Biomedical Engineering Reference
In-Depth Information
Most HTS assays have more than one principal component. A principal compo-
nent that is present at a concentration lower than that of all the other ones is called a
limiting component
; other principal components represent nonlimiting components.
For example, in an HTS assay employing an enzymatic reaction, the limiting and
nonlimiting components are the enzyme and its substrates, respectively. The signal
in the assay is directly proportional to the concentration of the limiting component;
thus, its concentration defines the S/B value of the assay. The lowest concentration
of the limiting component that is discernible from the assay background is called the
limit of detection
(LOD) [9]. Numerically, the LOD is equal to 3 standard deviations
of the background measures, divided by the slope of the linear dependence between
the assay signal and the concentration of the limiting component. For most screen-
ing applications, maintaining the concentration of the limiting component above
10
LOD is required.
At a high enough concentration of the limiting component, the linear dependence
between the measured signal and its concentration breaks, indicating either a change
in the limiting component or inability of the instrumentation to handle high signal.
The limit of linearity defines the concentration corresponding to an upper limit of
quantitation. The parameter may not be important for the identification of inhibitors,
as long as the concentration of the limiting compound in the assay is below the
upper limit of quantitation. On the other hand, this parameter can play a critical role
in assays aimed at the identification of activators, as it will define the upper limit
of an apparent activation observed in the assay. A project performed at the Conrad
Prebys Center for Chemical Genomics (CPCCG) at the Sanford-Burnham Medical
Research Institute (SBMRI) provides an example of a successful assay optimization
for concurrent identification of both inhibitors and activators in a single assay [10].
Interestingly, the time component of a steady-state enzymatic reaction possesses
the properties of a limiting component and requires similar optimization. The con-
centration of the limiting component translates directly into signal strength and is
critical for assay precision. On the other hand, concentrations of the nonlimiting
components define differential sensitivity of the assay in identifying modulators with
various MOAs. It is a common knowledge that uncompetitive inhibitors of enzymes
are more potent in the presence of a high concentration of substrates, while competi-
tive inhibitors are more potent at a low concentration of substrates. The concentration
of a nonlimiting component corresponding to its apparent
K
m
(or
K
d
in the case of
a binding ligand) provides the best overall sensitivity for identification of modulator
classes with various MOAs.
At a concentration of the nonlimiting component equal to
K
m
(
K
d
), the EC
50
value
of competitive and uncompetitive compounds is 2
×
K
i
, whereas the EC
50
value of
noncompetitive inhibitors is independent of the substrate concentration and equal
to
K
i
. Thus, the nonlimiting component concentration defines the accuracy of the
EC
50
values determined in the screening. Utilization of a nonlimiting component
concentration equal to
K
m
(
K
d
) at the primary HTS stage is desired especially in
screening DOS compounds, since it permits identification of compounds with diverse
MOAs in a single assay. A compound's MOA and the dissociation constant (
K
i
)are
usually established during the lead characterization stage.
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