritization of hits based on
MOA and could be cost-effective for screening campaigns with a high number of
primary hits. Performing the primary HTS in a single assay and using mHTS at the
hit confirmation stage may provide an acceptable compromise.
12.2.7 Principles of Assay Design
The goal of the assay development is to establish the most appropriate conditions
for HTS. The assays are optimized through varying different assay parameters and
aimed at improving assay performance. Precision and accuracy are two major param-
eters that evaluate the assay performance and sensitivity.
Precision
affects the repro-
ducibility of repeated measurements, while
accuracy
defines the degree of closeness
between the true values and those determined experimentally. Interestingly, condi-
tions that favor precision in HTS assays do not normally provide acceptable assay
accuracy. Thus, optimization of an HTS assay finds an acceptable balance between
precision and accuracy.
Each HTS assay employs numerous components that could be classified into
two main groups. Some of these components, such as enzyme, substrate, or cells,
are necessary in the assay and are called the
principal assay components
. Other
components, such as secondary buffer or cell media additives, could be omitted or
substituted and represent the supporting assay components of the assay.
