LSHM-CT-2007-037765
106743583
SIXTH FRAMEWORK PROGRAMME
PRIORITY 1
LifeSciHealth
Life sciences, genomics and biotechnology for health
LSHM-CT-2007-037765
PHOTOLYSIS
Specific Targeted Research Project
Development of flash photolysis for deep uncaging in vivo
and high throughput characterisation of neurotransmitter
gated ion channels in drug discovery
Publishable Final Activity Report
Period covered:
Date of preparation:
Start date of project:
Duration:
Project coordinator - Project coordinator
organisation name
Revision
March 1st 2007 – August 31st 2010
November 26th, 2010
March 1st 2007
3 years
David Ogden (France)
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1.- Projet Execution .................................................................................................. 3
1.1 Project Objectives: New developments in photolysis. ................................................................... 3
Introduction: background and state of the art. ................................................................................. 3
Development and availability of new probes: .................................. Error! Bookmark not defined.
New TP probes: ............................................................................... Error! Bookmark not defined.
x-ray Photolysis: .............................................................................. Error! Bookmark not defined.
Spatial definition of photolysis: ........................................................ Error! Bookmark not defined.
Photolysis in drug discovery: ........................................................... Error! Bookmark not defined.
Identifying targets for drug discovery: ............................................. Error! Bookmark not defined.
Neuroscience Research: ................................................................. Error! Bookmark not defined.
Overview of the Participating Groups and their field of research ........................................................ 6
Coordinator contact details: ................................................................................................................. 7
Photos to illustrate the project : ........................................................................................................... 7
Project public website: ......................................................................................................................... 7
2.- Dissemination and use ....................................................................................... 7
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1.- Projet Execution
1.1 Project Objectives: New developments in photolysis.
Introduction: background and state of the art.
Signalling within and between cells is usually with small, diffusible molecular messengers
that impart specificity, spatial range and direction to the signal propagation. Each messenger
binds to and activates one or a few kinds of receptor on the target cells. There are many
examples in cell-cell signalling: the neurotransmitters glutamate, GABA (gamma amino
butyric acid) and glycine mediate fast millisecond timescale synaptic transmission in the
brain; there are slower ‘neuromodulators’ that affect neuronal excitability; local hormones
mediate defense mechanisms in the immune system and vascular inflammatory responses;
and there are diffusible morphogens in development and long term cell control. Intracellular
signals include calcium ions which are involved in many secretory and contractile processes,
small nucleotide second messengers such as cAMP and cGMP, and the inositol phosphates.
The speed and efficiency of transmission is achieved with close apposition, on a micron
scale, of the release site and the target receptors. This presents the major technical problem
in cell physiology and neuroscience of mimicking the spatiotemporal scale of physiological
events in an experimental context. Photolysis is an experimental tool that has been
increasingly applied to cell physiology over 3 decades. The idea is straightforward, an inert
photolabile precursor of the physiological ligand – the ‘cage’- is equilibrated in the tissue and
the ligand released by a pulse of light. When combined with laser microscopy the
spatiotemporal requirements to mimic physiological events can in principle be met and the
implementation and development of this idea is fundamental to the Photolysis programme.
Areas identified for improvement were the photochemistry of two-photon excitation and the
availability of probes; the spatial and temporal modification of the excitation light; the
application of the method in drug discovery. The Photolysis consortium comprises
photochemists, photonics experts, high throughput specialists and neuroscientists to facilitate
the development and application of photolysis in neuroscience and cell physiology.
The specific aims were
(1) to apply current photochemistry to generate new tools for neuroscience research and
to develop new photochemistry to make better cages particularly for two-photon
photolysis
(2) to develop photonics methods to improve the efficiency of photolysis and to better
define the spatial dimensions
(3) to develop methods for applying photolysis in drug discovery, and
(4) to support research programmes in neuroscience. SMEs were involved with each
aspect; in the chemistry of uncaging, in drug discovery and in research.
Development and availability of new probes:
A number of new cages have been made available to the neuroscience and cell biology
communities as a direct consequence of the participation of Tocris-Cookson PLC (Partner 6).
The cages were collaboratively evaluated by neuroscience labs in Paris (Partner 1-CNRS),
London (consultant J. Corrie, MRC-NIMR) and Prague (Partner 7-DCN).
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Localised laser photolysis at 405 nm of cages based on nitroindoline and DMN
chromophores has been shown to be a more efficient alternative to photolysis with twophoton excitation by pulsed lasers and is substantially less expensive (Partner 1-CNRS and
MRC-NIMR).
Although two photon absorption and photolysis are intrinsically very inefficient an enhanced
implementation of TP photolysis would greatly improve the depth of uncaging in brain slices
and in vivo. Two approaches have been taken to achieve this. The photolysis of nitroindoline
cages was made more efficient by an intramolecular antenna triplet sensitizer. However, as a
consequence the resulting excitation has a TP absorption maximum in the red. A red pulsed
laser source has been implemented (Partner 3, the University of Strathclyde) and the
efficiency of photolysis of the sensitized cage has been evaluated at different pulse widths
(Partner3-UoS, Partner 1-CNRS). The efficiency of photolysis of the sensitized
nitroindolines increases at long pulse durations by up to100 fold.
New TP probes:
The problem of the inefficiency of TP photolysis has also been approached by developing
new photochemistry to improve TP absorption by Partner 1-CNRS. This has been based on
modular syntheses and, as far as is possible, with rational design. The programme has
generated dipolar and octupolar chromophores with large TP absorption cross-sections.
Further, in collaboration with mechanistic photochemist M Blanchard-Desce (consultant,
CNRS Rennes), we have compared the properties of several reagents with differing
structures in order to determine a rational basis of chromophore design for TP photolysis.
x-ray Photolysis:
Although TP photolysis has significantly improved penetration in tissue when compared to
one-photon, it is restricted at most to 1 mm from the irradiated tissue surface. A significant
spin-off from the development of novel cage photochemistry here has been the finding that
the use of tightly complexed lanthanides as intramolecular antennae generates cages that
are photolysed by x-rays, the energy from the electron rearrangements induced in the
lanthanide are coupled by local intramolecular energy transfer to the photolabile group,
producing photorelease. Because of the penetration of x-rays this development makes
photolysis possible in vivo for research applications in whole animal physiology. It has further
potential for drug delivery in cancer chemotherapy to allow the release of toxic agents in
target tissues with low doses of x-rays. Furthermore, the targeting of the cage can be
followed by using the strong MRI signal form the lanthanide.
Two European patent applications have been submitted based on the novel photochemistry
developed in the Photolysis programme.
Spatial definition of photolysis:
Photolysis excitation is generally applied by laser scanning microscopy where the spatial
dimension is addressed sequentially pointwise. An alternative is to use wide field holographic
methods which permit simultaneous excitation in defined areas of the field. This approach
has been developed by Partner 2-INSERM as part of this programme. Spatial light
modulators are used to pattern the illumination of the microscope field to address specific
areas. This method was applied initially to one-photon photolysis of nitroindoline cages. The
method as initially implemented showed marked laser speckle. Recently the method has
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been refined by using generalised phase contrast and temporal focusing of TP excitation to
excite channel rhodopsin targeted to particular neurons by Partner 2-INSERM.
Photolysis in drug discovery:
Many drugs acting on ion channels and neurotransmitter receptors show a use dependent
action. To see effects of this kind in primary screens and particularly to study drug effects on
rapidly desensitizing receptors, it is necessary to produce fast receptor activation. This can
be achieved by photolysis. The incorporation of photolysis in a high throughput screen has
been developed in the Photolysis programme by a collaboration between the Institute of
Photonics at the University of Strathclyde (Partner 3-UoS) and SME Flyion (Partner 5Flyion) who specialize in automated patch clamp for high throughput parallel patch clamp.
The technical problems associated with cost-effective light application by a near-UV LED and
the perfusion system required for application and removal of the cage have been solved and
a prototype apparatus has been implemented.
Identifying targets for drug discovery:
Ligand gated ion channels in the brain are important targets. Two of these, the NMDA
subtype of excitatory glutamate receptor and the vanilloid receptors important in pain
pathways, have been investigated in the Department of Cellular Neuroscience at the
Academy of Sciences of the Czech Republic, Prague (Partner 7-DCN).
The NMDA receptors are known to be important in neuronal plasticity. DCN Partner 7 have
shown important correlations between neuromodulatory actions of neurosteroids and also of
second messengers on the channels and their primary amino acid structure. Further, a
database of 90 mutations of the TRPA1 receptor has been generated that will serve as a
basis for HTS-P of potential TRP ligands effective in pain.
Neuroscience Research:
Four partners in the consortium have conducted research in neuroscience as well as
contributing to the development of photolysis techniques.
Partner 1-CNRS has published two studies;
(1) demonstrated a functional interaction between fast synaptic transmission and slow
metabotropic receptors at synapses between parallel fibres and Purkinje neurons of
the cerebellum mediated by protein tyrosine kinase/phosphatase.
(2) described a novel synaptic event in the presynaptic axon of cerebellar interneurons
that is due to the release of GABA at the same synaptic site. It is proposed that this
provides feedback involved in the development of synapses during cerebellar circuit
building.
Partner 2-Inserm has shown that patterned photorelease can produce additional information
on the distribution of receptors.
Partner 7 DCN has investigated the regulation of NMDA receptors and ankyrin TRP type 1
receptors. They have shown differences in the temperature sensitivity of NMDA receptors,
comparing receptors in situ and recombinant receptors expressed in cell lines. The structure
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– activity relation of endogenous neurosteroids in activating NMDA receptors was studied in
recombinant systems. The mode of activation of TRPA1 receptors by various ligands has
been investigated by pharmacological analysis of the polymodal gating and by comparison of
the primary structures of defined regions associated with gating in TRPa1 and related
channels.
Partner 8-GeneGrafts has developed a novel therapy, somatic cell replacement therapy,
that aims to control the overactivity of brain foci involved in parkinsons disease withput the
side-effects of drug therapy. They have examined the effect of fibroblasts transplanted to
areas responsible for motor dysfunction in hemi-parkinsonian rats, since bursting
synchronous discharges in internal segment of globus pallidus (GPi) are thought to be
partially responsible for the movement disorders of PD. Fibroblasts express gap junctions
and ion channels, and so, when transplanted to brain tissue, can potentially modulate
excessive electrical activity. They demonstrate in vitro that the introduction of fibroblasts into
a network of neurons does not interfere with overall functional measures of activity, while
moderately altering the characteristics of synchronous neuronal discharge. In rats with
unilateral 6-hydroxydopamine lesions of the nigro-striatal dopaminergic pathway,
apomorphine-induced rotations were reduced by more than 60% following ipsilateral
transplantation of fibroblasts to the GPi. L-Dopa-induced dyskinesia was also significantly
reduced. Fibroblast transplantation is shown to be a potential alternative treatment strategy
for the parkinsonian patient.
-
Overview of the Participating Groups and their field of research
Participant
name
1
2
3
Centre National de la
Recherche Scientifique
Institut National de la
Santé et de la Recherche
Médicale
University of
Strathclyde
Part.
short
name
CNRS
David
Ogden
INSERM
Serge
Charpak
UoS
5
Flyion GMBH
FL
6
Tocris - Cookson
TC
8
Dept of Cellular
Neurophysiology, Institute
of Physiology,AS CR,
v.v.i
GeneGraft
9
Inserm-Transfert
7
Scientific
team
leader
DCN
GG
IT
Gail
McConnell
Albrecht
LeppleWienhues
Duncan
Crawford
Ladislav
Vyklicky
Yair Feld
Isabelle
Geahel
Expertise
Channel biophysics, synaptic
transmission and optical imaging
in neuroscience. Synthetic
organic chemistry Photochemistry
Optical imaging in vivo
Optical methods
in neuroscience
Photonics and laser science in
Biomedicine
High throughput patch clamp
platforms for drug discovery.
Synthesis of ligands for
neurotransmitter receptors
Functional characterization of
receptors and ion channels
Somatic cell therapy
Project management/ business
development
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Country
France
France
UK
Germany
UK
Czech
Republic
Israel
France
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Coordinator contact details:
Prof. David Ogden
CNRS UMR 8118 Physiologie Cerebrale,
Universite Rene Descartes Paris 5,
45 Rue des Saints Peres, 75006 France
Photos to illustrate the project :
Project logo:
Project public website:
http://www.photolysis.eu/
2.- Dissemination and use
A number of significant advances have been made in all aspects of the initial proposal, and it
is clear that the deviations from the initial proposals have followed good scientific practice.
Two patents have been awarded for novel photochemistry.
10 new probes are available to neuroscience and cell physiology research communities.
Significant improvements have been made photonics applied to microscopy and photolysis,
in the sources available for excitation and the spatial distribution of excitation in full field
microscopy.
Through the Photolysis research project Flyion has now achieved the ability to implement
uncaging capabilities with optical means. To this day this feature is completely unique in the
area of automated patch clamp systems.
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The output of the results the study on the Modification of neural network discharge by
transplantation of cells over-expressing ion channels provided insight on the understanding
of the mechanism of action of fibroblasts on neuronal network, and specifically on the
behavior of parkinsonian rats when transplanted to the GPi. The experimental results
enabled Genegraft company to move forward with non-human primate study.
Finally, significant results have been obtained in neuroscience research with a total of close
to twenty papers published.
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