New Approaches for the Identification of Drug Targets in Protozoan Parasites - International Review of Cell and Molecular Biology - page 368

2.3. Identification by Drug-Target Affinity
From the perspective of an investigator, the ideal situation for elucidating
the mode of action of antiparasitic drugs consists in binding of a given
compound to a target molecule (DNA, RNA or protein), and this interac-
tion then results in the inhibition of essential cellular functions. In the case
of DNA, intercalating agents are identified via their ability to slow down
DNA migration in conventional gels upon electrophoresis, or via changes
in their fluorescence properties (e.g. ethidium bromide is an intercalating
agent). In addition, they interfere in DNA replication and RNA synthesis
and/or processing and thereby inhibit protein biosynthesis. Since DNA
is ubiquitous, intercalating agents are not specific and, therefore, prone to
side effects on part of the host cells. Therefore, the relevance of a DNA-
binding agent as an antiparasitic drug resides in other—more species spe-
cific—properties. These can include the preference for circular DNA (like
the genome of mitochondria in trypanosomatids and the apicoplast in
apicomplexan parasites), differential uptake or metabolism, or the pres-
ence of other targets besides DNA. These targets are identified by applica-
tion of other methods.
Soluble protein targets may be identified using affinity chromatogra-
phy. In such cases, the drug molecule is covalently linked to a matrix, pro-
teins from crude parasite extracts or defined fractions are allowed to bind,
and bound proteins are eluted by competition after extensive washes using
unbound drug, and/or by unspecific conditions such as high salt concentra-
tions or low pH. In order to minimize artifacts, pull-downs are performed
in parallel on a mock-coupled matrix or, better, on a matrix coupled to
an irrelevant compound of similar structure ( Knockaert et al., 2000 ). The
eluted proteins are conveniently identified by mass spectroscopy. The rel-
evant proteins are then cloned, overexpressed in a suitable bacterial or
eukaryotic system and purified. These expressed proteins are employed to
perform binding studies or activity assays (in the case of proteins with enzy-
matic functions) in the presence of the drug and analogous compounds.
The advantage of this approach is that it is “hypothesis-free”, i.e. we are
not working with a predefined class of potential target proteins. The method
can be extended to membrane proteins or even cytoskeletal proteins by
adapting the extraction conditions. The disadvantage of such an approach
is that irrelevant proteins may be identified, which bind to the drug matrix
by mere structural coincidence but without being affected in their function.
Moreover, detoxification enzymes or transporters, which preferentially bind