Download In vivo target function

In vitro target function
Functional autoradiography:
Incorporation of [35S]-GTPγS
X
[35S]GTPγS
In vitro target function
A case study SP1999
Functional autoradiography: incorporation of [35S]-GTPγS
(Laitinen et al 2001)
In vitro target function
A case study SP1999
Time-lapse confocal microscopy
Stimulation of hippocampal slices with ADP induces
microglia process extension and cell migration
(Haynes et al. 2006)
In vitro target function
A case study SP1999
Linking gene to function: platelet aggregation
A patient with a phase shift mutation in the gene
showed a reduced and reversable platelet aggregration
after ADP stimulation
(Hollopeter et al 2001 and Humbert et al 1996)
In vivo target function
Functional imaging in vivo
• Light imaging
– Bioluminescence
– Fluorescence
• Magnetic resonance
– MRI
• fMRI
• Poistron emission tomography (PET)
In vivo target function
A case study SP1999
Two photons laser microscopy in vivo
Extension of microglia processess after ATP
injection (a) or focal laser ablation (c)
(Haynes et al 2006)
Target validation
SP1999 target validation summary
Receptor identification
•
SP1999 is a receptor for ADP, belong to the purinergic receptor family and has been named P2Y12
Expression profile
•
mRNA is present in brain and platelets (northern blot, PCR, and in situ hybridization)
•
The protein is expressed in several brain region and is localized in microglia cells (immunocytochemistry)
•
Protein expression is increased in a disease models of chronic pain (immunocytochemistry)
Functional activity
• Activation of Gi protein (cAMP and GTPγS)
• Modulation of microglia activation (live cell imaging)
• Role in platelet aggregration (genetic analysis and in
vitro assay)
In vivo target function
Linking a target to a pathology:
in vivo function
• Genetic manipulation
• Pharmacological manipulation
To perform these studies it is necessary
to develop appropriate disease models
In vivo target function
Animal Models
“A laboratory animal model is a model in which basic biology
or behavior can be studied, or in which a spontaneous or
induced pathological process can be investigated
(including the therapeutic effect of drugs), and in which the
phenomenon in one or more respects resembles the same
phenomenon in humans or other species of animal.”
Animal models are used in various phases
of the drug discovery process
TARGET
I
D
E
A
Exploratory
research
CANDIDATE
Therapeutic
research
Exploratory
development
Use of animal models
• Target validation
• Screening/optimization/efficacy profile
• Evaluation of tolerability and toxicity
POC
Full
development
D
R
U
G
In vivo target function
Type of animal models
Experimentally induced disease.
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Behavior (e.g stress)
Pharmacology (e.g. CFA)
Genetic manipulation (e.g. transgenics, knockouts, knock-ins)
Surgery (e.g. nerve cuts, vessels’ ligature)
Spontaneous disease
Naturally occurring with similar mechanisms
to human diseases
(e.g. asthma in cat; Factor VIII deficiency in Irish setter equivalent to
hemophilia A, etc..)
Negative
The disease is not developed by the animal
“why DOESN’T - models”.
(e.g.no atherosclerosis in dog, no AIDS in HIV infected chimpanzees )
In vivo target function
Essential features of an animal
disease model
• Predictive validity
The effects of a test drug in this model correspond to that of
clinically effective drugs.
• Face validity
Phenomenological similarities between the model and the clinic.
• Construct validity
The primary cause of the disease is similar in man
In vivo target function
Learning and memory: Water Maze
In vivo target function
Lomotor activity and coordination
Rotarod
Activity Cage
This model is used to assess motor
coordination, balance, and motor learning.
This model is used to assess horizontal and
vertical locomotor activity.
In vivo target function
Target Knockdown
In vivo target function
The central tenet
X
No gene
X
No protein
X
No phenotype
In vivo target function
Techniques to KO a gene product
Gene KO
Oligoantisense
RNA Interference
Aptamers
Deletion of
target gene
Reduction of
protein
transcription
Reduction of
protein activity
In vivo target function
A case study SP1999
readout
Mechanical or tactile allodynia
von Frey hair test
P2Y12 KO mice do not develop
tactile allodynia after
L5 spinal nerve transection
(Tozaki-Saitoh et al 2008)
In vivo target function
A case study SP1999
(Foster et al 2006)
KO mice showed a reduced
platelet aggregration and a
prolonged bleeding time
Target validation
SP1999 target validation summary
Receptor identification
• SP1999 is a receptor for ADP, belong to the purinergic receptor family and has been named
P2Y12
Expression profile
• mRNA is present in brain and platelets (northern blot, PCR, and in situ hybridization)
• The protein is expressed in several brain region and is localized in microglia cells
(immunocytochemistry)
• Gene expression is increased in some disease models (in situ hybridization)
Functional activity
• Activation of Gi protein (GTPγS)
• Modulation of microglia activation (live cell imaging)
• Role in platelet aggregration (Genetic analysis and in vitro assay)
Potential role in diseases
• Bleeding disorder/Thrombosis
• Chronic pain