Pharmacological insights into C-­‐C motif chemokine receptor 5 mediated chemotaxis

Jacques, Richard (2013) Pharmacological insights into C-­‐C motif chemokine receptor 5 mediated chemotaxis. Doctoral thesis, University of East Anglia.

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Abstract

Aim:
Despite
being
well
validated
as
a
therapeutic
target,
no
chemokine
receptor
antagonists
to
be
used
as
therapeutic
agents
in
inflammatory
or
metastatic
disease
have
made
it
to
market.
This
is
in
part
due
to
receptor
redundancy,
but
also
a
lack
of
understanding
with
regard
to
cytoplasmic
signal
transduction
linking
activated
chemokine
receptors
to
chemotaxis.
Resolving
signal
transduction
pathways
in
model
chemokine
receptor
systems
may
allow
intracellular
drug
targets
to
be
identified,
bypassing
the
difficulties
associated
with
extracellular
chemokine
receptor
blockade.
Methodology:
Experimentation
was
undertaken
in
THP-­‐1
monocytes
expressing
the
chemokine
receptors
CCR5
and
CCR1
and
in
stably
CCR5-­‐transfected
HeLa
and
CHO
cell
lines.
Small
molecule
inhibition
and
protein
overexpression
was
used
before
chemotaxis
and
calcium
release
assays
to
measure
cellular
responses.
Immunocytology
was
used
to
determine
the
effect
of
protein
blockade
on
receptor
internalisation,
protein
localisation
and
the
formation
of
cellular
structures
associated
with
migration.
Experiments
were
also
performed
in
activated
primary
tissue
for
comparative
analysis
and
validation
of
results
in
normal
human
tissue.
Results:
A
systematic
blockade
of
signalling
proteins
by
small
molecule
means
revealed
that
Gβγ,
ERK1/2,
p38
and
PI3K
are
not
required
for
CCL3
stimulated
monocyte
migration.
GRK2
and
PKC
inhibition
along
with
internalisation
blockade
showed
antagonistic
effects
on
the
ability
of
cells
to
migrate,
suggesting
arrestin
dependent
signalling
was
involved
in
chemotaxis.
Inhibition
of
dynamin,
Grb2
and
non-­‐receptor
tyrosine
kinases
were
equally
effective
at
blocking
migration
in
THP-­‐1
cells
but
less
effective
at
blocking
CXCL11
stimulated
migration
in
activated
PBLs.
Conclusions:
This
study
has
shown
that
CCL3
stimulated
chemotaxis
through
CCR5
does
not
occur
through
typical
G-­‐protein
mediated
signalling,
but
maybe
therapeutically
accessible
by
inhibition
of
dynamin
and
Grb2.
Additionally
the
differences
in
dynamin
inhibitor
efficacy
suggest
that
the
production
of
migration
specific
dynamin
inhibitors
may
be
possible.
Overall
the
research
in
this
thesis
has
identified
novel
targets
for
therapeutic
intervention
in
diseases
where
dysregulation
of
chemokine
receptor
mediated
migration
are
causative.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Pharmacy
Depositing User: Mia Reeves
Date Deposited: 16 May 2013 12:02
Last Modified: 16 May 2013 12:02
URI: https://ueaeprints.uea.ac.uk/id/eprint/42415
DOI:

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