Biomedical Engineering Reference
In-Depth Information
of compounds could be accommodated through the reconfirmation stage. Lower
Z
-factor values simply increase the chance of missing the active compounds and/or
enriching the pool of active compounds with those that appear active for stochastic
reasons, the situation correctable through replicate samples.
12.2.3 Primary Hit Selection Criteria
Compounds with a desired activity in primary HTS assay are called
primary hits
or
primary positives
. The primary screening process is usually designed as a process for
decreasing the number of compounds for the follow-up work; it is aimed at discarding
out unwanted compounds while retaining those that appear to genuinely affect the
target. This hit identification process relies on selection criteria defined by several
factors: the size of the screened library, the overall level of efficacy demonstrated by
the compounds within the library, and the throughput of the most critical assay at
the follow-up stage. In majority of HTS campaigns, compounds are selected using
either relative efficacy or statistical significance as the main selection criterion of
their effect in the assay.
Primary HTS is usually designed to establish apparent compound efficacy at a set
concentration utilized in screening. Relative compound efficacy is usually calculated
using positive and negative control values and expressed as a percent response:
−
X
NC
relative efficacy
=
NC
·
100%
(12.2)
−
PC
where
X
is the experimental value of activity corresponding to the compound well
within a plate; NC and PC are mean values of negative and positive control wells of
the plate, respectively. Compound efficacy is expected to correlate with its potency,
which is usually designated as EC
50
, in an inverse nonlinear fashion, such that a
50% response corresponds to the EC
50
value equal to the screening concentration.
Therefore, the primary hits could be selected based on their relative efficacy being
above a certain preset cutoff value. For example, 50% cutoff would select compounds
with EC
50
values below the concentration used in primary HTS.
The second approach relies on the statistical significance of the compound effect,
measured through the standard score, also known as the Z-score, parameter values
[4]. The value of the Z-score parameter correlates the compound differential effect
expressed in units of the assay dispersion:
X
−
Z-score
=
(12.3)
where
X
is the experimental value of activity corresponding to the compound well
within a plate;
are the mean and standard deviation of the population of
inactive samples. Since the great majority of compounds are expected to be inactive in
the primary HTS, routinely,
and
are calculated using compound wells. Utilization
of negative control wells for calculation of
and
and
values is more appropriate for
