New Approaches for the Identification of Drug Targets in Protozoan Parasites - International Review of Cell and Molecular Biology - page 369

to xenobiotics, may be identified besides the “true” target. In some cases,
susceptibility to a drug is triggered by the ability to convert an ineffective
prodrug to the effective drug. In these instances, the searches for drug tar-
gets have to be performed with the effective molecules rather than with
the prodrug in order to exclude that the transforming enzyme is identified
rather than the target itself.
2.4. Identification of Drug Targets by Analysis of
Resistant Parasites
A typical study of this type is performed by (i) creating resistant mutants
of a drug-sensitive parasite, either by continuous breeding in the presence
of increasing amounts of the drug or by mutagenesis followed by selection
of resistant populations or clones in the presence of lethal concentrations
of the drug; (ii) comparison of the resistant clones to the corresponding
drug-sensitive wildtype on the genomic, transcriptomic and/or proteomic
levels, and selection of differentially expressed genes/proteins; (iii) the func-
tional analysis of differentially expressed genes by overexpression and/or
silencing and after heterologous expression in a suitable system. Since point
mutations causing the presence or absence of a single amino acid or nucleo-
tide (in the case of rRNA as a target) discriminate between resistance or
susceptibility, whole genome sequencing of resistant clones is necessary.
The advantage of this approach is that it is—again—hypothesis-free. The
disadvantage is that it may not lead to the direct target, but to proteins
involved in transport or metabolism of the (pro-)drug. An example for this
is given by spiroindolone resistance in P. falciparum due to a mutation in a
cation transporter ( Rottmann et al., 2010 ). Moreover, due to the flexibility
of gene expression in protozoa (e.g. G. lamblia ), it is possible that the true
target is hidden by resistance formation in resistant clones due to compen-
satory changes in gene expression ( Müller et al., 2008a ).
2.5. Identification of Drug Targets by Genome Mining
For many drugs in the market, the relevant target molecules are known.
Due to amazing genome sequencing efforts in the years 1990-2010, whole
genome sequences of parasites are available in interactive databases on the
World Wide Web (e.g. www.genedb.org ). These genomes can be searched
for the presence or absence of potential target molecules. Following over-
expression of the targets, drug-binding studies can be performed in order
to obtain information on the potential activity of a given compound. Drug
derivatives with a better selectivity for parasite vs host enzymes can be