Target-Based Antimicrobial Drug Discovery - Bacterial Pathogenesis: Methods and Protocols - page 256

interest but have acceptable amino acid sequence divergence from human gene
products.
Development of broad-spectrum antibiotics should minimally include a
comparison of genomic content of bacteria involved in respiratory tract, surgical
site, and urinary tract infections (6) . Bioinformatics procedures can also provide
clues into a protein's function and aid in assay development to a certain
extent. For instance, one can determine whether a putative target contains ATP
hydrolysis domain, which can in turn be exploited when developing an assay
using purified protein (i.e., ATPase activity assay). While in silico techniques
provide a starting point for identifying and perhaps prioritizing putative targets
for drug discovery, wet laboratory tactics are needed to definitively determine
whether a gene is essential for cellular survival. This can be accomplished
through both indirect or, preferably, direct approaches (7) . The former involves
using random mutagenesis with agents such as nitrosoguanidine to identify and
characterize conditional lethal mutants. Another indirect means of assigning
essentiality of putative novel targets involves analyzing members of a trans-
poson mutant library (8) . Transposon library members actually contain inser-
tions into non-essential genes, which can be subtracted from the genetic compo-
sition of a bacterial genome to indicate which genes are essential for in vitro
survival. Transposon subtractive analysis has successfully identified genes that
are essential for the growth of Escherichia coli and Helicobacter pylori (9,10) .
One can also directly determine whether a putative target is essential through
targeted gene disruptions, through various methods the description of which
are beyond the scope of this discussion [reviewed in (7) ]. Nonetheless, the
overall goal is to establish that an essential gene cannot be completely knocked
out. Below, we describe drug discovery efforts in these three “classes” of
targets (characterized and poorly characterized essential genes, and virulence
determinants).
3. Types of Targets
3.1. Oldies but Goodies
Until very recently, antibacterial targets could be clustered into a limited
number of categories. Historically, antibiotics have been natural fermentation
products derived from organisms seeking to protect themselves from microbial
attack and tended to be complex molecules targeting specific macromolecular
structures or pathways (11) . Interestingly, recent analysis of essential gene
products analyzed by several genomic and proteomic methods recurrently
identified the same components as antimicrobial targets (5,12) . As is so often
true in the case of antimicrobial strategy, nature appears to know best.