Biomedical Engineering Reference
In-Depth Information
vector was introduced into a wild-type yeast strain. The individual transformants
can be tested for resistance to the inhibitory compound, and resistant strains can
be selected as able to survive a lethal concentration of the compound. As observed
for approaches described previously, in this case also the main limitation to drug
target identification is that only a subset of potentially interesting compounds has
a growth-inhibitory phenotype in yeast. Nevertheless, this approach was useful for
the identification of the molecular targets of several known antifungals, such as
ketoconazole [43] and soraphen [44]. A yeast library consisting of strains transformed
with plasmids of over 80% (5800 ORFs) of all yeast ORFs, whose expression is
controlled by a galactose-inducible promoter, was created [45]. The supplementation
of galactose in the culture medium induces the high-level expression of the ORF
included in the plasmid. Although the considerable advantage of using an inducible
promoter is that the expression can be induced at specific times during the course of
the experiment, galactose induction often exceeds the endogenous gene expression
level, thus resulting in toxicity to the host cell [46]. It is worth noting that 15% of
yeast genes are toxic to the cell when overexpressed. Nevertheless, it is possible to
take advantage of these toxic effects, despite their apparent disadvantage. Indeed, the
toxic genes can give useful insights into how regulation of the gene products can alter
the cell physiology, and they can be used as a starting point for chemical suppressor
screens to identify compounds that suppress the genic toxicity. This approach, called
chemical suppression
, has been employed successfully to identify human genes that,
when expressed in yeast, result in a growth defect [47]. Tugendreich et al. suggested
that an overexpression of the human
p38
, aMAP kinase suggested to play an important
role in the development of AIDS and HIV-associated neurocognitive disorders [48],
induces yeast cell death. Thus, the ability of novel molecules to nullify the toxic
gene effect can be exploited as potential drugs for HIV treatment when looking for
p38 inhibitors.
14.2.2 Reverse Chemical Genetics
Reverse chemical genetics is based on a process that starts with the selection of
a specific protein. Chemical libraries are then screened for a ligand of the protein
chosen, and the ligand is finally used to determine the phenotypic consequences of
altering the function of the protein in a cellular context (Figure 14.3b).
14.2.2.1 Yeast Two-Hybrid Assay
The yeast two-hybrid (Y2H) method was first
described in 1989 by Fields and Song [49], and later developed to the high-throughput
level [50]. This method allows for the assessment of functional protein-protein or
protein-DNA interactions. The premise behind the assay is the activation of a reporter
gene through the binding of a transcription factor to a reporter-specific promoter
(a DNA sequence located upstream and regulating the expression of the coding
sequence). In the two-hybrid method, the transcription factor is divided into two
fragments: the binding domain (BD, responsible for the DNA binding) and the acti-
vating domain (AD, responsible for the activation of transcription) (Figure 14.6).
