Guest Blogger Lynn Silver of LL Silver Consulting, LLC
The three main
screening paradigms are 1) in vitro screens for enzyme inhibition, ligand
binding, interruption of protein-protein interactions, etc. 2) phenotypic whole
cell screens that select for inhibitors or effectors of specific cellular
targets and 3) empirical, whole cell kill-the-bug-screens.
To start with the
last - for most grants I’ve seen, empirical screening seems mostly applied to
natural products – but this is very inefficient if not coupled with a rapid
dereplication system that can identify knowns, since a very high percentage of
Actinomycete and fungal fermentation broths contain antibacterial agents and a
minuscule number of those will be novel.
Note that most Pharma chemical libraries, and, likely, most other
publicly available libraries, have been screened empirically already. Empirical screens could still be useful if
libraries were previously unscreened, or, for natural products, if the
producing organisms are themselves novel.
But any empirical screening will require extensive follow-up to
establish mechanism and safety.
In vitro assays
for inhibition of enzyme activity (or other readout) are prevalent in grant
applications. Standard rules for HTS
obtain (in establishing z-factors, etc.), but also important is setting up
assays to reflect as well as possible intracellular conditions. For example, if
ATP is present in the cell at 2 mM and you use 100 ?M in the assay – you may
find ATP-competitive agents that will not show activity intracellularly.
With many screens
there will be no true positive control – but that shouldn’t stop you. It is very important, however, to run many
negative controls, antibacterials,
possible interfering compounds – as false positives and high hit rates will
complicate screening and require a good deal of counterscreening to
eliminate. In writing the grant,
discuss planned controls, possible pitfalls and remedies. As Dave noted in his initial write-up, many
in vitro screening hits will not have the physicochemical properties necessary
to enter the cell and will lack antibacterial activity. This is especially true for Gram-negatives
which have a tougher permeability barrier and more contribution from efflux
pumps than do Gram-positives. What will
you do about it? How do you plan to
optimize in vitro hits to gain antibacterial activity – and, importantly, show
that any antibacterial activity you do develop is related to enzyme
inhibition? In the same vein – you may
find that your hit has antibacterial activity – but you must, very early on,
establish that the antibacterial activity is indeed due to inhibiting your
selected target – because this is very rarely true with screening of synthetic
libraries. Some pointers on connecting
in vitro to whole cell activity may be found in O’Neill and Chopra (2004. Exp.
Opin. Invest. Drugs 13:1045-1063) and Silver (2011. Clin. Microbiol. Rev.
74:71-109).
One way of making
the connection is to have a phenotypic assay which will give a whole cell
readout that indicates a specific kind of effect on cellular physiology. For example, inhibitors of cell wall
synthesis should cause Gram-negative bacteria to form spheroplasts in
osmotically stabilized medium. And
extraneous toxic properties of the hit should prevent spheroplast formation –
so spheroplast formation is a good indication that a step (or steps) in cell
wall synthesis has been inhibited. In
fact, the spheroplasting assay was used at Merck and at other pharmaceutical
companies from the early 1960’s as a primary screen for cell wall
inhibitors. Thus phenotypic assays may
be used as secondary to primary in vitro screens but also as another primary
screening paradigm. And primary
screening with a whole cell phenotypic assay has the great benefit of selecting
for compounds with activity against the whole cell.
Other phenotypic
screens may use engineered bacterial strains to turn on a reporter gene if a
specific target or pathway is inhibited – for example by taking advantage of
stress regulons (see Fischer et al. 2004. Genome Res. 14:90-98), by
specifically downregulating a desired target (for example by turning on
antisense; see Singh et al. Curr. Opin. Drug Disc. Devel. 10:160-166) in order to sensitize to
inhibitors or using a pair of strains engineered so that one should be
sensitive to an inhibitor while the other is resistant (see Testa. 2003. Curr.
Pharm. Biotechnol.4:248-259 for a screen for inhibitors of the non-mevalonate
isoprenoid synthetic pathway). Concepts
of cell based screening are discussed in Mills and Dougherty (2012. pp.
901-929. In Antibiotic Discovery and Development. Eds. Dougherty and Pucci. Springer.) As with any antibacterial screening
methodology, hits must followed up with tests of specificity (hitting the
desired target[s] only), selectivity (hitting bacteria and not their hosts) and
testing and optimization for the many layers of necessary pharmacological
requirements for development (which presumably Dave or another guest blogger
will discuss.
In writing
screening grants, first read as much of the literature on screening as you can
and recognize that there has been much of it done over the past 50 or more
years. It raises the reviewers’ hackles
to read a dismissive introduction that says antibacterial screening has always
been done empirically and now you will do it rationally. It has been done rationally – BUT it must be
noted that almost all the classes of antibacterial drugs in clinical use were
FOUND by empirical and not rational methods.
Thus it would behoove us all to consider why such approaches haven’t
worked so well (see Silver. 2012. Clin.
Microbiol. Rev. 74:71-109 and Gwynn et al., 2011. Ann. N. Y. Acad. Sci. 1213:
5-19) and approach new projects and grant-writing with an aim of improving on
the past.
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