Biomedical Engineering Reference
In-Depth Information
(i.e., genes involved in cancer insurgence). In the latter case, background knowledge
of the gene function is required. The use of small molecules in a chemical genetic
mindset gives another striking advantage to the common advantages arising from the
approach itself. Indeed, these molecules usually act rapidly at the intracellular level,
thanks to their low chemical hindrance, which makes them more prone to permeate
the cell membrane with respect to bigger molecules possessing similar polarity.
14.2.1 Forward Chemical Genetics
As in forward genetics, forward chemical genetics entails the introduction of a pertur-
bation on a cell. In the first case, the perturbation is represented by a genetic mutation;
in the latter, this effect is given by a small molecule. A molecule able to induce a
chosen phenotype is then selected, and the entity (i.e., protein or DNA) whose tar-
geting by the molecule induces the phenotypic effect is identified (Figure 14.4a).
The use of
S. cerevisiae
with a forward chemical genetics mindset to discover novel
compounds able to induce a desired phenotype is favored by the possibility of easily
treating a large number of different cell cultures simultaneously and of monitoring
the phenotype selected. By using multiple-well parallelized cultures, it is possible to
monitor simultaneously the effects of large numbers of compounds in large numbers
of yeast strains, thus briefly obtaining a wide amount of input-effect combinations.
To that aim, all forward and reverse chemical genetics approaches can be scaled to
the high-throughput level.
14.2.1.1 Haploinsufficiency Profiling
The principle behind drug-induced hap-
loinsufficiency is that lowering the dosage of a single gene targeted by a chemical
compound could make the cell sensitized to the molecule [38]. As a preliminary test,
Giaever et al. constructed six heterozygous
S. cerevisiae
strains, each one deleted
in a gene encoding a known drug target [38]. Whereas the wild-type yeast strain
growth is affected by treatment with the compound, the heterozygous strain deleted
in one copy of the gene coding the targeted protein will be more susceptible to the
treatment with the same compound (Figure 14.5). On the basis of previous investi-
gations, indicating that
ALG7
overexpression confers resistance to the glycosylation
inhibitor tunicamycin, Giaever et al. [38] generated a heterozygous
alg7
/
ALG7
strain
and evaluated the effects of treatment with tunicamycin on its growth rate with
respect to the wild-type strain. As expected, the treatment decreased the growth rate
of the deletant strain, thus giving the proof of principle for this assay. The same
results were achieved when treating with other compounds (fluconazole, benomyl, 3-
aminotriazole, and hydroxyurea), the strains deleted for the relative molecule targets,
further confirming use of the method with a broad range of molecules. Later, the test
was expanded to a larger number of deletant strains [39], again confirming the utility
of this approach for drug target identification. In their work, Baez et al. monitored by
O.D. measurement the growth rate of a set of more than 5000 heterozygous
S. cere-
visiae
deletant strains treated with dihydromotuporamine C (dhMotC, an inhibitor of
angiogenesis and metastasis) or with DMSO (the drug diluent) as the control [39].
Thanks to this method, it was possible to identify the sphingolipid metabolism as
