Principles and Practice of Pharmaceutical Medicine

with the binding of the transcription regulatory
complex to DNA.
A key decision in each lab is when to incur the
expense, and time to clone the molecular target and
set up the robotizedin vitro assays which can
screen compounds with a high rate of throughput.
The best assays are those which relate directly to
cellular events, which allow screening of huge
numbers of chemical compounds and which pre-
dictin vivoresponses. Other assays during this
exploratory stage may be used as secondary
screens for candidates identified by the first one,
if at rather slower throughput.

Genomics and molecular biological
approaches

The Human Genome Project has had a significant
effect on target identification. One by-product was
that gene expression profiling technologies were
invented which allowed for direct comparisons of
mRNA levels in normal and diseased cells (e.g.
‘gene microarrays’ or ‘gene chips’; Cunningham,
2000; Clarkeet al., 2001). Technologies such as
these allow the pharmaceutical researcher to com-
pare the expression levels of nearly all the genes in

thegenome in one experiment, and in an automated

fashion. Gene expression profiling is useful not

only in target identification as described here but

also in finding significant use in later stages of drug

development such as toxicology, surrogate marker

generation and mechanism of action studies (see

Figure 4.3).

‘Antisense oligonucleotides’ are short, single-

stranded DNA molecules that are complementary

to a target mRNA (Baker and Monia, 1999;

Crooke, 1999; Kolleret al., 2000). Once bound to

the mRNA of interest, it is targeted for cleavage

and degradation resulting in a loss of protein

expression. There are several naturally occurring

catalytic RNAs including ‘hammerhead’, ‘hairpin’

and ‘hepatitis delta virus’ introns and the RNA

subunit of RNAase P (Khan and Lal, 2003). Cata-

logues exist where the researcher can simply look

up which genes a particular antisense sequencewill

map to, and the use of fluorescent tags can then be

used to probe the location of disease-producing

mutants.

But the pharmaceutical researcher should not

rely entirely on gene expression profiling for target

identification, even though the technology is very

powerful. Gene expression does not automatically

lead to predictable protein synthesis. Protein

Figure 4.3 Drug screening flowchart

4.2 DESIGNING A DRUG DISCOVERY PROJECT 47