Functional autoradiography:Incorporation of [35S]-GTPγS

In vitro target function

[35S]GTPγS

X

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

Learning and memory: Water Maze

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

Target Knockdown

In vivo target function

The central tenetIn vivo target function

No geneX

No protein

X

No phenotypeX

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