New Approaches for the Identification of Drug Targets in Protozoan Parasites - International Review of Cell and Molecular Biology - page 365

the medium reduces resazurin and thus causing high background values
( Nillius et al., 2011 ). In drug efficacy studies, vitality assays may lead to
false-positive results due to metabolically inactive stages of the parasite
that may survive the treatment and resurrect after the drug pressure has
been removed.
Complementary to vitality assays are methods that monitor cell dam-
age. As a general principle for such assays, cultures of parasites are treated
with compounds, and after given time points, medium is harvested and
the release of intracellular enzymes such as, e.g. nucleoside hydrolase
(NH) from G. lamblia ( Kang et al., 1998 ) or phosphoglucose isomerase
from the helminth Echinococcus multilocularis ( Stadelmann et al., 2010 ) is
quantified.
2.1.2. Quantification of Drug Effectiveness
In the first round, a screen with a high concentration of test compounds
allows to identify effective compounds. In the next step, the efficacy of
the positively tested compounds has to be quantified in terms of inhibi-
tion constants that allow a comparison with other compounds. The con-
stants that are most widely used are the concentration corresponding to
50% growth inhibition of the parasite (IC 50 ) and the minimum inhibitory
concentration (MIC) corresponding to the minimal concentration of the
compound at which growth of the parasite cannot be detected anymore
( Andrews, 2001 ). More rarely, values corresponding to 90% inhibition are
also presented (IC 90 ). These three constants are summarized in Fig. 7.1A
representing an ideal sigmoid plot of a relative growth parameter p (con-
trol = 1) vs the concentration of a test compound.
From an inhibition curve of this shape, the IC 50 value can be precisely
determined by a linear logit transformation of p ( x -axis) and the log trans-
formation of the concentration ( y -axis). The logit, ln( p /(1 s− p )), is 0 for
p = 1 − p , or p = 0.5. The intersection of the line with the y -axis (ln of con-
centration) thus gives the IC 50 value. By using a standard least square tool,
the intersection value and standard errors can be determined. The logit-log
plot of Fig. 7.1A is presented in Fig. 7.1B . The IC 50 value is a constant that
allows comparisons between different compounds in the same test system,
different test systems or even with other constants such as binding constants
of potential target proteins.
The MIC is more difficult to determine but is a useful indicator for subse-
quent in vivo or clinical trials of the antiparasitic compound. If the MIC value
of a given compound in vitro is much higher than the concentration that is