Target-Based Antimicrobial Drug Discovery - Bacterial Pathogenesis: Methods and Protocols - page 262

make fine adjustments to create perfect assay conditions. Large amounts of
materials are required because of large void volumes, and ideal temperatures
may not be maintained. Assay components must be stable over long periods of
time. Nevertheless, high-throughput screening undoubtedly offers a staggering
improvement in assaying large collections of potential inhibitors.
In vitro assays in which the target has been purified or partially isolated are
widely used in high-throughput screens. Advantages to having purified compo-
nents are obvious. One can adjust parameters to optimize substrate concen-
trations and conditions (38) . Activity kinetics can be characterized, and often
binding curves can be generated to thoroughly understand the mechanism of
inhibition and optimize inhibitory measurements. However, assay development
time can be longer than a cell-based screen in that purified materials are required
and may be difficult to obtain. Ideal conditions must be determined, and those
conditions may not reproduce the cellular milieu in a biologically meaningful
way. In addition, in vitro assays for antimicrobial agents may not ultimately
be active in vivo if the inhibitor cannot penetrate or are modified by the cell.
Lipinski's rules are often utilized to predict whether an identified inhibitor will
have a chance of actual “drug-like” activity (39) . It should be noted that in
vitro schemes for identifying inhibitors require secondary assays with alter-
native detection methods to ensure that target modulation and biological activity
correlate (40,41) .
In vitro assays are often designed to detect inhibitors of enzyme activity
and are ideal if the purified enzyme is available. Depending on reaction rate,
continuous detection or stopped reaction times may be used. Rate-based assays
are highly sensitive, and the generation of multiple fluorescent probes has
provided exceptional tools for their development (37) . Often, product detection
is the measure used to determine inhibition. In vitro assays may not always
only measure the activity of a single enzyme—it is quite possible to examine
the activities of linked components of a pathway. A complicating component of
fluorescent readout assays that must be considered is that compounds (putative
inhibitors) may alter the fluorescent properties of the probe being measured
necessitating the need for secondary assays (as described above).
In vitro inhibition assays need not be only for enzymatic targets. Purified
structural components may also be investigated although assay development
and interpretation of results may be more challenging. Assays to determine
affinity of binding, both of protein-protein and nucleic acid interactions, have
been developed and can be used in moderate throughput (42) . These can
include nuclear magnetic resonance or mass spectrophotometric techniques, as
well as fluorescent modulation or calorimetric methods. Obvious drawbacks to
these methods are the need for pure and stable components and challenges in
identifying inhibitors capable of disrupting high-affinity interactions that will