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. » » » » 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