Alma Mater Studiorum – University of Bologna CHEMICAL AND GENOMICS-BASED STRATEGIES

1. Alma Mater Studiorum – Universityof Bologna CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS Toll-likeReceptor Signalling Pathway: Dual Luciferaseapproach in thediscovery ofnoveltherapeuticopportunities Dr. Monica Baiula Bologna - June 25th 2013

2. INNATE IMMUNE RESPONSES TO INFECTIONS AND TOLL-LIKE RECEPTORS Innate immunity is evolutionarily ancient and selected mechanisms are conserved TLRs play a cucial role in innate immune responses to infection CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

3. INNATE IMMUNE RESPONSES TO INFECTIONS AND TOLL-LIKE RECEPTORS The family ofTLRsrepresent the major microbe-sensing system in mammals Different cell types may express a diverse set of TLRs The selective engagement of adaptors defines the mode of signal transduction CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

4. TOLL-LIKE RECEPTORS: FUNCTIONAL ROLE TOLL-LIKE RECEPTORS (TLRs) PATTERN RECOGNITION RECEPTORS (PRRs) expressed in many different cell types TLRsactasSENTINELSagainst a wide rangeofPathogens-AssociatedMolecularPatterns (PAMPs) and Danger-AssociatedMolecuar Pattern molecules (DAMPs) WIDE RANGING IMPACT ON SEVERAL DISEASE SETTINGS CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

5. TOLL-LIKE RECEPTORS IN HEALTH AND DISEASE BROAD EXPRESSION PROFILE TLRsrecognizeconservedstructuresofmicrobes and endogenous (host-derived) molecules TLRs = PPR pattern recognitionreceptor Pathogen-associated Molecular Patterns (PAMPs) Lipoproteins, lipids, dsRNA, ssRNA, un-methylated CpG TLRs are both KEY HOST DEFENCE MECHANISMS and RAPID RESPONSE MECHANISM TO LOCAL TISSUE DAMAGE Danger-associated Molecular Patterns (DAMPs) Intracellular proteins, ECM proteins, purine metabolites, heparin sulfate, fatty acids, DNA CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

6. TOLL-LIKE RECEPTORS: CRITICAL IMMUNE SENSORS TOLL-LIKE RECEPTORS (TLRs) PATTERN RECOGNITION RECEPTORS (PRRs) belonging to Innate Immune System Immediate first line of defense against a diverse repertoire of invading microbial pathogens TLRsabilitytoengagedifferentintracellularsignallingmolecules and cross-talk withotherregulatorypathwaysiscrucial in shapingTYPE, MAGNITUDE, DURATIONofinflammatoryresponse CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

7. TARGETING TOLL-LIKE RECEPTORS: NOVEL POTENTIAL THERAPEUTICS TOLL-LIKE RECEPTORS (TLRs) fullfillmanyof the criteriathat are requiredtobeconsideredpotential THERAPEUTIC TARGETS • TLRs are over-expressed in several diseases • TLRs knock-out mice are RESISTANT to disease in disease models • Genetic differences in TLRs or their SIGNALLING PROTEINS correlate with increased risk of disease • Many polymorphisms in genes that encode TLRs and their signaling molecules have been associated with human disease progression and susceptibility • Hennessey EJ et al. Nature Reviews Drug Discovery 2010 CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

8. Extracellular Leucine-rich Repeats Toll/IL-1 receptor (TIR) signalling domains TOLL-LIKE RECEPTORS: STRUCTURAL FEATURES TOLL-LIKE RECEPTORS (TLRs): Type I integral transmembrane glycoproteins To date more than a dozen of TLRs TLR 1 -9 conserved among humans and mice TLR10 selectively expressed in humans CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

9. TLRs are located on the outer cell membrane or on endosomes Each TLR detect a specific set of ligands All TLRs mediate the production of inflammatory cytokines TLR3, TLR4, TLR7, TLR8 and TLR9 stimulate the production of type I Interferons CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

10. CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

11. TARGETING TOLL-LIKE RECEPTORS: WHEN IT COULD BE USEFUL? • TLRs activation promotes maturation of APCs which in turn direct the induction of adaptive immune responses => TLRs AGONISTS (cancer, infections) • TLRs activation promotes inflammatory cytokines production and play a pathogenic role in many diseases with inflammatory basis => TLRs ANTAGONISTS (asthma, atherosclerosis, multiple sclerosis, rheumatoid arthritis, systemic lupus eritematosus, diabetes) • TLRs activation is reported in different pahtological states within CNS => MODULATION OF TLRs (opioid-resistant chronic pain, neurodegenerative diseases) • Xenobiotics and drugs may mis-activate TLRs => ADVERSE EFFECTS (Opioids, Antidepressants) CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

12. TLR AGONISTS AND CANCER TLR-mediatedstimulationofadaptiveimmunity and activationofNKs and cytotoxicT-cellsmediatesantitumoureffects • In the 19th Century William Coley observed antitumour effects of repeated injections of toxins from dead Gram+ and Gram- bacteria => activation of TLR4 and TLR9 • Imiquimod, one of the first and most successful drugs targeting TLRs, determines a TLR7-dependent secretion of the antiviral and antitumour cytokine IFNa • Individuals with breast cancer carrying a loss-of-function allele of TLR4 relapsed much quicker following radiotherapy and chemotherapy than their wild-type counterparts • Hennessey EJ et al. Nature Reviews Drug Discovery 2010 QUEST OF TLRs AGONISTS FOR NOVEL ANTICANCER THERAPIES CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

13. TLR AGONISTS AND CANCER: Drugspresently under investigation CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

14. TLR AGONISTS AND CANCER: Perspectives and drawbacks POLY-TLR AGONISTS Cadi-05 (autoclavedmycobacteriatargetingseveralTLRs) isbeingevaluatedfor the treatment ofadvanced melanoma, prostate and bladdercancer => in phase I trial 5/9 becameasymptomatic and showed no diseaserecurrence at 2 years LACKING OF COMPELLING RATIONALE IN HUMAN CLINICAL TRIALS MANY CANCER PATIENTS ARE IMMUNOSUPPRESSED CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

15. TLR ANTAGONISTS Inappropriately overactive immune system TLR ANTAGONISTS • HCQ (TLR9 antagonist, TLR7 and TLR8 weak antagonist) is the current treatment for SLE • CPG-52364 (TLR7, TLR8, TLR9 antagonist) is being evaluated in a Phase I clinical trial • TLR7 – TLR9 dual antagonists are being developed for the treatment of SLE, rheumatoid arthritis, multiple sclerosis, colitis, psoriasis. • Eritoran (TLR4 antagonist) decreased by 6.4% the mortality due to sepsis in a Phase II clinical trial; Phase III is presently under initiation • Ibudilast (TLR4 antagonist) is being studied in advanced clinical trials for the treatment of chronic pain states and addiction withdrawal and TLR4 antagonistic antibodies are very promising anti-inflammatory drugs in animal models of colitis • Poly I:C (dsRNA molecule; TLR3 agonist) => treatment of Hepatitis B, Hepatitis C, HIV, influenza CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

16. TOLL-LIKE RECEPTORS AND CENTRAL NERVOUS SYSTEM TLRs are differentially expressed by the various cell type within the CNS CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

17. NEURON-GLIA INTERACTIONS Glia crucial role in the maintenance of neuronal homeostasis Neurogenesis Myelination Synaptic plasticity Neuronal migration, proliferation and differentiation ASTROCYTES Trophic support to neurons Clearing cell debris Taking up released transmitters MICROGLIA Active immune surveillance CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

19. TLRs AND CENTRAL NERVOUS SYSTEM TLRs in the CNS not only control host-defense responses but play also important roles in tissue development, cellular migration and differentiation, limiting inflammation and mounting repair processes following trauma Type, duration and strength of TLRs activation Neuroprotection Neurodegeneration CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

20. NEUROPATHICPAIN PAIN MORPHINE-MEDIATED ACTIVATION OF TLR4!!! FIRST STEP: damage to neuronal fibers Skrik, Edvard Munch • SECOND STEP: • Glial cell activation (crucial role of Toll-like receptors) • Release of pro-inflammatory mediators which acts on neurons and glial cells Chronic pain condition in which opiod analgesics are ineffective or detrimental CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

21. NEURON-TO-GLIA SIGNALS IN NEUROPATHIC PAIN: maladaptiveplasticitywithin the nociceptive system Maladaptiveplasticity and activationofglialcells

22. TLR4: A NOVEL TARGET FOR NEUROPATHIC PAIN THERAPY? TLR4 ANTAGONISTS combined with opioids OPIOID AGONISTS that do not bind to TLR4 CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

23. TLRs MIS-ACTIVATION: A SOURCE OF DRUG-MEDIATED SIDE EFFECTS? Xenobiotics and drugs may mis-activate TLRs OPIOIDS TLRs as novel drug targets: Finding AGONISTS Finding ANTAGONISTS Checking if drugs not directed to TLRs may activate them ANTIDEPRESSANTS OTHERS ? CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

24. TARGETING TOLL-LIKE RECEPTORS: EXPERIMENTAL STRATEGIES Screening molecules to evaluate their activity towards TLRs Library of putative antagonists Library of putative agonists TLR agonist TLR agonist Library of miRNA or small molecules BLOCK OF RECEPTOR ACTIVATION BLOCK SPECIFIC SIGNALLING RECEPTOR ACTIVATION CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

25. IS THERE IN TLRs INTRACELLULAR SIGNALLING AN EFFECTOR WICH IS COMMONLY ACTIVATED? NF-kB

26. SCREENING OF TLR LIGANDS: MeasuringNF-kBactivation Screening molecules to evaluate their activity towards TLRs Library of putative antagonists Library of putative agonists TLR agonist TLR agonist Library of miRNA or small molecules MODULATION OF NF-kB ACTIVATION BLOCK OF NF-kB ACTIVATION NF-kB ACTIVATION CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

27. EVALUATING NF-kB ACTIVATION: Traditionalapproaches Nuclear translocation by WB Binding to responsive elements by EMSA IkB phosphorylation by WB INDIRECT, NON HOMOGENEOUS, TIME CONSUMING; BETTER FOR RESEARCH THAN FOR SCREENING OF LIBRARIES CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

28. EVALUATING NF-kB ACTIVATION: Innovative strategies NF-kB reporter vectors QUICK, SENSITIVE, OMOGENEOUS SUITABLE FOR AUTOMATION AND HTS APPLICATIONS CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

29. ENZYME Promoter RNApol STIMULUS NF-kB NF-kB RE + Minimal Promoter REPORTER VECTORS: Generalmechanismofaction TF TF REPORTER GENE CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

30. Reporter Reporter Drug NF-kB RE NF-kB RE NFkB Enzyme RNApol IkB S TLR Nucleus CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

31. Normalising results - Dual Reporters First reporter (e.g. luc) Experimentalplasmid Cells Control plasmid Second reporter(e.g. CAT, ß-Gal) Normalised = Experimental reporterresponse Control reporter CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

32. Current Dual Reporter Systems • Cells may have endogenous -Gal activity. • CAT, -Gal assays are endpoint assays. • Can’t combine luciferase, CAT, -Gal in a single-tube format that is equally sensitive and rapid for both reporter assays. CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

33. Cells Dual-Luciferase Reporter Assay Experimentalplasmid FF LUC Control plasmid R LUC • Two luciferase reporter enzymes • Sequential quantification of Firefly luciferase and Renillaluciferase • Distinct evolutionary origin CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

34. Firefly luciferase reaction Luciferases: Monomeric, 61 kDa Co-substrates: ATPMg2+, CoA Luciferin: Photinus pyralis Luciferase Oxyluciferin +AMP+ PPi + CO2 + light Luciferin + ATP + O2 CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

35. Luciferase Renilla luciferase reaction Sea Pansy (Renilla reniformis) Coelenteramide + CO2 + light Coelenterazine + O2 Luciferases: -Monomeric, 36 kDa (Renilla) Coelenterazine: CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

36. 1. Rapid(30s manual assay,7s automated assay) 2. Sensitive 3. Linear 4. Precise(both measurementsfrom a single sample) Dual-Luciferase Assay Format Light Output (Renilla) Stop & GloTM Reagent Light Output (Firefly) Luciferase assay reagent Sample CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

37. Firefly LUC NF-kB RE Renilla LUC TK TARGETING TOLL-LIKE RECEPTORS: NF-kBDualLuciferasesystems • PRO • Physiological TLR expression levels • Specific cell-type features • Reporter enzyme sequentially detectable in lysates => OMOGENEUS CELL LINES EXPRESSING TLRs TO BE TRANSFECTED WITH THE REPORTER SYSTEM OF INTEREST • CONS • Reporter enzyme accumulates => less sensitivity and kinetics hardly measurable CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

38. Destabilized Luciferase Genes Intracellular Reporter Pool Non-destabilized Newly expressed Reporter Signal Destabilized Signal Newly expressed Reporter CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

39. Greater induction Reporter intracellular reporter pool Destabilized Reporter intracellular reporter pool Induction detection sooner Destabilized Luciferase Genes CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

40. CHRONIC PAIN, TLRs AND DRUG DISCOVERY: Chronic pain condition in which opiod analgesics are ineffective or detrimental because of opioid-mediated TLR4 activation!!! SEARCH OF OPIOID ANALGESICS THAT DO NOT ACTIVATE TLR4 IN GLIAL CELLS CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

41. CHRONIC PAIN, TLRs AND DRUG DISCOVERY: OUR CELL MODEL AND REPORTER VECTORS FFLuc U-87 MG HUMAN GLIOBLASTOMA CELL LINE • Physiologicalexpressionlevelsof TLR4 • Goodmodelofhuman glia RLuc CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

42. CHRONIC PAIN, TLRs AND DRUG DISCOVERY: OUR TREATMENTS LPS from E. Coli TLR4 agonist POSITIVE CONTROL Morphine The prototypical OPIOID ANALGESIC DAMGO – peptidic opioid agonist CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

43. CHRONIC PAIN, TLRs AND DRUG DISCOVERY: OUR DUAL-LUCIFERASE ASSAY CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

44. CHRONIC PAIN, TLRs AND DRUG DISCOVERY: OUR EXPECTED RESULTS LPS DAMGO Morphine TLR4 ? CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS

45. TARGETING TOLL-LIKE RECEPTORS: NOVEL POTENTIAL THERAPEUTICS • TLRs are crucial for both host defense against pathogens and for a rapid response to local tissue damages. • Because of TLRs involvement in immune responses as well as in several diseases related to inflammation, targeting TLRs may represent a novel and effective therapeutic opportunity. • NF-kB Dual Luciferase Reporter systems represent an useful experimental tool to screen library of putative TLRs ligands in a rapid, sensitive, linear and precise way. CHEMICAL AND GENOMICS-BASED STRATEGIES IN THE DISCOVERY OF NOVEL DRUG TARGETS