Peptide-Based Drug Design

162 Scocchi et al.
membrane interaction, leading to subsequent membrane damage and/or peptide
translocation into the cytoplasm where they can interact with molecular targets.
In most cases, this membrane interaction is directed to anionic phospholipids,
driven by electrostatic effects (4). In some instances, however, as for example
bacterial lantibiotics or some plant defensins, specific membrane components
are involved (respectively, lipid II and sphingomyelins) (5). In any case, direct
interaction with the membrane is a principal factor in the mode of action, subsequently
leading to membrane compromising or peptide translocation.
The proline-rich antimicrobial peptides (PRPs) are thought to act by a
specific two-stage mechanism (6–8): (i) they have the ability to cross biological
membranes without altering them, and so penetrate the bacterial cytoplasm in
which (ii) they bind to, and functionally modify, one or more specific targets (9).
In this respect, one such target has been identified as the molecular chaperone
DnaK for the insect PRP pyrrhocoricin (10,11). It has also been proposed that
the binding of the porcine proline-rich PR-39 to DNA may be involved in its
killing mechanism (12).
A unique characteristic of both insect and mammalian PRPs is that the all-
D enantiomers of the natural peptides are poorly, or not at all, active (6). This
implies that either or both the internalization and target inactivation mechanisms
require a stereospecific molecular interaction. Studies to identify the
mode of action of PRPs must thus take this into account. One procedure is that
of using peptide-based affinity columns to which bacterial lysates are applied
(10). In principle, only tightly binding species (the putative molecular target)
should be retained on the column. In practice, it is difficult to detect other than
cytoplasmic targets and only if these are abundantly expressed. These shortcomings
can be avoided by combining affinity-binding and genetic methods
(13). These are based on selection of bacterial isolates that are more resistant
or more susceptible to PRPs than the parental strain, following isolation and
identification of the genes underlying the altered susceptibility. These methodologies
can be quite potent as they can lead to the identification of (i) proteins
involved in membrane translocation of peptides, (ii) target proteins that mediate
the antibacterial activity of PRPs, or (iii) proteins involved in PRP clearance
(pumps, proteases, etc.) that may be responsible for resistance to peptide. The
following chapter describes a method to select Escherichia coli clones resistant
to the bovine PRP Bac7 that led to the identification of a membrane protein
(SbmA) that may be involved in peptide internalization (14) and of a protease
(OpdB) that could be implicated in PRP resistance.
Clearly, identification of resistance mechanisms and of potential intracellular
targets all lead to information which would be very useful for the development
of novel anti-infective drugs by peptide-based design, once the structure of
these targets is known. In addition, identification of transport proteins that may