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 Our work on cationic antimicrobial
peptides arose from the convergence of two lines of research. The first was
our work on self promoted uptake for which we had demonstrated that
polycationic antimicrobials such as polymyxin B and aminoglycosides are
taken up across the outer membrane by a novel pathway that involves
binding of these polycations to the divalent cation binding sites on LPS, and disruption
of these sites, leading to an increased permeability of the outer membrane
to probe molecules, but more significantly to the polycation itself. It
occurred to us as early as 1984 that cationic antimicrobial peptides such as
insect cecropins and mammalian neutrophil defensins might also access this
pathway. The second line of research involved a series of studies on the
mechanism of killing of Pseudomonas aeruginosa by phagocytic cells,
and our resultant investigations on the mechanism of action of rabbit
defensins from macrophages and neutrophils which we showed in 1986 were able
to access the self promoted uptake system. The difficulties that we
experienced in isolating substantative quantities of defensins led us to
attempt to devise a recombinant DNA method for manufacturing such
antimicrobial cationic peptides. After about 4 years we managed to devise a
procedure for general manufacture of recombinant cationic peptides in
sensitive bacteria. This technology, and several classes of peptides, were
spun out into
Micrologix Biotech Inc (now renamed Migenix), a Toronto Stock Exchange
listed company (trading symbol: MGI) established in Vancouver around this
technology. Micrologix has now taken peptides into clinical trials and one
of these has just started a phase IIIb clinical trial to confirm efficacy in
preventing colonization and tunnel infections associated with the
implantation of central venous catheters. Another company that has licensed
in a large amount of lab technology in the antimicrobial peptide arena is
Helix Biomedix. |
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 The lab has been studying four structural
classes of cationic peptides: (1) amphipathic alpha- helices based on a
fusion of silk moth cecropin and bee melittin, (2) extended peptides based
loosely on the structure of the cattle neutrophil peptide indolicidin, (3)
beta-sheet peptides including beta-hairpin peptides based on the horseshoe
crab peptide polyphemusin, and cyclic beta-sheet peptides synthesized by our
collaborator, Dr. Bob Hodges from the University of Alberta in Edmonton,
based on the general structure of bacterial gramicidin S, and (4) loop
peptides cyclized with a single cysteine disulphide based on cattle
neutrophil bactenecin. We have studied the mechanism of action of
representatives of most of these classes. Basically these peptides interact
with the surface of Gram negative bacteria and are taken up by self-promoted
uptake. They then insert into the cytoplasmic membrane under the influence
of the transmembrane electrical potential gradient (which in bacteria is
about -150 mV oriented internal negative so as to electrophorese the
cationic peptides towards the membrane). They assemble in the membrane into
multi-state channels which we have described via the “aggregate model”, and
in many cases cross the cytoplasmic membrane to access cytoplasmic targets,
or in some cases permeabilize the cytoplasmic membrane barrier. It should be
stated that in the past many people in the antimicrobial peptide field
favoured the latter mechanism for most peptides; however, our own published
evidence appears to be more consistent with cytoplasmic targets for many
peptides. The better cationic peptides act very rapidly (within minutes) to
kill cells and have very broad ability to kill microbes including the most
important Gram negative and Gram positive pathogenic bacteria as well as
fungi like Candida albicans. They are by and large unaffected by the
most common clinical mechanisms of antibiotic resistance, and in our hands
do not easily select resistant mutants even after multiple passages on
sub-MIC doses of cationic peptides. We have been able to demonstrate that
certain a-helical
peptides are effective against systemic infections of mice by P.
aeruginosa, and are also effective against chronic rat infections when
delivered by aerosol.
The ability of these peptides to access the self
promoted uptake system and permeabilize the outer membrane explains in part
one of their more useful properties. Since the outer membrane is normally a
semi permeable barrier to conventional antibiotics, we would predict that
cationic peptides, in overcoming this barrier, would promote the activity of
such conventional antibiotics. We have demonstrated this in vitro for
selected cationic peptides, and further demonstrated that one peptide,
CP-26, can reverse all of the major clinically important mechanisms of
antibiotic resistance in P. aeruginosa including de-repressed beta-lactamase,
DNA gyrase mutations and efflux-mediated multiple antibiotic resistance. The
peptides also demonstrate synergy with the host defence molecule lysozyme
and different peptides demonstrate synergy with each other.
Our current studies are concentrating on solving the
three dimensional structures of selected peptides, rational
computer-assisted design of new peptide variants for structure activity
relationship studies and random design of variant peptides by peptide array
procedures. We recently solved the structure of several peptides by NMR. We
are also interested in the ability of peptides to interact with mammalian
cells as described in more detail under innate
immunity studies, and in particular are interested in the ability of
peptides to cross membranes, an activity that in mammalian has been ascribed
to a group of peptides termed cell penetrating peptides. Studies on the
mechanisms of action of the peptides against bacteria and viruses and on
mechanisms of resistance continue in the lab.
We have evaluated different methods for MIC determination for cationic
peptides and have proposed a standard method as described in our
Methods section.
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