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 13:02
    Last Modified: 16 May 2013 13:02
    URI: https://ueaeprints.uea.ac.uk/id/eprint/42415
    DOI:

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