Target-Based Antimicrobial Drug Discovery - Bacterial Pathogenesis: Methods and Protocols - page 263

actually be antibacterial and non-toxic at the end of the day. These methods are,
however, highly useful in the characterization of the inhibitor-target interface.
In vivo or whole-cell assays have certain advantages over in vitro assays
in that the complexities of biological systems can be addressed directly (2) .
While targets with multiple components can be assayed simultaneously in vitro,
assessments of antibacterial activity and target accessibility are intrinsic to a
whole-cell method. However, issues plague this assay scheme as well. “Off
target” activity is extremely possible (or even likely). Determining selectivity
of inhibition is a challenge. Just as in vitro assays require secondary assays
to ensure target modulation is the root cause of antibacterial activity, in vivo
screens require multiple secondary assays to confirm mechanism of action (43) .
Readouts for in vivo drug discovery are multiple and diverse. Spectrophoto-
metric methods to assay calorimetric reagents, changes in metabolites, or optical
density have been used. Reporter gene fusion systems in which modulation
of a target or target expression results in a detectable change in, for example,
nutrient requirements or color, have been used repeatedly (2) . For pathway
screens in which inhibition of any one of a number of gene products can result
in antibacterial activity, induction of a reporter signal generated downstream in
the pathway is ideal. One parameter that must be considered for these screens
is the timing of signal detection versus cell death, as death will be the ultimate
outcome of successful target inhibition and may inherently result in signal loss.
A final type of screening is of course virtual, in which the scientist utilizes
known structural information to predict what type of molecule will bind to a
given target and presumably alter its function (44,45) . Prediction programs and
advances in crystallographic methods have made this a useful method, where
reagents, time, or other resources may be limiting. Virtual screening is also
often used to take advantage of a known inhibitor and model structure-activity
relationships. As attractive as this method can be, it cannot replace the wet
laboratory approach and any predicted structures need to be tested at the bench
for at the least antimicrobial activity if not target interaction.
5. Concluding Thoughts
This discussion has attempted to provide a top-level view of target types
currently and historically under investigation and some thoughts on various
methods for targeting them. While the fact that the genomic revolution has
provided a plethora of targets known to be essential for bacterial viability is
undeniable, it is not clear to date how many of them will provide any use in drug
discovery. Have all the low-hanging fruit been gathered such that the search
for new targets must continue? As noted before, it is interesting that many of
the targets identified and currently under investigation are the same as those