1. Drug Discovery Process Massimiliano Beltramo, PhD

2. 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

3. Target validation flow-chart Biology of the target Expressio n Profile Expression in relevant areas Expression in pathologic state Expression consistent between human and animal model Functional role Role in human disease In vivo studies In vitro studies Scientific rationale definition Predicted side effect profile Predicted additional indications

4. Platelet activation cascade X Your drug Scientific rationale

5. Increase expression in microglia after induction of chronic pain Reduction of pain in P2Y12 KO mice What is the link between microglia and pain reduction?? Chronic pain

6. Do we need an agonist or an antagonist? Gi AC ATP cAMP Stimulate AC with forskolin ↓ Add test compound ↓ Detect modulation of cAMP level fsk stimulation [Drug] cAMP level agonist 100% inactive/ antagonist inverse agonist

7. A New Paradigm for Drug Discovery Gene of Unknown Function Determination of Cellular Function Validation as an Attractive Drug Target High-Throughput Screening Assays Established against Target Protein Expression/ Protein Purification Identify Lead Inhibitor Chemical Structure- Activity Analysis Therapeutic Index Pharmacokinetics - Gene-to-Function -

8. R&D process for a new drug Exploratory development Full development IDEAIDEA DRUGDRUG CANDIDATE POCTARGET Therapeutic research Exploratory research Hit to lead

9. Discovery of new active molecules Biological target Assay Test to assess target biological activtiy Chemical compound library Hit Lead Drug candidate HIGH THROUGHPUT SCREENING (HTS) Hit to lead

10. How to maximize the chance of success: High diversity of chemical library compounds Capacity to screen rapidly a large number of compounds Robust and standardized assay Low false positive and false negative rate Hit to lead

11. Chemical library  Derived from combinatorial chemistry  Collection from previous project  Commercially available Hit to lead

12. The Old Way –One chemist, one compound, 1 gram recrystallized and stored forever A chemist might make 50-100 compounds a year Combinatorial Synthetic Methods –“Mix and Split” Solid Phase Synthesis – Multi-Parallel Synthesis and Purification – Automation of Synthetic and Analytical Methods A chemist today might make > 10,000 compounds/year Compound synthesis Hit to lead

13. Combinatorial chemistry R1 R2 R3 R4 Scaffold or pharmacophore 20 R1 20 R2 13 R3 10 R4 20 X 20 X 13 X 10 = 52,000 compounds !! Substituents N Hit to lead

14. High Throughput Screening (HTS) is the process through which a high number of small molecules (>50,000-100,000 per day) are tested on the target of interest. To perform an HTS is necessary an adequate in vitro assay High throughput screening Hit to lead

15.  Require the expression of the target:  recombinant cell lines  native systems In vitro assay development  Two types: 1. “cell-free” 2. “cell-based” Hit to lead

16. General requirements - Target pharmacology must be preserved - Signal have to be: 1. robust 2. reproducible 3. easy to measure - experiment should require minimal handling - miniaturized - automatized - low cost In vitro assay development Hit to lead