Mode-of‐action of PAF26 and the discovery of more active and stable cyclic PAF26 derivatives
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Zhao2017.pdf (53.55Mb)
Date
07/07/2017Item status
Restricted AccessEmbargo end date
31/12/2100Author
Zhao, Can
Metadata
Abstract
The
significance
of
fungal
infections
has
been
grossly
underestimated.
Only
a
few
drugs
are
clinically
available
to
treat
life-‐threatening
fungal
infections,
and
resistance
against
these
drugs
is
rising.
Antifungal
peptides
(AFPs)
are
being
actively
explored
as
novel
pharmaceuticals.
PAF26
is
a
de
novo
designed
hexapeptide
possessing
N-‐terminal
cationic
and
C-‐terminal
hydrophobic
regions.
Previously
the
roles
of
each
of
these
motifs
in
the
antifungal
mode-‐of-‐action
of
PAF26
have
indicated
that
it
involves
three
stages:
interaction
with
the
plasma
membrane,
internalisation,
and
cell
killing.
The
overall
aim
of
my
project
was
to
obtain
further
insights
into
its
mode-‐of-‐action
and
develop
more
active
antifungal
derivatives
of
PAF26.
Three
experimental
fungal
systems
were
used
in
this
study:
the
model
Neurospora
crassa,
the
human
pathogen
Aspergillus
fumigatus
and
the
plant/human
pathogen
Fusarium
oxysporum.
The
first
objective
of
the
study
was
to
evaluate
the
impact
of
different
fluorescent
labels
on
the
intracellular
localisation
and
antifungal
properties
of
PAF26.
For
this
purpose
a
library
of
PAF26
labelled
with
13
different
fluorophores
was
synthesised.
This
library
contained
PAF26
conjugates
of
broad
chemical
and
spectral
diversity.
These
fluorescent
PAF26
conjugates
were
analysed
by
live-‐cell
imaging
and
tested
for
their
antifungal
activities.
The
different
fluorescent
labels
were
found
to
have
significant
impacts
both
on
intracellular
localisation
and
antifungal
activities.
TMR,
carboxyfluorescein,
NBD
and
DMN
were
found
to
be
the
best
labels
for
live-‐cell
imaging
because
they
had
the
least
influence
on
the
intracellular
localisation
and
antifungal
activity
of
PAF26.
The
second
objective
was
to
identify
target
proteins
of
PAF26
in
N.
crassa
cells.
A
large
number
of
proteins
were
identified
as
binding
to
PAF26
from
a
protein
pull-‐down
and
mass
spectroscopy
analysis
using
TMR-‐
and
fluorescein-‐labelled
PAF26.
One
of
these
proteins
was
the
highly
abundant
plasma
membrane
ATPase
PMA-‐1.
An
in-‐silico
analysis
showed
that
PMA-‐1
is
likely
to
be
a
major
target
protein
of
PAF26.
The
final
objective
was
to
develop
novel
antifungals
based
on
PAF26
with
improved
activities
and
stability.
Novel
cyclic
derivatives
of
PAF26
were
designed
in-‐silico
against
PMA-‐1.
These
peptides
were
synthesised
and
tested
against
N.
crassa,
A.
fumigatus
and
F.
oxysporum
and
were
found
to
have
higher
activities
(at
the
sub-‐micromolar
level)
and
greater
stability
than
the
linear
PAF26.
Overall
this
study
has
provided
novel
mechanistic
insights
into
the
mode-‐of-‐action
of
PAF26
and
discovered
novel
highly
active
antifungal
peptides
with
clinical
potential
as
therapeutics.
