1. Functional autoradiography: Incorporation of [ 35 S]-GTP γ S In vitro target function [ 35 S]GTPγS X

2. In vitro target function A case study SP1999 (Laitinen et al 2001) Functional autoradiography: incorporation of [ 35 S]-GTPγS

3. 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

4. A case study SP1999 In vitro target function 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)

5. Functional imaging in vivo Light imaging –Bioluminescence –Fluorescence Magnetic resonance –MRI fMRI Poistron emission tomography (PET) In vivo target function

6. 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)

7. 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) Target validation

8. 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

9. 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

10. Exploratory development Full development IDEAIDEA DRUGDRUG CANDIDATE POCTARGET Therapeutic research Exploratory research 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

11. 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..) NegativeThe 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

12. 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

13. Learning and memory: Water Maze In vivo target function

14. 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 coordination In vivo target function

15. Target Knockdown In vivo target function

16. The central tenet In vivo target function No gene X No protein X No phenotype X

17. Techniques to KO a gene product Reduction of protein activity Aptamers Reduction of protein transcription Oligoantisense RNA Interference Gene KO Deletion of target gene In vivo target function

18. A case study SP1999 Mechanical or tactile allodynia von 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

19. A case study SP1999 In vivo target function (Foster et al 2006) KO mice showed a reduced platelet aggregration and a prolonged bleeding time

20. 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 Target validation