Upload
lee-bates
View
220
Download
1
Embed Size (px)
Citation preview
In vitro target function
A case study SP1999
(Laitinen et al 2001)
Functional autoradiography: incorporation of [35S]-GTPγS
In vitro target function
A case study SP1999
(Haynes et al. 2006)
Stimulation of hippocampal slices with ADP induces microglia process extension and cell migration
Time-lapse confocal microscopy
A case study SP1999
In vitro target function
Linking gene to function: platelet aggregation
A patient with a phase shift mutation in the geneshowed a reduced and reversable platelet aggregration
after ADP stimulation
(Hollopeter et al 2001 and Humbert et al 1996)
Functional imaging in vivo
• Light imaging– Bioluminescence
– Fluorescence
• Magnetic resonance– MRI
• fMRI
• Poistron emission tomography (PET)
In vivo target function
Two photons laser microscopy in vivo
In vivo target function
A case study SP1999
(Haynes et al 2006)
Extension of microglia processess after ATP injection (a) or focal laser ablation (c)
SP1999 target validation summaryReceptor 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)
Target validation
Linking a target to a pathology: in vivo function
• Genetic manipulation
• Pharmacological manipulation
In vivo target function
To perform these studies it is necessary
to develop appropriate disease models
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.”
In vivo target function
Exploratorydevelopment
Fulldevelopment
IDEA
DRUG
CANDIDATE POCTARGET
Therapeuticresearch
Exploratoryresearch
Use of animal models
• Target validation• Screening/optimization/efficacy profile• Evaluation of tolerability and toxicity
Animal models are used in various phases of the drug discovery process
Type of animal models
Experimentally induced disease. » 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
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.
Lomotor activity and coordinationIn vivo target function
Techniques to KO a gene product
Reduction of protein activityAptamers
Reduction of protein transcription
Oligoantisense
RNA Interference
Gene KO Deletion oftarget gene
In vivo target function
A case study SP1999
Mechanical or tactile allodyniavon Frey hair test
readout
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
In vivo target function
(Foster et al 2006)KO mice showed a reduced platelet aggregration and aprolonged bleeding time
SP1999 target validation summaryReceptor 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
Target validation